Brief Summary of Minutes of Annual Meeting

BRIEF SUMMARY OF MINUTES OF ANNUAL MEETING Annual meeting of the technical committee, October 9-10, 2008 Hilton Waikaloa, Hawaii. The annual meeting was called to order at 0800 Thursday morning, October 9th. After brief introduction and discussion of the meeting format and agenda, the annual reports were presented. The regular business portion of the meeting commenced Thursday afternoon after the annual report presentations. Calistoga was selected as the site for the 2009 meeting and it was agreed that the 2009 meeting would meet Thursday the 8th and Friday the 9th of October. For 2009, David Williams, Oregon State University, would serve as Secretary, Pratibha Nerurkar (University of Hawaii) as Chair-elect, and Mike Denison (University of California, Davis) as Chair. The responsibilities of the Secretary, Chair-elect and Chair were discussed and it was agreed that the Chair writes the report and chairs the meeting, the Chair-elect organizes the next years meeting working closely with the local host and also assists the Chair in writing the report, the Secretary writes the minutes. Submission of the Report and the minutes are the responsibility of the Chair and the Secretary. The meeting was adjourned at around 5:00 pm on 9 October.

ACCOMPLISHMENTS 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. Poultry was developed as a model for sensitivity to dietary carcinogens, like aflatoxin B1 (AFB1), and responsiveness to chemopreventives such as phenolic antioxidants. Modern commercial turkeys are extremely sensitive to the toxic effects of AFB1, a condition associated with a combination of efficient cytochrome P450 (CYP)-mediated bioactivation of AFB1 to the reactive exo-AFB1-8,9-epoxide (AFBO), and a deficiency of AFBO detoxifying glutuathione S-transferases. Conversely, wild and heritage turkeys, which are relatively resistant to AFB1, possess hepatic GSTs that can detoxify AFBO. This profound difference in GST activities may be a result of the loss of allelic variation in commercial turkeys from intensive breeding. Thus, GSTs appear to play a central role in AFB1 susceptibility and resistance. Using 5-3 RACE, we cloned and sequenced four alpha-class GSTs from the livers of domestic turkeys. Single nucleotide polymorphisms (SNPs) have mapped the GST gene cluster to turkey chromosome MGA2. Based on the chicken sequence, we identified several transcription factors in the 5-regulatory elements of these GSTs, and their significance in GST regulation is under investigation. This genomic approach is aimed at identifying genetic markers related to AFB1 susceptibility and resistance in turkeys with the ultimate goal of re-introducing AFB1-protective alleles back into the genetic stock of commercial turkeys. In humans, hepatic cytochromes P450 (CYP) 1A2 and 3A4 bioactivate AFB1 to exo-AFB1-8,9-epoxide (AFBO) which is a requisite step in the toxic and carcinogenic action of this mycotoxin. In turkeys, AFB1 is bioactivated by human homologues CYP3A37 and CYP1A5. Heterologously expressed turkey CYP1A5 metabolizes AFB1 to AFBO and the detoxified metabolite aflatoxin M1 (AFM1) as human CYP1A2. Turkey CYP3A37 cloned from liver RNA was 76% identical to human CYP3A4, and the E. coli expressed protein CYP3A37 possessed metabolic similarities to human CYP3A4. Like CYP3A4, CYP3A37 efficiently metabolized AFB1 to exo-AFBO and the detoxified metabolite aflatoxin Q1 (AFQ1). Formation of exo-AFBO was inhibited by the specific CYP3A4 inhibitor 17b-ethynylestradiol (IC50 = 40.18 µM), while prototype inhibitors to other mammalian isoforms (1A2, 2D, 2E and 3A1) either slightly inhibited this activity, or not at all. Like its human homologue, CYP3A37 also had nifedipine oxidase activity. The gene, which is organized into 13 exons and 12 introns, has an ORF of 1512 bp, and the protein is predicted to be 504 amino acids with 97% homology to chicken CYP3A37. A single nucleotide polymorphism in the 11th intron was used to assign CYP3A37 to turkey linkage group 10. Because of the importance of P450s in the extreme sensitivity of turkeys to the toxic effects of AFB1, this study may be useful in identifying allelic variants of this gene in poultry. Polyclonal anti-CYP3A37 antibody directed against specific active-site peptides inhibited both AFBO formation and nifedpine oxidase activity. The comparative kinetic constants for exo-AFBO formation of expressed CYP3A37 and CYP1A5 lead us to believe that CYP3A37 may be the predominant AFB1 epoxidating enzyme in turkey liver. Ongoing immunoinhibition studies will examine this hypothesis. (Utah State University) The addition of the phytochemical, indole-3-carbinol (I3C), found in high concentrations in cruciferous vegetables, to the maternal diet of mice during pregnancy and lactation, markedly protected her offspring from transplacental carcinogenesis well into their adult lives. These studies were carried out in collaboration with Dr. Len Bjeldanes at the University of California, Berkeley. We found similar, although not as striking, results if green tea was utilized. Chlorophyllin, the derivative of the plant pigment chlorophyll, was very effective in inhibition of transplacental carcinogenesis due to administration of the polycyclic aromatic hydrocarbon (PAH), dibenzo[a,l]pyrene (DBP) to the pregnant mouse. (Oregon State University) Analysis of a variety of naturally occurring flavonoids and a large number of synthetic flavonoids and benzoflavones for their ability to affect nuclear receptor signaling pathways, particularly those of the Ah receptor (AhR). Utilizing recombinant cell lines that we have developed that express firefly luciferase in an AhR-dependent manner, our screening demonstrated that the majority of the naturally occurring flavonoids were weak agonists of the AhR, more than 20 synthetic flavonoids were identified as 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, provides strong support for differential ligand binding of chemicals to the AhR. Whether the differential interaction of flavonoids with the AhR contributes to the lack of toxic and biological effects by these chemicals, compared to those observed with TCDD and related chemicals, remains to be determined. (University of California, Davis). Breast cancer is the most commonly diagnosed cancer in women. Seventy five percent of breast cancers occur in postmenopausal women and the majority of these are estrogen dependent, which respond to antiestrogen like tamoxifen. It is observed that the dietary isoflavone supplements have been consumed by more American women, including the postmenopausal women with breast cancer by assuming that soy intake would be a safe alternative to hormone replacement therapy. Genistein, a predominant isoflavone in soy, acts as an estrogen agonist at physiologically relevant dosages and has shown to stimulate growth of estrogen-responsive breast cancer cells. If the breast cancer patients with tamoxifen treatment consume considerable isoflavone supplements, the potential combined effect of tamoxifen and genistein may result in significant undesirable outcome on mammary tumors. In this study, we investigated interaction of three dietary dosages of genistein with tamoxifen on the growth of estrogen-dependent breast cancer (MCF-7) cells implanted in ovariectomized athymic mice. We found that 250ppm and 500ppm genistein induced mammary tumor growth over the inhibitory effect of tamoxifen in a dose dependent manner. However at higher dosage, 1000ppm genistein did not negate the effect of tamoxifen. It is important to note that high incidence of tumor was observed in the 1000ppm treatment group. The high rate of apoptosis in this group may explain the small tumor size. Mice tumor characterization using primary culture indicates these tumors retain the estrogen dependent phenotype. These studies demonstrate that the interaction of genistein with tamoxifen is complex, involving multiple mechanisms that are dose-dependent. (University of Illinois) The consumption of the trichothecene mycotoxin deoxynivalenol (DON) induces interleukin-6 (IL-6)-dependent IgA nephropathy (IgAN) in mice and this effect can be prevented by feeding long chain n-3 polyunsaturated fatty acids (PUFAs) found in fish oil. Identificaiton of the signal transduction pathways by which DON upregulates IL-6 in the peritoneal macrophage and how consumption of fish oil enriched with the n-3 PUFA, docosahexaenoic acid (DHA), suppresses these processes is critical. Incubation with DON induced IL-6 expression in naïve macrophages maximally at 3 h. Knockdown of the transcription factor cAMP response element-binding protein (CREB) or pharmacologic inhibition of the CREB kinases, Akt1/2, MSK1 and RSK1, downregulated this expression. Inhibition of double-stranded RNA-activated protein kinase (PKR) suppressed not only IL-6 expression but also phosphorylation of CREB and its upstream kinases, Akt1, MSK1 and RSK1. Phosphorylations of PKR, CREB kinases and CREB were markedly impaired in peritoneal macrophages isolated from mice that consumed DHA-enriched fish oil for 6 to 8 wk. DHAs effects were not explainable by increased activity of protein phosphatase 1 and 2A since both were suppressed in mice consuming the DHA diet. Although cells cultured directly with DHA expressed less IL-6 compared to cells cultured with arachidonic acid (AA), neither fatty acid treatment affected DON-induced protein phosphorylation. Furthermore, DHA and AA similarly inhibited cell-free protein kinase activity. These data suggest that DON-induced IL-6 expression is CREB-mediated and PKR-dependent and that requisite kinase activities for these pathways were suppressed in macrophages from mice fed DHA for an extended period. (Michigan State University) Obesity-associated diabetes and hyperlipidemia are escalating worldwide. Changes in life style, such as dietary recommendations form the cornerstone of treatment. Our specific aim is to investigate the signaling mechanisms involved in the anti-obesity, anti-diabetic and hpolipidemic properties of Momordica charantia (bitter melon, BM). Traditionally, BM is used to treat diabetes and its complications. Our studies demonstrate that BM also reduces adiposity and plasma and hepatic lipids in mice fed high fat diet (HFD). We further demonstrated that BM lowers plasma apolipoprotein B (apoB) by improving hepatic insulin receptor phosphorylation and its downstream signaling molecules. (University of Hawaii) OBJECTIVE 2. Identify mechanisms of action and biomarkers of natural and induced toxicants in food for human risk assessment and disease prevention. Collaborative studies with scientists at the University of Nebraska Medical Center have focused on determination of the possible role of fumonisin-induced elevation of sphingoid base 1-phosphates, ligands for S1P Receptors (S1P1-5) as a risk factor for neural tube defects in mice. 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 could be explored in Guatemala. In support of developing mechanism-based biomarkers for detecting fumonisin-induced effects in humans, we also found that human consumption of food products containing low levels of fumonisins (below the USFDA guidelines of 4 ppm) could cause easily detectable elevation of fumonisin B1 in human urine but at the levels consumed had no significant effect on sphingoid base 1-P levels in blood. In collaboration with researchers in Guatemala, a questionnaire was developed to identify heavy consumers of maize products in two areas of Guatemala. A study was initiated to determine if urinary FB could be used as an exposure marker. (USDA-ARS, Athens) A possible role for 1-dexoxysphingoid bases and 1-deoxydihydroceramides in FB hepatotoxicity has been examined. In cultured cells, inhibition of ceramide synthase (CerS) by FB1 increases sphinganine (Sa), Sa 1-phosphate and a previously unidentified metabolite, believed to be a novel sphingoid base. In this study, the metabolite was analyzed by mass spectrometry (MS) and assigned a m/z of 286.3123 in positive ionization mode, consistent with a 1- or 3-deoxysphinganine (deoxySa), C18H40NO. Mass spectrometry suggested that that the putative deoxySa was most likely formed from the utilization of alanine by serine palmitoyl transferase (SPT), the first and rate-limiting enzyme in sphingolipid biosynthesis. Inhibition of SPT with myriocin blocked the formation of both Sa and 1-deoxySa. Labeling studies using L-alanine-U-13C3 and L-serine-U-13C3 showed the preferential incorporation of alanine into 1-deoxySa, confirming that 1-deoxySa arises from condensation of alanine with palmitoyl-CoA via SPT. In LLC-PK1 cells, Vero cells, and other cell lines, 1-deoxySa accumulated to high levels after treatment with FB. 1-DeoxySa was also detected in liver and kidney of P53N5 mice and increased in mice fed FB diets. Cells treated with FB plus 10 µM 1-deoxySa showed a 25-fold elevation in 1-deoxySa compared to the FB-only treated cells and a 42-fold elevation compared to the cultures treated with only 10 µM 1-deoxySa. Conversely, in LLC-PK1 cells treated with 10 µM 1-deoxySa, the level of total N-acyl-1-deoxySa (i.e., 1-deoxydihydroceramides) went from 32.2 to 4,634 pmol/mg protein. Co-treatment with FB reduced the increase to 396 pmol/mg protein, indicating that the endogenous 1-deoxySa is acylated by CerS. 1-Deoxydihydroceramides were also easily detected in mouse liver. These findings implicate 1-deoxy sphingoid bases in diseases caused by FB, and they may play important roles in cell regulation. (USDA-ARS, Athens) PAHs are of increasing environmental concern. They are formed from the burning of organic materials, especially fossil fuels. With the large demand for energy production throughout the world, increasingly PAHs are being generated from the burning of coal, gasoline, diesel, etc. These PAHs get deposited on food and the majority of human exposures to PAHs are dietary. We have conducted studies employing Cyp1b1 knockout mice to demonstrate that transplacental cancer (primarily lymphoma) is DBP-dependent transplacental induction of lymphoma is dependent upon fetal expression of Cyp1b1. These results provide us with a new gene target for future cancer chemoprevention studies. In subsequent work, utilizing a cross-foster approach, where litters are switched after birth between control mothers and those exposed to DBP, that the majority of cancer in the offspring was from the short (2-3 day) in utero exposure rather than the 3 weeks of nursing. These studies have identified a critical window for prevention efficacy. (Oregon State University) The frequent presence of DON in cereal-based foods and the high intake of these foods by children raises particular concerns about the relative susceptibility of this subpopulation to adverse effects evoked by this mycotoxin. We tested the hypothesis that both toxicokinetics and proinflammatory cytokine gene expression following a oral DON exposure at 5 mg/kg bw differ between weanling (3-4 wk) and young adult (8-10 wk) female mice. DON was rapidly taken up with maximum plasma concentrations reaching 1.0 g/ml in adult mice at 15 min, whereas DON levels were approximately twice as much in weanling mice at these times. DON was rapidly cleared in both weanling and adult mice with concentrations being reduced by 78 and 81% of the peak levels, respectively, after 2 h. DON accumulation and clearance in spleen, liver, lung and kidney followed similar kinetics to that of plasma with tissue burdens also reaching twice that of adult mice. When TNF-, IL-1b and IL-6 mRNAs in spleens (a primary source of systemic proinflammatory cytokines) were used as biomarkers of the effects of DON, expression of these mRNAs was two to three times greater in weanling than adult mouse. However, differences in proinflammatory cytokine expression were less robust or not apparent in the liver or lung. Accordingly, these data suggest 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. (Michigan State University) Translational inhibitors such as DON and ribosomal inhibitory proteins (RIPs) induce mitogen-activated protein kinase (MAPK)-driven chemokine and cytokine production by a mechanism known as the ribotoxic stress response (RSR). Double-stranded RNA-activated protein kinase (PKR) associates with the ribosome in close proximity to the peptidyl transferase center making it uniquely positioned to sense 28S rRNA damage and initiate the RSR. We have previously shown that PKR mediates DON-induced MAPK phosphorylation in macrophages and monocytes. The purpose of this study was to test the hypothesis that PKR is essential for induction of IL-8 expression in monocytes by DON and two prototypical RIPs, ricin and Shiga toxin 1 (Stx-1). Preincubation of human monocytic U937 cells with the PKR inhibitors C16 and 2-aminopurine (2-AP) blocked DON-induced expression of IL-8 protein and mRNA. Induction of IL-8 expression was similarly impaired in U937 cells stably transfected with a dominant negative PKR plasmid (UK9M) as compared to cells transfected with control plasmid (UK9C). NF-kB binding, which has been previously shown to be a requisite for DON-induced IL-8 transcription, was markedly reduced in UK9M cells as compared to UK9C cells. As observed for DON, ricin- and Stx-1-induced IL-8 expression was suppressed by the PKR inhibitors C16 and 2-aminopurine as well as impaired in UK9M cells. Taken together, these data indicate that PKR plays a common role in IL-8 induction by DON and the two RIPs, suggesting that this kinase might be a critical factor in RSR. (Michigan State University) OBJECTIVE 3. Discover bioactive compounds that have beneficial or adverse effects on human health. Perfluorinated compounds, typified by perfluorooctanoic acid (PFOA), are ubiquitous environmental pollutants. These compounds have a number of industrial uses, included the manufacture of Teflon and similar coatings. We have found that dietary exposure of trout to PFOA resulted in alterations in gene expression very similar to the pattern obtained with estrogens; hence, PFOA, at least in fish, is a xenoestrogens. As with other estrogens, PFOA is also a promoter of carcinogen-initiated liver cancer in this model. (Oregon State University) Liquiritigenin is a plant-derived highly selective estrogen receptor beta (ERb) agonist. After the Women's Health Initiative found that the risks of hormone therapy outweighed the benefits, a need for alternative drugs to treat menopausal symptoms has emerged. We explored the possibility that botanical agents used in Traditional Chinese Medicine for menopausal symptoms contain ERb-selective estrogens. We previously reported that an extract of 22 herbs, MF101, has ERb-selective properties. In this study we 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. The ERb-selectivity of liquiritigenin was due to the selective recruitment of the coactivator steroid receptor coactivator-2 to target genes. In a mouse xenograph model, liquiritigenin did not stimulate uterine size or tumorigenesis of MCF-7 breast cancer cells. Our results demonstrate that some plants contain highly selective estrogens for ERb. (University of California, Berkeley) Novel estrogenic therapies are needed that have the bone-sparing effects of endogenous estrogens but do not promote breast or uterine cancer. Recent evidence suggests that selective activation of the ERb subtype inhibits breast cancer cell proliferation. We showed previously that DIM has estrogenic activity. We investigated whether the estrogenic activities of DIM are ERb-selective. DIM promoted ERb but not ERa activation of an estrogen response element upstream of the luciferase reporter gene. DIM also selectively activated multiple endogenous genes through ERb. The activation of ERb induced by DIM allows ERb to bind regulatory elements and recruit the GRIP1 coactivator leading to the activation of ER target genes. DIM did not bind to ERb, indicating that it activates genes by a ligand-independent mechanism. Our results demonstrate that DIM 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. (University of California, Berkeley) The AhR regulates the expression of a battery of genes in a wide range of species and tissues and is responsible for mediating the toxic and biological effects of TCDD and related halogenated aromatic hydrocarbons (HAHs) and polycyclic aromatic hydrocarbons (PAHs). Differences in the toxic potency of HAHs and PAHs, the latter not producing AhR-dependent toxicity, suggested that these chemicals may differentially bind to and activate the AhR. We have demonstrated that the prototypical HAH (TCDD) and PAH (beta-naphthoflavone (BNF)) can differentially interact with the binding pocket of Ah receptor. This was accomplished by our identification of a novel chemical (MSD237) from high-throughput screening analysis that could inhibit the ability of TCDD, but not BNF, block to bind to AhR and activate AhR-dependent signal transduction pathway. Like that of other AhR ligands, the binding of MSD237 to the AhR ligand binding pocket was essentially irreversible. These results provide insights into the structural diversity of AhR ligands as these ligands may differentially interact with the ligand binding pocket and suggest that differential interactions of HAHs and PAHs with the AhR may contribute to differences in their ability to produce AhR-dependent toxicity. In addition, the use of MSD237 as a potential therapeutic agent to block the biological and toxicological effects of HAHs is being examined. (University of California, Davis) We have further developed our 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. The essential role of selected residues was further evaluated using molecular docking simulations of TCDD to both wild-type and mutant mAhRs. Further refinement of the docking simulation with TCDD and other known AhR ligands will facilitate the development of a model that can be used for screening purposes to identify new AhR ligands. Taken together, our results allow for a more complete understanding of the molecular determinants of TCDD binding and provides a basis for future studies directed toward rationalizing the observed species differences in AhR sensitivity to TCDD and understanding the mechanistic basis for the dramatic diversity in AhR ligand structure. (University of California, Davis) Anti-diabetic properties of BM, have been attributed to their high antioxidant properties due in part to phenols, flavonoids, isoflavones, terpenes, anthroquinones, and glucosinolates. Momordicosides and triterpenoids specifically demonstrated hypoglycemic effects in diabetic mice. Most of the studies, demonstrating an effect on body weights, and lipids were conducted using whole BM juice (BMJ) and the active ingredients remain unknown. We chemically fractionated BMJ and tested in mouse and human adipocyte cell cultures as well as human liver cell cultures to test the effect on lipid accumulation in the cells. Our results demonstrate that whole BMJ was more potent in lowering cellular lipids in mice adipocytes and human liver cells than any fraction alone. (University of Hawaii) OBJECTIVE 4. Develop strategies to increase beneficial or decrease adverse effects of bioactive food constituents and microbial contaminants. Fumonisin translocation and disruption of sphingolipid metabolism in maize seedling, developing strategies for resistance to Fusarium verticillioides maize seedling diseases. The ability of maize seedlings to translocate pure fumonisins from soil into aerial plant parts (leaves) was assessed. It was found that in maize seedlings watered with pure fumonisin B1, pure fumonisin B2 or the combination of B1 and B2, fumonisin B1 was preferentially accumulated in root tissue but was not significantly translocated into leaf tissue. Conversely, the fumonisin that entered roots caused significant elevation in sphingoid bases and sphingoid base 1-phosphates in roots and these were translocated into leaf tissues. This finding was different than what was seen when seedlings were grown from seeds inoculated with the fungus that produces fumonisins. In this case both fumonisin B1 and B2 accumulated in roots whereas only fumonisin B1 was appreciably translocated into leaf tissue. The results suggest that the fungal/plant/soil interaction contributes to the ability of fumonisins to enter the roots and be translocated into leaf tissues. This is important because plant debris 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. (USDA-ARS, Athens) We 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. (USDA-ARS, Albany) 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. (USDA-ARS, Albany) In a collaborative study, we found that cinnamaldehyde, cinnamon oil, carvacrol, oregano oils, 2,5-dihydroxybenzaldeyde, and 2-hydroxy-2-methoxyenzaldehyde inactivated Mycobacterium avium paratuberculosis that infects cattle causing Johns diseases 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. (USDA-ARS, Albany) A collaborative modeling study has 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. (USDA-ARS, Albany) We developed HPLC and LC/MS assays for flavonoids present in fresh, light exposed, and home-processed onions, and in commercial onion products. Improved HPLC and LC/MS assays for determining the content of flavonoids in onions, phenolic compounds in potatoes, and piperamides in peppercorns were also developed. Together, these studies facilitate ongoing efforts to relate content biologically active compounds in plant extracts to inhibitory activities against food-borne pathogens and bacterial toxins. (USDA-ARS, Albany)

PUBLICATIONS Ahn, K.C., Zhao, B., Chen, J., Cherednichenko, G., Sanmarti, E., Denison, M.S., Lasley, B., Pessah, I.N., Kultz, D., Chang, D.P., Gee, S.J. and Hammock, B.D. (2008) In vitro biological activities of the antimicrobials triclocarban, its analogs, and triclosan in bioassay screens: receptor-based bioassay screens, Environ. Health Perspect. 116: 1203-1210. Amuzie, C. J., Harkema, J. R., and Pestka, J. J. (2008) Tissue distribution and proinflammatory cytokine induction by the trichothecene deoxynivalenol in the mouse: comparison of nasal vs. oral exposure. Toxicology 248: 39-44. Bae, H. K., and Pestka, J. J. (2008) Deoxynivalenol induces p38 interaction with the ribosome in monocytes and macrophages. Toxicol Sci 105: 59-66. Beli, E., Li, M., Cuff, C., and Pestka, J. J. (2008) Docosahexaenoic acid-enriched fish oil consumption modulates immunoglobulin responses to and clearance of enteric reovirus infection in mice. J Nutr. 138: 813-819. Bohonowych, J.E. and Denison, M.S. (2007) Persistent binding of ligands to the aryl hydrocarbon receptor, Toxicol. Sci. 98: 99-110. Brown, D. J., Orelien, J., Gordo, J. D., Chu, A. C., Chu, M. D., Murata, H. J., Kayama, F., Denison, M. S. and Clark, G. C. (2007) Mathematical model developed for environmental samples: prediction of GC/MS dioxin TEQ from XDS-CALUX bioassay data, Environ. Toxicol. Chem. 41: 4354-4360. Burns, T.D., Snook, M.E., Riley, R.T. and Voss, K.A. (2008) Fumonisin concentrations and in vivo toxicity of nixtamalized Fusarium verticillioides culture material: Evidence for fumonisin-matrix interactions. Food Chem Toxicol 46: 2841-2848. Carter, O., Dashwood, R.H., Wang, R., Dashwood, W.M., Orner, G.A., Fisher, K.A., Löhr, C.V., Pereira, C.B., Bailey, G.S. and Williams, D.E. (2007) Comparison of White Tea, Green Tea, Epigallocatechin-3-Gallate and Caffeine as Inhibitors of PhIP-Induced Colonic Aberrant Crypts. Nutr Cancer 58: 60-65. Castro, D.J., Löhr, C., Fischer, K., Waters, K., Webb-Robertson, B.-J., Dashwood, D.H., Bailey, G.S. and Williams, D.E. (2008) Identifying Efficacious Approaches to Chemoprevention with Chlorophyllin, Purified Chlorophylls and Freeze-Dried Spinach in a Mouse Model of Transplacental Carcinogenesis. Carcinogenesis 30: 315-320. Castro, D.J., Baird, W.M., Pereira, C., Giovanini, J., Löhr, C., Fischer, K., Yu, Z., Gonzalez, F.J., Krueger, S.K. and Williams, D.E. (2008) Fetal Cyp1b1 in Mice and Transplacental Carcinogenesis From Maternal Exposure to Dibenzo[a,l]pyrene. Cancer Prev Res 1: 128-134. Castro, D.J., Yu., Z., Löhr, C.V., Pereira, C.B., Giovanini, J., Fischer, K.A., Orner, G.A., Dashwood, R.H., and Williams, D.E. (2008) Chemoprevention of Dibenzo[a,l]pyrene Transplacental Carcinogenesis in Mice Born to Mothers Administered Green Tea: Primary Role of Caffeine. Carcinogenesis 29: 1581-1586. Castro, D.J., Yu, Z., Dashwood, R.H., Bailey, G.S., Baird, W.M. and Williams, D.E. (2008) A Model for the Study of Maternal Dietary Inhibition of Transplacental Carcinogenesis. In Recent Advances in Carcinogenesis, (R.M. Mohan, Ed.). Castro, D.J., Löhr, C.V., Fischer, K.A, Pereira, C. and Williams, D.E. (2008) Lymphoma and Lung Cancer in Offspring Born to Pregnant Mice Dosed with Dibenzo[a,l]pyrene: The Importance of In Utero Versus Lactational Exposure. Toxicol Appl Pharmacol 233: 454-458. Chang, E.C., Charn, T.H., Park, S.H., Helferich, W.G., Komm, B., Katzenellenbogen, J.A. and Katzenellenbogen, B.S. (2008). Estrogen Receptors alpha and beta as determinants of gene expression: influence of ligand, dose, and chromatin binding. Mol Endocrinol 22: 1032-1043. Coulombe, R.A. and Yip, S.M. (2007) Heterologous expression of a cytochrome P450 from turkey liver that activates aflatoxin B1. In Poisonous Plants: Global Research and Solutions (K. Panter, T. Wierenga, J. Phister, eds.) CAB International, London. pp. 82-88. Du, W.-X., Olsen, C.E., Avena-Bustillos, R.J., McHugh, T.H., Levin, C.E. and Friedman, M. (2008) Antibacterial activity against E. coli O157:H7, physical properties, and storage stability of novel carvacrol-containing edible tomato films. J Food Sci 73: M378-M383. Du, W.-X., Olsen, C.W., Avena-Bustillos, R.J., McHugh, T.H., Levin, C.E. and Friedman, M. (2008) Storage stability and antibacterial activity against Escherichia coli O157:H7 of carvacrol in edible apple films made by two different casting methods. J Agric Food Chem 56: 3082-3088. Dvorak, N., Riley, R.T., Harris M. and McGregor, J.A. (2008) Fumonisin mycotoxin contamination of corn-based foods consumed by potentially pregnant women in Southern California. Journal Reprod Mede 53: 672-676. Friedman, M. (2008) Dietary significance of processing-induced lysinoalanine in food. In "Process-Induced Food Toxicants: Occurrence, Formation, Mitigation, and Health Risks", Stadler, R.H. and Lineback, D.R., Eds., John Wiley & Sons, New York, Chpt. 6.1, pp. 477-510. 2009. Friedman, M. (2008) Dietary significance of processing-induced D-amino acids. In "Process-Induced Food Toxicants: Occurrence, Formation, Mitigation, and Health Risks", Stadler, R.H. and Lineback, D.R., Eds., John Wiley & Sons, New York, Chpt. 6.2, pp. 511-537. 2009. Friedman, M. and Levin, C.E. (2008) Review of methods for the reduction of dietary content and toxicity of acrylamide. J Agric Food Chem 56: 6113-6140. Friedman, M., C. E. Levin, C.E., Lee, S.-U., Lee, J.-S., Ohnisi-Kameyama, M. and Kozukue, N. (2008) Analysis by HPLC and LC/MS of pungent piperamides in commercial black, white, green, and red whole and ground peppercorns. J Agric Food Chem 56: 3028-3036. Glenn, A.E., Zitomer, N.C., Zimeri, A.M. , Williams, L.D., Riley, R.T. and Proctor, R.H. (2008) Transformation-mediated complementation of a FUM gene cluster deletion in Fusarium verticillioides restores both fumonisin production and pathogenicity on maize seedlings. Molec Plant-Microbe Interact 21: 87-97. Gray, J. S., Bae, H. K., Li, J. C., Lau, A. S., and Pestka, J. J. (2008) Double-stranded RNA-activated protein kinase mediates induction of interleukin-8 expression by deoxynivalenol, Shiga toxin 1, and ricin in monocytes. Toxicol.Sci 105, 322-330. Guarisco, J.A., Hall, J.O. and Coulombe, R.A., Jr. (2007) Butylated hydroxytoluene reduces aflatoxin B1 bioavailability and hepatic adduct formation in turkeys. In Poisonous Plants: Global Research and Solutions (K. Panter, T. Wierenga, J. Phister, eds.) CAB International, London. pp. 197-202. Guarisco, J.A., Hall, J.O. and Coulombe, R.A., Jr. (2008) Butylated hydroxytoluene chemoprevention of aflatoxicosis - effects on aflatoxin B1 bioavailability, hepatic DNA adduct formation, and biliary excretion. Food Chem Toxicol 46: 3727-3723. Guarisco, J.A., Hall, J.O. and Coulombe, R.A., Jr. (2008) Mechanisms of butylated hydroxytoluene chemoprevention of aflatoxicosis - inhibition of aflatoxin B1 metabolism. Toxicol Appl Pharmacol 227: 339-346. Heemstra, J. M., Kerrigan, S. A., Doerge, D. R., Helferich, W. G. and Boulanger, W. A. (2007). Total synthesis of (S)-equol. Org Lett 8:5441-5443. Helferich, W.G., Andrade, J.E. and Hoagland, M.S. (2008). Phytoestrogens and breast cancer: a complex story. Inflammopharmacology : 219-226. Higdon, J.V., Delage, B., Williams, D.E. and Dashwood, R.H. (2007) Cruciferous Vegetables and Human Cancer Risk: Epidemiologic Evidence and Mechanistic Basis. Pharmacol Res 55: 224-236. Hu, W., Sorrentino, C., Denison, M.S., Kolaja, K. and Fielden, M. (2007) Induction of Cyp1a1 is a Non-Specific Biomarker of Aryl Hydrocarbon Receptor Activation: Results of Large Scale Screening of Pharmaceuticals and Toxicants In Vivo and In Vitro, Molec. Pharmacol. 71: 1475-1486. Im, W.I., Suh, B.-S., Lee, S.-U., Kozukue, N., Ohnisi-Kameyama, M., Levin, C.E. and Friedman, M. (2008) Analysis of phenolic compounds by high-performance liquid chromatography and liquid chromatography/mass spectrometry in potato plant flowers, leaves, stems, and tubers and in processed potatoes. J Agric Food Chem 56:3341-3349. Islam, Z., Hegg, C. C., Bae, H. K., and Pestka, J. J. (2008) Satratoxin G-induced apoptosis in PC-12 neuronal cells is mediated by PKR and caspase independent. Toxicol.Sci 105, 142-152. Jiang, X., Patterson, N.M., Ling, Y., Xie, J. Helferich, W.G. and Shapiro, D.J. (2008). Low Concentrations of the Soy Phytoestrogen Genistein Induce Proteinase Inhibitor 9 and Block Killing of Breast Cancer Cells by Immune Cells. Endocrinology 149: 5366-5373. Ju, Y.H., Doerge, D. and Helferich, W.G. (2008). A dietary supplement for female sexual dysfunction, Avlimil, stimulates the growth of estrogen-dependent breast tumors (MCF-7) implanted in ovariectomized athymic nude mice. Food Chem Toxicol 46: 310-320. Ju, Y.H., Doerge, D.R., Woodling, K.A., Hartman, J.A., Kwak, J. and Helferich, W.G. (2008). Dietary Genistein Negates the Inhibitory Effect of Letrozole On The Growth Of Aromatase-expressing Estrogen-Dependent Human Breast Cancer Cells (MCF-7Ca) In vivo. Carcinogenesis 29: 2162-2168. Juneja, V.K. and Friedman, M. (2008) Carvacrol and cinnamaldehyde facilitate thermal destruction of Escherichia coli O157:H7 in raw ground beef. J Food Prot 71: 1604-1611. Lee, S.U., Lee, J.H., Choi, S.H., Lee, J.S., Ohnisi-Kameyama, M., Kozukue, N., Levin, C.E. and Friedman, M. (2008) Flavonoid content in fresh, home-processed, and light-exposed onions and in dehydrated commercial onion products. J Agric Food Chem 56: 8541-8548. Li, M., and Pestka, J. J. (2008). Comparative induction of 28S ribosomal RNA cleavage by ricin and the trichothecenes deoxynivalenol and T-2 toxin in the macrophage. Toxicol.Sci 105, 67-78. Lim, S., Dragull, K., Tang, C.S., Bittenbender, S. and Nerurkar, P.V. (2007) Effects of Kava Alkaloid, Pipermethystine (PM), and Kavalactones on Oxidative Stress and Hepatic Cytochrome P450 in F344 Rats. Toxicol Sci 97: 214-221. Loniewski, K., Shi, Y., Pestka, J., and Parameswaran, N. (2008) Toll-like receptors differentially regulate GPCR kinases and arrestins in primary macrophages. Mol Immunol. 45, 2312-2322. Mersereau, J.E., Levy, N., Staub, R.E., Baggett, S., Zogric, T., Chow, S., Ricke, W.A., Tagliaferri M., Cohen, I., Bjeldanes, L.F. and Leitman, D.C. (2008) Liquiritigenin is a plant-derived highly selective estrogen receptor beta agonist. Mol Cell Endocrinol 283: 49-57. Mottram, D.S. and Friedman, M. (2008) Preface - Symposium on the Chemistry and Toxicology of Acrylamide. J Agric Food Chem 56: 5983. Nerurkar, P.V., Lee, Y.-K., Motosue, M., Adeli, K. and Nerurkar, V.R. (2008) Momordica charantia (bitter melon) reduces plasma apolipoprotein B-100 and increases hepatic insulin receptor substrate and phosphoinositide-3 kinase interactions. British J Nutri 100: 751-759. Nerurkar, P.V. and Nerurkar, V.R. (2008) Is Sirt1 the magic target for diabetes? Cell Science Reviews, 2008; 4: 50-56. Nerurkar, P.V. (2008) Cancer and Sirt1 regulation Cell Science Reviews, 4: 82-96. Nording, M., Persson, Y., Spinnel, E., Baston, D., Denison, M.S. and Haglund, P. (2007) Analysis of dioxins in contaminated soils using CALUX and CAFLUX bioassays, an immunoassay and gas chromatography - high resolution mass spectrometry, Environ. Toxicol. Chem. 26: 1122-1129. Pestka, J. J., Yike, I., Dearborn, D. G., Ward, M. D., and Harkema, J. R. (2008). Stachybotrys chartarum, trichothecene mycotoxins, and damp building-related illness: new insights into a public health enigma. Toxicol.Sci 104, 4-26. Pestka, J. J., and Amuzie, C. J. (2008) Tissue distribution and proinflammatory cytokine gene expression following acute oral exposure to deoxynivalenol: comparison of weanling and adult mice. Food Chem Toxicol. 46, 2826-2831. Pestka, J. J. (2008) Mechanisms of deoxynivalenol-induced gene expression and apoptosis. Food Addit.Contam 1-13. Pestka, J. J., Islam, Z., and Amuzie, C. J. (2008) Immunochemical assessment of deoxynivalenol tissue distribution following oral exposure in the mouse. Toxicol.Lett 178: 83-87. Putt, K.S., Chen, G.W., Pearson, J.M., Sandhorst, J.S., Hoagland, M.S., Kwon, J.T., Hwang, S.K., Jin, H., Churchwell, M.I., Cho, M.H., Doerge, D.R., Helferich, W.G. and Hergenrother, P.J. (2007) Small-molecule activation of procaspase-3 to caspase-3 as a personalized anticancer strategy. Nat Chem Biol 10: 543-550. Ravishankar, S., Zhu, L.,Law, B., Joens, L. and Friedman, M. (2008) Plant-derived compounds inactivate antibiotic-resistant Campylobacter jejuni strains. J Food Prot 71: 1145-1149. Reed, K.M., Mendoza, K.M. and Coulombe, R.A., Jr. (2007). Structure and genetic mapping of the Cytochrome P4501A5 gene in the turkey (Meleagris gallopavo). Cytogenetics and Genome Research 116: 104-109. Riby, J.E., Firestone, G.L. and Bjeldanes, L.F. (2008) 3,3'-Diindolylmethane reduces levels of HIF-1alpha and HIF-1 activity in hypoxic cultured human cancer cells. Biochem Pharmacol 75: 1858-67. Rochester, J.R., Klasing, K.C., Stevenson, L., Denison, M.S., Berry, W. and Millam, J.r. (2008) Dietary red clover (Trifolium pratense) induces oviduct growth and decreases ovary and testes growth in Japanese quail chick, Reprod. Toxicol. 27: 63-71. Shi, Y., and Pestka, J. J. (2008). Mechanisms for suppression of interleukin-6 expression in peritoneal macrophages from docosahexaenoic acid-fed mice. J Nutr Biochem in press. Simonich, M.T., Egner, P.A., Robuck, B.D., Orner, O., Jubert, C., Pereira, C., Groopman, J.D., Kensler, T.W., Dashwood, R.H., Williams, D.E. and Bailey, G.S. (2007) Natural Chlorophyll Inhibits Aflatoxin B1 Induced Multi-Organ Carcinogenesis in the Rat. Carcinogenesis 28: 1294-1302. Simonich, M.T., McQuistan, T., Jubert, C., Pereira, C., Hendricks, J.D., Schimerlik, M., Zhu, B., Dashwood, R., Williams, D.E. and Bailey, G.S. (2008) Low-Dose Dietary Chlorophyll Inhibits Multi-Organ Carcinogenesis in the Rainbow Trout. Food Chem Toxicol 46: 1014-1024. Sirk, T.W., Brown, E.F., Sum, A.K. and Friedman, M. (2008) Molecular dynamics study on the biophysical interactions of seven green tea catechins with lipid bilayers of cell membranes. J. Agric. Food Chem 56: 7750-7758. Snee, L.S., Nerurkar, V.R., Dooley, D., Efird, J., Shovic, A., Nerurkar, P.V. (2008) Feasibility study to increase palatability and consumption of Momordica charantia (Bitter Melon). Appetite, submitted. Tilton, S.C., Hendricks, J.D., Orner, G.A., Pereira, C.B., Bailey, G.S. and Williams, D.E. (2007) Gene Expression Analysis During Tumor Enhancement by the Dietary Phytochemical and Supplement, 3,3-Diindolylmethane, in Rainbow Trout. Carcinogenesis, 28: 1589-1598. Tilton, S.C., Orner, G.A., Benninghoff, A.D., Carpenter, H.M., Hendricks, J.D., Pereira, C.B. and Williams, D.E. (2008) Genomic Profiling Reveals an Alternate Mechanism for Hepatic Tumor Promotion by Perfluorooctanoic Acid in Rainbow Trout. Environ Hlth Perspect 116: 1047-1055. Torres, O.A., Palencia, E., Lopez de Pratdesaba, L., Grajeda, R, Fuentes, M., Speer, M., Merrill, A.H., Jr., ODonnell, K., Bacon, C.W., Glenn, A.E. and Riley, R.T. (2007) Estimated fumonisin exposure in Guatemala is greatest in consumers of lowland maize. J Nutrition 137: 2723-2729. Voss, K.A., Riley, R.T. and Gelineau van-Waes, J.B. (2007) Fetotoxicity and neural tube defects in CD1 mice exposed to the mycotoxin fumonisin B1. Mycotoxins 57: 67-72. Wang, R., Dashwood, W.M., Löhr, C.V., Fisher, K.A., Pereira, C.B., Louderback, M., Nakagama, H., Bailey, G.S., Williams, D.E. and Dashwood, R.H. (2008) Protective Versus Promotional Effects of White Tea and Caffeine on PhIP-Induced Tumorigenesis and b catenin expression in the Rat. Carcinogenesis 29: 834-839. Wang, R., Dashwood, W.-M., Löhr, C.V., Fischer, K.A., Nakagama, H., Williams, D.E. and Dashwood, R.H. (2008) Beta-Catenin is Strongly Elevated in Rat Colonic Epithelium Following Short-Term Intermittent Treatment with 2-Amino-1-Methyl-6-Phenyimidazo[4,5-b]Pyridine (PhIP) and a High-Fat Diet. Cancer Sci 99: 1754-1759. Wang, V.C., Sable, H.J., Ju, Y.H., Allred, C.D., Helferich, W.G., Korol, D.L. and Schantz, S.L. (2008). Effects of chronic estradiol treatment on delayed spatial alternation and differential reinforcement of low rates of responding. Behav Neurosci 122: 794-804. Watterson, T.L., Sorenson, J., Martin, R., and Coulombe, R.A., Jr. (2007). Effects of PM2.5 Collected from Cache Valley Utah on Genes Associated with the Inflammatory Response in Human Lung Cells. J Toxicol Environ Health 70: 1731-1734. Wong, S.Y., Grant, I.R., Friedman, M., Elliott, C.T. and Situ, C. (2008) Antibacterial activities of naturally occurring compounds against Mycobacterium avium subsp. paratuberculosis. Appl Environ Microbiol 74: 5986-5990. Xue, L., Pestka, J. J., Li, M., Firestone, G. L., and Bjeldanes, L. F. (2008). 3,3'-Diindolylmethane stimulates murine immune function in vitro and in vivo. J Nutr.Biochem 19, 336-344. Zitomer, N. C., Glenn, A. E., Bacon, C.W. and Riley, R.T. (2008) A single extraction method for the analysis by liquid chromatography/tandem mass spectrometry of fumonisins and biomarkers of disrupted sphingolipid metabolism in tissues of maize seedlings. Anal Bioanal Chem 391: 2257-2263.