Progress 05/14/01 to 02/07/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? This is the final progress report (10/2005 through 04/2006). Overview of problem. Over $100 billion of exported food commodities are susceptible to mycotoxin contamination. It has been estimated that in the United States economic crop loss due to mycotoxins is between $418, 000,000 and $1,660,000,000 annually. Animal losses are estimated to be $6,000,000 per year. Human health effects have been documented for several food-borne mycotoxins, including aflatoxins, ergot alkaloids, and trichothecenes. In the United States, it is well established that farm animal diseases are caused by Fusarium mycotoxins that occur in major cereal grains, including corn and wheat. Because Fusarium mycotoxins are known causes of farm animal disease and suspected causes of human disease, their presence can
adversely affect the marketability and export of crops and agricultural products. The current basis for setting international tolerance levels for mycotoxins in agricultural products is science based risk assessment. Failure to accurately assess the hazards associated with mycotoxin exposure can result in over regulation of US exports such as corn, wheat and nuts. For example, a recent study funded by RAND Science and Technology estimates that imposing a 0.5 ppm fumonisin international standard on corn would result in a $170,000,000 loss of US corn exports. Likewise, at the EU standard of 4 ppb of aflatoxins in peanuts, US export losses would be about $120,000,000. Reducing the uncertainty in mycotoxin hazard assessment will reduce the likelihood of the over regulation of US agricultural exports. Objectives. The long-term goal of this project is to provide the basis for identifying and reducing hazards to animals and man related to mycotoxin exposure. The objectives are: a)
define the biochemical, molecular and physiological mechanisms by which mycotoxins act including factors that modify susceptibility to disease; b) predict the consequences of exposure to combinations of mycotoxins and determining the extent to which mycotoxins modify the response of animals or humans to infectious agents; and c) evaluate cooking and processing procedures with the intent to minimize mycotoxin exposure to consumers of commodity- based food products. Approaches. The following approaches are being used to accomplish the objectives: a) identify, evaluate and apply biochemical and molecular mechanisms of action as biomarkers and criteria to evaluate control and decontamination methods, as well as to provide information required for risk analysis; b) identify and evaluate the ability of fumonisin to modify the biological activity of other toxins and infectious agents; c) evaluate the effects of cooking and processing on the distribution and biological activity of fumonisin
in corn-based food products. Who will benefit and impact. Relevant products resulting from accomplishment of the objectives will include: basic and applied knowledge documented by scientific publications, as well as patented processes based on mechanisms of action; diagnostic methods; bioassay methods; analytical techniques; decontamination procedures; therapeutic agents; research tools; and scientifically sound risk assessments by regulatory agencies. The public will benefit from this research through the continued availability of safe food. Expected users of the findings from the proposed studies will include the food and feed industry since one objective will be to determine the fate of mycotoxins during processing. In addition, scientists and regulatory agencies will benefit from the research defining toxicity levels, effects of combined toxins as occur in naturally contaminated crops, and the biochemical mechanisms by which toxic effects are produced. Producers, extension
personnel, and consumers will also benefit by learning levels of mycotoxins and combinations of mycotoxins that produce adverse effects. 2. List by year the currently approved milestones (indicators of research progress) Year 1 (FY 2001) 1. Define the biochemical, molecular and physiological mechanisms by which mycotoxins act including factors that modify susceptibility to disease. Commence the execution of studies to determine role of disrupted sphingolipid metabolism as a mechanism of plant disease, develop screening methods for identifying fungal inhibitors of ceramide synthase and serine palmitoyl transferase using in vitro models, evaluate the role of cytokines in fumonisin-induced liver toxicity using animal models, and conduct studies to assess the role of peroxisome proliferation in fumonisin induced liver toxicity. 2. Predict the consequences of exposure to combinations of mycotoxins and determine the extent to which mycotoxins modify the response of animals or humans to
infectious agents. Commence the execution of studies to determine the ability of fumonisin to promote aflatoxins- initiated liver tumors, the possible additive cardiovascular toxicity of moniliformin and fumonisin in rodents, the ability of fumonisin to modulate response to infectious agents (T. cruzi), and the ability of fumonisin to disrupt glycosphingolipid (toxin receptors) biosynthesis in intestinal epithelia. 3. Evaluate cooking and processing procedures with the intent to minimize mycotoxin exposure to consumers of commodity-based food products. Commence the execution of studies to determine the fate of fumonisins during processing, fermentation, and survey the presence of fumonisins in Guatemala. Year 2 (FY 2002) 1. Commence the execution of studies to determine if differences in tissue metabolism of sphingoid bases play a role in the tissue specificity of fumonisins in animal models, and conducted, completed and documented studies to determine the ability of TNF to modulate
LPS response in fumonisin-treated cells. Continue the conduct of studies to determine role of disrupted sphingolipid metabolism as a mechanism of plant disease, development of screening methods for identifying fungal inhibitors of ceramide synthase, evaluation of the role of cytokines in fumonisin-induced liver toxicity using animal models, and assessment of the role of peroxisome proliferation in fumonisin induced liver toxicity.. Completed and documented studies for screening for serine palmitoyl transferase inhibitors using in vitro models, 2. Commence the execution of studies to determine the interaction between deoxynivalenol and zearalenone in animal models and commence studies to determine if fumonisins can modulate the response of liver to inducers of mixed function oxidase activity. Continued studies on the possible additive cardiovascular toxicity of moniliformin and fumonisin in rats, the ability of fumonisin to modulate response to infectious agents (T. cruzi), and the
ability of fumonisin to disrupt glycosphingolipid (toxin receptors) biosynthesis in intestinal epithelia of mice and poultry. Completed and documented the conduct of studies to determine the ability of fumonisin to promote aflatoxin-initiated liver tumors using trout. 3. Continue the conduct of studies to determine the fate of fumonisins during processing including traditional and commercial processing methods, beer fermentation using traditional methods, and survey the presence of fumonisins and Fusarium in the highland and lowlands of Guatemala. Year 3 (FY 2003) 1. Continue the conduct of studies to determine the role of differences in tissue metabolism of sphingoid bases in the tissue specificity of fumonisins in rats. Continue studies on the role of disrupted sphingolipid metabolism as a mechanism of plant disease using corn seedlings, develop screening methods for identifying fungal inhibitors of ceramide synthase, evaluate the role of cytokines in liver fumonisin- induced
liver toxicity using animal models, and assess the role of peroxisome proliferation in fumonisin induced liver toxicity. 2. Terminated studies to determine the interaction between deoxynivalenol and zearalenone in animal models and studies to determine if fumonisins can modulate the response of liver to inducers of mixed function oxidase activity due to the retirement of the SYs. Completed studies on the possible additive cardiovascular toxicity of moniliformin and fumonisin in rats. Completed and documented the ability of fumonisin to modulate response to infectious agents (T. cruzi), and the ability of fumonisin to disrupt glycosphingolipid (toxin receptors) biosynthesis in intestinal epithelia of mice. The studies in poultry were completed but have yet to be documented. 3. Continue the conduct of studies to determine the fate of fumonisins during processing including traditional and commercial processing methods, beer fermentation using traditional methods, and survey the
presence of fumonisins and Fusarium in Guatemala. Year 4 (FY 2004) 1. Completed and documented the conduct of studies to determine the role of differences in tissue metabolism of sphingoid bases in the tissue specificity of fumonisins in rats. Commenced studies to evaluate sphingoid base 1-phosphates as mediators of renal and liver toxicity in rat. Continue studies on the role of disrupted sphingolipid metabolism as a mechanism of plant disease using corn seedlings and commence studies to determine the fate of fumonisins in soils. Complete studies to develop screening methods for identifying fungal inhibitors of ceramide synthase. Completed and documented initial studies to evaluate the role of cytokines in fumonisin-induced liver toxicity using animal models and commenced additional studies using transgenic mouse models. Completed studies to assess the the role of peroxisome proliferation in fumonisin induced liver toxicity. 2. Documented studies on the possible additive
cardiovascular toxicity of moniliformin and fumonisin in rats. 3. Completed and documented the results of studies to determine the fate of fumonisins during commercial and traditional processing methods. Continued studies of fate during beer fermentation using traditional methods. Continued to survey the presence of fumonisins and Fusarium in Guatemala. Year 5 (FY 2005) 1. Continued studies to evaluate sphingoid base 1-phosphates as mediators of renal and liver toxicity in rat. Continue studies on the role of disrupted sphingolipid metabolism as a mechanism of plant disease using corn seedlings and completed and documented studies to determine the fate of fumonisins in soils. Documented studies on screening methods for identifying fungal inhibitors of ceramide synthase. Completed and documented initial studies to evaluate the role of cytokines in fumonisin- induced liver toxicity using using transgenic mouse models. Completed and documented studies to assess the the role of
peroxisome proliferation in fumonisin induced liver toxicity. 2. All milestones under objective 2 were completed in FY 2004. 3. Commenced studies to develop a better understanding of conditions that can mask detection of fumonisins during processing. Studies on the fate of fumonisin during beer fermentation using traditional methods were discontinued due to a redirection at another agency that was part of the collaboration. Documented sampling and analystical methods for analysis of fumonisins in Guatemala and continued to survey for the presence of fumonisins in the highlands and lowlands of Guatemala. 4a List the single most significant research accomplishment during FY 2006. Improved understanding of the mechanistic basis for the sensitivity of rat kidney to fumonisin toxicity: The relative sensitivity of male rat kidney and liver is most likely a consequence of differences in the mechanisms responsible for both fumonisin uptake/clearance and sphinganine metabolism. In a
feeding study, we showed for the first time that the sensitivity of rat kidney to fumonisin is a result of preferential accumulation of fumonisin by kidney and the fact that kidney accumulates much greater amounts of sphingoid bases and sphingoid base 1- phosphates compared to liver. This finding is important because the rat kidney is the critical target organ used in the fumonisin risk analysis and upon which the recommended fumonisin tolerable daily intakes are based. Until now, the reason that rat kidney is more sensitive than liver to fumonisin toxicity was not known. Understanding the underlying mechanistic basis for organ/species specific toxicity will allow us to predict the risk in humans. 4b List other significant research accomplishment(s), if any. Recommendations made to reduce fumonisin exposure in Guatemala:. The results of the five year fumonisin survey in Guatemala (objective 3.3) and a preliminary fumonisin exposure assessment for Guatemala was developed and the
results were presented to representatives of the Guatemala Ministry of Health, Codex Guatemala and the Consejo Nacional de Cienca y Technologia (28-31 August 2005). The team of scientists working on this project from the Instituto de Nutricion de Centro America y Panama and USDA-ARS were asked by the Consejo Nacional de Cienca y Technologia to provide a draft proposal of policies and recommendations to minimize fumonisin exposure in Guatemala. The purpose of these recommendations is to provide the basis for a public policy intended to minimize the health risks associated with fumonisins in human foods in Guatemala. The draft document included recommendations for guidance levels, consumer education, commercial processing and agronomic practices which if implemented would reduce fumonisin exposure in Guatemala. Food Processing - Deoxynivalenol: Samples of cookies, crackers, cereal, pretzels, and bread were analyzed to determine the effect of the cooking methods on deoxynivalenol
concentrations. Only donuts showed significantly lower concentrations, compared to the starting material, and some products showed increased concentrations. Further experiments showed that the apparent increases (or lack of reduction). Food Processing - Fumonisins: In cooperation with University of Nebraska and FDA scientists, chemical and bioassay studies were done utilizing products obtained from extrusion cooking of fumonisin-contaminated corn grits. Diets were fed to rats and tissues were collected and processed for analysis. Preliminary results suggest that extrusion products produced with glucose supplementation were less toxic. Histological examination of the tissues and statistical analysis of the data are pending. 4d Progress report. There are several areas where additional work is needed in order to fill knowledge gaps in the risk assessments for fumonisins and deoxynivalenol that are the basis for regulatory action. First, it has been proposed that fumonisins could be
a risk factor for the high incidence of neural tube defects in humans in areas where corn consumption is high. Second, it has been suggested that fumonisin intake is underestimated because of the presence of hidden fumonisins in foods. Third, gaps in the toxicology database for deoxynivalenol have resulted in proposed action limits that could adversely impact US cereal exports; these include knowledge gaps in our understanding of the mechanism of action, pharmacokinetics, and dose-response studies. For both fumonisins and deoxynivalenol, lack of understanding of the dose-response relationships and mechanistic relevance in humans could increase the safety factors used in future risk assessments and thus adversely impact US agriculture. 5. Describe the major accomplishments to date and their predicted or actual impact. (4/2001 through 5/2006) Milestones completed under objective 1 of this project plan addressed National Program Action Plan sections 1.1.1.2, 1.1.2.1, 1.1.7.1, 1.1.7.2,
and 1.1.7.3. Specifically, used cultured cells, plant models, and genetically modified mice to determine specific molecular endpoints of fumonisin-induced disruption of sphingolipid biosynthesis and downstream mechanisms of action. Specifically, using cell lines demonstrated that cell-specific responses (growth vs. death) to fumonisins are determined by the relative activity of enzymes in the de novo biosynthetic and turnover pathways for sphingolipids; in corn seedlings fumonisin-induced inhibition of root growth is mediated by disruption of sphingolipid metabolism; using gene knockout and transgenic mouse strains demonstrated the close correlation between inhibition of sphingolipid metabolism and the incidence and severity of liver toxicity, that alterations in cytokine expression and downstream signaling pathways play important roles in hepatoxicity, that hepatocellular apoptosis and mitosis are not mediated by peroxisomal proliferators activated receptor alpha (PPAR() mediated
pathways and that exposure to F. verticillioides culture materials and purified FB1 elicit similar gene expression profiles; developed patented in vitro and in vivo mechanism (ceramide synthase inhibition) based bioassays to use for testing of cooking and processing methods and ability of various fungal strains to produce fumonisin-like activity. The results of these studies were the basis for developing mechanism based bioassays for fumonisin like activity in foods and other matrices and provided critical data for science-based risk assessments worldwide, thereby enabling US and international regulatory decisions to protect consumers while insuring the continued availability of high quality commodities both for US consumption and export to foreign markets. Milestones completed under objective 2 of this project plan addressed National Program Action Plan sections 1.1.7.2 and 1.1.7.3. Specifically, demonstrated that dietary fumonisin can modulate aflatoxin and N-methyl-
N'-nitro-nitrosoguanidine tumorigenicity in long-term feeding studies using rainbow trout and that the fumonisin promotion of aflatoxin carcinogenicity is closely correlated with evidence of disruption of sphingolipid metabolism; that dietary fumonisins modulate host resistance to infectious agents, specifically showed increased resistance of mice to infection by the parasite Trypanosoma cruzi and that the mechanism of resistance was related to increased nitric oxide production and altered sphingolipid biosynthesis; using mice, rats and chicks and cell lines showed that fumonisin could modulate glycosphingolipid biosynthesis in intestinal and other tissues and thus alter microbial receptor expression in tissues; found that fumonisin inhibition of ceramide biosynthesis protected renal cells exposed to E. coli endotoxins under conditions where the cytokine TNF-alpha was increased. The results from the aflatoxin/fumonisin interaction study shows that efforts to reduce aflatoxin in corn
should also carefully consider the effect of management strategies on the fumonisin levels. Similarly, the effectiveness of animal vaccines and the response of farm animals and humans to infectious agents will be affected by consumption of fumonisin contaminated feeds and foods. Milestones completed under objective 3 of this project plan addressed National Program Action Plan sections 1.1.1.2, 1.1.7.1, 1.1.7.3. Specifically, identified the critical step that significantly reduces fumonisin levels in masa and tortilla chips prepared from contaminated corn under commercial conditions; using a mechanism-based in vitro bioassay, showed that chemical analyses accurately predicted biological activity of the masa and tortilla chips; using a rat feeding bioassay and validated toxicological endpoints, showed that frying and baking did not significantly affect measurable fumonisin levels or toxicity of fumonisin- contaminated cornmeal; using chemical analysis and fumonisin-like activity using
an in vitro mechanism-based bioassay determined that traditional Mayan tortilla production methods reduce total fumonisins, conducted a five year survey of fumonisins in corn grown in Guatemala, and used the combined data to produced an exposure assessment for corn consumers in Guatemala; using a combination of in vivo and in vitro bioassays showed that high levels of fumonisins were present in joala, a home-brewed beer consumed in Africa, made from F. verticillioides contaminated corn but that another potentially important novel genotoxic, DNA adduct forming compound made by the fungus is not transferred to the joala. The results showed that the processing methods could reduce fumonisins in foods and that the chemical analysis and the biological activity based on the biomarkers developed in objective 1 was in close agreement and indicated that hidden fumonisins are not produced in significant amounts during cooking or frying of highly contaminated cornmeal, nor are they produced
during traditional household processes for producing tortillas. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Unit scientists have been requested to share their expertise in numerous national and international food safety forums and to serve as members of organizations that shape the international thinking on the regulation of and human risks associated with mycotoxin contaminated foods. Examples of activities in the period 10/05 to 5/06 include presentations, participation in workshops, development of position papers, design of research programs and contributions to task forces assembled to address specific food safety issues. These activities have occurred at numerous venues including the USDA-ARS 5nd Fungal Genomics, 6th Fumonisin Elimination
and 18th Aflatoxin Elimination Workshops, The 45rd Annual Meeting of the Society of Toxicology, The 40th United States-Japan Joint Panel on Toxic Microorganisms, The W-1122 Multistate Research Project, the EU-Australia Symposium on Mycotoxins in Foods, and the Southern Section of AOAC International 20th Annual Meeting. Unit scientist is serving as Co-chair for the upcoming Gordon Research Conference on Mycotoxins and Phycotoxins. The conference is a major international venue for exchanging the latest scientific and technical information on mycotoxins by ARS and other scientists, setting research priorities related to existing "data gaps" and emerging problems, and promote collaborations to address these research needs. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Fumonisin expert for Study supports corn toxin as culprit in birth defects
Evidence backs theory that diet rich in tortillas caused border epidemic Laura Beil/The Dallas Morning News, 5 February, 2006. Several (>20) slightly different versions of The Dallas Morning News article with quotes that were also slightly different were published over the period 2/2006 to 3/2006.
Impacts
(N/A)
Publications
- Corton, J., Riley, R.T., Dunn, C., Voss, K.A. 2006. The role of tumor necrosis factor alpha and the peroxisome proliferator-activated receptor alpha in moldulating the effects of fumonisin in mouse liver. Toxicology 222:165-174.
- Voss, K.A., Gelineau-Van Waes, J., Riley, R.T. 2006. Fumonisins: current research trends in developmental toxicology. Mycotoxin Research 22(1):61- 69.
- Riley, R.T., Voss, K.A., Speer, M., Stevens, V.L., Gelineau-Van Waes, J. 2006. Fumonisin inhibition of ceramide synthase: a possible risk factor for human neural tube defects. In: Hirabayashi, U., Igarashi, I., Merrill, Jr., A.H., editors. Sphingolipid Biology. Heidelberg, Germany: Springer. p. 345-361.
- Stauber, A.J., Brown-Borg, H., Liu, J., Waalkes, M.P., Laughter, A., Staben, R.A., Coley, J.C., Swanson, C., Voss, K.A., Kopchick, J.J., Corton, J.C. 2004. Constitutive expression of peroxisome proliferator-activated receptor alpha-regulated genes in dwarf mice. Molecular Pharmacology. 67:681-694.
- Riley, R.T., Voss, K.A. 2006. Differential sensitivity of rat kidney and liver to fumonisin toxicity: organ specific differences in toxin accumulation and sphingoid base metabolism. Toxicological Sciences. 92(1) :335-345.
- Williams, L.D., Glenn, A.E., Bacon, C.W., Smith, M.A., Riley, R.T. 2006. Fumonisin production and bioavailability to maize seedlings grown from seeds inoculated with fusarium verticillioides and grown in natural soils. Journal of Agricultural and Food Chemistry 54(15):5694-5700.
- Riley, R.T., Torres, O.A., Palencia, E. 2006. International shipping of fumonisins from maize extracts on C18 sorbent. Food Additives and Contaminants 23(8):826-832.
- Cousin, M.A., Riley, R.T., Pestka, J.J. 2005. Foodborne mycotoxins: chemistry, biology, ecology, and toxicology. In: Fratamico, P.M., Bhunia, A.K., editors, Foodborne Pathogens: Microbiology and Molecular Biology. Norfolk, United Kingdom: Horizon Scientific Press, Ltd. p. 164-226.
- Voss, K.A., Liu, J., Anderson, S.P., Dunn, C., Miller, J., Owen, J.R., Riley, R.T., Bacon, C.W., Corton, J. 2006. Toxic effects of fumonisin in mouse liver are independent of the peroxisome proliferator-activated receptor alpha. Toxicological Sciences. 89(1):108-119.
- Voss, K.A., Riley, R.T., Gelineau-Van Waes, J. 2005. Trends in fumonisin research: recent studies on the developmental effects of fumonisins and fusarium verticillioides. Journal of the Japanese Association of Mycotoxicology/Mycotoxins 55(2):91-100
- Sharma, R.P., He, Q., Riley, R.T. 2005. Lupus-prone NZBWF1/J mice, defective in cytokine signaling, are resistant to fumonisin hepatotoxicity despite accumulation of liver sphinganine. Toxicology. 216:59-71.
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Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Overview of problem. Over $100 billion of exported food commodities are susceptible to mycotoxin contamination.NB It has been estimated that in the United States economic crop loss due to mycotoxins is between $418, 000,000 and $1,660,000,000 annually.NB Animal losses are estimated to be $6,000,000 per year.NB Human health effects have been documented for several food-borne mycotoxins, including aflatoxins, ergot alkaloids, and trichothecenes.NB In the United States, it is well established that farm animal diseases are caused by Fusarium mycotoxins that occur in major cereal grains, including corn and wheat.NB Because Fusarium mycotoxins are known causes of farm animal disease and suspected causes of human disease, their presence can adversely affect the marketability and export of crops and agricultural
products. The current basis for setting international tolerance levels for mycotoxins in agricultural products is science based risk assessment.NB Failure to accurately assess the hazards associated with mycotoxin exposure can result in over regulation of US exports such as corn, wheat and nuts.NB For example, a recent study funded by RAND Science and Technology estimates that imposing a 0.5 ppm fumonisin international standard on corn would result in a $170,000,000 loss of US corn exports.NB Likewise, at the EU standard of 4 ppb of aflatoxins in peanuts, US export losses would be about $120,000,000.NB Reducing the uncertainty in mycotoxin hazard assessment will reduce the likelihood of the over regulation of US agricultural exports. Objectives. The long-term goal of this project is to provide the basis for identifying and reducing hazards to animals and man related to mycotoxin exposure. The objectives are:NB 1. Define the biochemical, molecular and physiological mechanisms by which
mycotoxins act including factors that modify susceptibility to disease. 2. Predict the consequences of exposure to combinations of mycotoxins and determine the extent to which mycotoxins modify the response of animals or humans to infectious agents. 3. Evaluate cooking and processing procedures with the intent to minimize mycotoxin exposure to consumers of commodity-based food products. Approaches. The following approaches are being used to accomplish the objectives: a) identify, evaluate and apply biochemical and molecular mechanisms of action as biomarkers and criteria to evaluate control and decontamination methods, as well as to provide information required for risk analysis; b) identify and evaluate the ability of fumonisin to modify the biological activity of other toxins and infectious agents; c) evaluate the effects of cooking and processing on the distribution and biological activity of fumonisin in corn-based food products. Who will benefit and Impact. Relevant products
resulting from accomplishment of the objectives will include: basic and applied knowledge documented by scientific publications, as well as patented processes based on mechanisms of action; diagnostic methods; bioassay methods; analytical techniques; decontamination procedures; therapeutic agents; research tools; and scientifically sound risk assessments by regulatory agencies.NB The public will benefit from this research through the continued availability of safe food.NB Expected users of the findings from the proposed studies will include the food and feed industry since one objective will be to determine the fate of mycotoxins during processing.NB In addition, scientists and regulatory agencies will benefit from the research defining toxicity levels, effects of combined toxins as occur in naturally contaminated crops, and the biochemical mechanisms by which toxic effects are produced.NB Producers, extension personnel, and consumers will also benefit by learning levels of mycotoxins and
combinations of mycotoxins that produce adverse effects. 2. List the milestones (indicators of progress) from your Project Plan. 1. FY 01-02: Determine the dose, the mechanism of action and effects on enzymes and their kinetics for the fumonisin mycotoxin: Fully met. 2. FY 02-03: Determine exposure levels and alterations to animals due to fumonisin exposure: Developed functional biomarkers and patents based on biomarkers and mode of action for the fumonisins; developed effects of the fumonisins on plants, as well as plant disease, and nature fumonisin binding to soil types; 3. FY 04-05: Determined under CRADAs that cooking and processing fumonisin-contaminated tortilla and corn chips renders the chips fumonisin-free; extended the mode of action of the fumonisin to include more detailed physiological parameters and extended the biomarker patents for the process; determined additional effects of the fumonisins on plant performance and soil interactions. 3a List the milestones
that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Determine the fumonisin content and using rodent toxicological studies determine the nature of toxicity resulting from consuming processed and cooked fumonisin-contaminated tortillia chips. Milestone Fully Met 2. Extended the nature of mechanisms of action for the fumonisin and its application as biomarkers for toxicity by this class of compounds as well as the fumonisins, with patent application for this extension. Milestone Fully Met 3. Determine the uptake and resulting effects of fumonisins on corn seedlings - Partially met. Milestone Substantially Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? 2006 to 2007: Studies will be conducted to determine the role of fumonisin-induced
ceramide synthase inhibition in corn seedling disease and the genetic basis for resistance/susceptibility. The role of soil type in modulating the plant response to fungal infection will be addressed using the elevation in sphingoid bases and their 1-phosphates as a biomarker of exposure and also for the ability of fumonisins to be translocated to and be biologically active in aerial tissues (leaf and stems). IMPACT: These studies will be of great interest to environmental toxicologists, agronomists and plant pathologist because they will provide a validated biomarker for detecting fumonisin effects on corn seedlings in the field and will resolve the debate as to whether or not fumonisin can enter soil ecosystems and be taken up by plants or cause plant disease. In addition understanding the mechanistic basis for susceptibility will aid in the selection for resistant corn varieties. 2006 to 2008: Studies will continue that will define the mechanistic basis for the organ specific
toxicity of fumonisins in different animal species. Using our mass spectrometer we will determine the dose- and time dependent changes in sphingolipid biomarkers of toxicity during feeding studies in laboratory animals. In particular we will determine the kinetics of changes in sphingoid base 1-phosphates and possibly ceramides in target organs including liver, kidney, and heart and also serum. The success of these additional studies will provide an improved biomarker for assessing the organ specific effects of fumonisin and will also improve the sensitivity of the biomarker in anticipated studies to determine fumonisin effects in human populations where corn consumption is high. IMPACT: An improved biomarker will improve the quality of the international fumonisin risk assessment and will provide useful information for the USFDA and other regulatory agencies. Reducing the uncertainty in the fumonisin risk assessment will reduce the chance of over regulation of US corn exports.
2006 to 2008: Reproductive studies in mice will determine the dose response relationships for fumonisin-induce neural tube defects; including studies to determine if folate in the diet protects against (dietary) fumonisin-induced neural tube defects or if mice adapt to fumonisin exposure in a manner offering protection against the mycotoxin's neural tube defect effect. This work will be conducted in collaboration with scientists from the University of Nebraska and will focus on the possible environmental, nutritional and genetic factors that might predispose humans to birth defects known as neural tube defect in countries were corn consumption is high. There is evidence in parts of the US (Texas), China, southern Africa and Guatemala that people consuming large amounts of corn-based foods have a higher incidence of neural tube defects. These studies will help to answer whether or not fumonisin exposure could contribute to these defects. IMPACT: The findings will be used by risk
assessors and regulators worldwide to determine the levels of exposure which might pose a risk to consumers and will protect the US corn industry from overregulation due to flawed hazard identification. 2006 to 2008: Determine the extent to which hidden fumonisins and deoxynivalenol (unknown or bound forms that are not detected by routine chemical analysis) occur in cooked products and evaluate, using bioassay methods, their potential toxicity to animals. There is accumulating evidence that hidden fumonisins occur in foods with the implication that fumonisin amounts in cooked products might be underestimated by routine analytical methods. IMPACT: The studies in progress will show the extent hidden fumonisins and DON might be formed under various cooking conditions and whether the toxicity of the cooked products are underestimated by routine chemical analyses. 4a What was the single most significant accomplishment this past year? Fumonisins and Neural Tube Defects in Mice - In 2005
we determined that fumonisin B1 causes NTD in the CD1 strain of mouse when given according to the protocol of Gelineau-van Waes et al. (2005; in publications).NB This is important because it establishes that NTDs in mice are not, as previously believed, a unique response of the highly inbred and NTD susceptible LM/Bc strain; rather, the experiment shows that a robust, outbred strain commonly used in teratology and toxicology studies are also vulnerable.NB The findings also rule out inherent strain-related physiological differences as the reason that FB1 did not cause NTDs in CD1 mice when tested according to a standard teratology (FDA Segment II) protocol.NB Further feeding studies using both the LM/Bc and CD1 strains have been done in which the mice were fed diets containing fumonisins (provided by adding F. verticillioides culture material to the diets) prior to mating and throughout gestation.NB These results indicate that the no effect level for NTDs in both strains is likely > 50
ppm, that NTDs only occur at maternally toxic doses, that fumonisins cross the placenta of LM/Bc mice at doses at which no transplacental transfer of fumonisins occurs in CD1 mice--suggesting the placenta is plays an important in NTD susceptibility.NB Together the findings will improve our understanding of NTD induction by fumonisins and decrease uncertainty in regard to risk assessment and thus will protect the US corn industry from overregulation due to flawed hazard identification. NB 4b List other significant accomplishments, if any. Fumonisin Exposure in Guatemala.NB In FY 2005 maize samples (>120) from the 2004 crop were analyzed from highland (> 1700 m) and lowland (< 360 m) markets in Guatemala; as part of a collaborative study with the Instituto de Nutricion de Centro America y Panama and Duke University.NB The results show that lowland maize, highly contaminated with fumonisin, is sold in highland markets in Departments where neural tube defect (NTD) incidence is often very
high.NB Thus, fumonisin exposure in high risk areas will be greatest in groups that obtain their maize from the market place since we have shown that maize that is grown in the highlands contains very low levels of fumonisins.NB Mechanisms of Fumonisin Renal Toxicity.NB A collaboration was established between Emory University scientists and TMRU and confirmed the identity of a new sphingoid base metabolite in renal cells treated with fumonisin B1.NB The new sphingoid base is definitely a deoxysphinganine (mw=285.2).NB Its role in fumonisin toxicity is still unknown; however, it could play an important role in fumonisin renal toxicity. NB Improved Biomarkers of Fumonisin Exposure.NB Feeding studies have been completed to determine the time and dose dependent effects of fumonisins on changes in sphingoid base 1-phosphates in liver and kidney of rats.NB The results indicate that elevation in sphingoid base 1-phosphates in kidney is quite marked, whereas, elevation in liver is minimal.NB This
finding suggests that the liver and kidney metabolize free sphingoid bases quite differently and that sphingoid base 1-phosphates may play an important role in the renal toxicity in rats.NB This is important because it will allow regulatory agencies to accurately assess the risk posed to humans by fumonisins in corn-based foods, thus reducing the uncertainty in the current risk assessment and preventing overregulation of the corn industry.NB NB Processing and Deoxynivalenol.NB Baking cookies and crackers does not appreciable destroy DON.NB We also found that, in contrast, DON appears to be degraded or otherwise "lost" during the frying of donuts.NB The data ultimately will be used for setting new or confirming current guidances for DON in commodities and foods. NB Mechanism of Fumonisin Liver Toxicity.NB Additional experiments were carried out in cooperation with JC Corton, Chemical Industry Institute of Toxicology to determine the role of peroxisome proliferators in liver disease.NB This was
part of our project to determine if fumonisin-induced hepatotoxicity is mediated through peroxisome proliferator responsive pathways, which are known to mediate proliferation and cancer induction in mice given compounds known as peroxisome proliferators or, as suggested in the literature, by sphingoid bases, which act as ligands for a receptor for peroxisome proliferator compounds.NB As part of our studies on peroxisome proliferation activity by fumonisins, we established that gene expression in liver of mice exposed (diet) to FB1 and F. verticillioides are very similar to each other - but different from that in mice exposed to a model peroxisome proliferator compound.NB The studies are important for understanding fumonisin's mode of action for hepatotoxicity and carcinogenicity in mice. 4d Progress report. There are several areas where additional work is needed in order to fill knowledge gaps in the risk assessments for fumonisins and deoxynivalenol that are the basis for regulatory
action.NB First, it has been proposed that fumonisins could be a risk factor for the high incidence of neural tube defects in humans in areas where corn consumption is high.NB Second, it has been suggested that fumonisin intake is underestimated because of the presence of hidden fumonisins in foods.NB Third, gaps in the toxicology database for deoxynivalenol have resulted in proposed action limits that could adversely impact US cereal exports; these include knowledge gaps in our understanding of the mechanism of action, pharmacokinetics, and dose-response studies.NB For both fumonisins and deoxynivalenol, lack of understanding of the dose-response relationships and mechanistic relevance in humans could increase the safety factors used in future risk assessments and thus adversely impact US agriculture. NB 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Milestones completed under objective 1 of this project plan addressed
National Program Action Plan sections 1.1.1.2, 1.1.2.1, 1.1.7.1, 1.1.7.2, and 1.1.7.3.NB Specifically, used cultured cells, plant models, and genetically modified mice to determine specific molecular endpoints of fumonisin-induced disruption of sphingolipid biosynthesis and downstream mechanisms of action.NB Specifically, using cell lines demonstrated that cell-specific responses (growth vs. death) to fumonisins are determined by the relative activity of enzymes in the de novo biosynthetic and turnover pathways for sphingolipids; in corn seedlings fumonisin-induced inhibition of root growth is mediated by disruption of sphingolipid metabolism; using gene knockout and transgenic mouse strains demonstrated the close correlation between inhibition of sphingolipid metabolism and the incidence and severity of liver toxicity, that alterations in cytokine expression and downstream signaling pathways play important roles in hepatoxicity, that hepatocellular apoptosis and mitosis are not
mediated by peroxisomal proliferators activated receptor alpha (PPAR*) mediated pathways; developed patented in vitro and in vivo mechanism (ceramide synthase inhibition) based bioassays to use for testing of cooking and processing methods and ability of various fungal strains to produce fumonisin-like activity.NB The results of these studies were the basis for developing mechanism based bioassays for fumonisin like activity in foods and other matrices and provided critical data for science-based risk assessments worldwide, thereby enabling US and international regulatory decisions to protect consumers while insuring the continued availability of high quality commodities both for US consumption and export to foreign markets. NB NB Milestones completed under objective 2 of this project plan addressed National Program Action Plan sections 1.1.7.2 and 1.1.7.3.NB Specifically, demonstrated that dietary fumonisin can modulate aflatoxin and N-methyl- N'-nitro-nitrosoguanidine tumorigenicity in
long-term feeding studies using rainbow trout and that the fumonisin promotion of aflatoxin carcinogenicity is closely correlated with evidence of disruption of sphingolipid metabolism; that dietary fumonisins modulate host resistance to infectious agents, specifically showed increased resistance of mice to infection by the parasite Trypanosoma cruzi and that the mechanism of resistance was related to increased nitric oxide production and altered sphingolipid biosynthesis; using mice, rats and chicks and cell lines showed that fumonisin could modulate glycosphingolipid biosynthesis in intestinal and other tissues and thus alter microbial receptor expression in tissues; found that fumonisin inhibition of ceramide biosynthesis protected renal cells exposed to E. coliNB endotoxins under conditions where the cytokine TNF-alpha was increased.NB The results from the aflatoxin/fumonisin interaction study shows that efforts to reduce aflatoxin in corn should also carefully consider the effect
of management strategies on the fumonisin levels.NB Similarly, the effectiveness of animal vaccines and the response of farm animals and humans to infectious agents will be affected by consumption of fumonisin contaminated feeds and foods. NB Milestones completed under objective 3 of this project plan addressed National Program Action Plan sections 1.1.1.2, 1.1.7.1, 1.1.7.3. Specifically, identified the critical step that significantly reduces fumonisin levels in masa and tortilla chips prepared from contaminated corn under commercial conditions; using a mechanism-based in vitro bioassay, showed that chemical analyses accurately predicted biological activity of the masa and tortilla chips; using a rat feeding bioassay and validated toxicological endpoints, showed that frying and baking did not significantly affect measurable fumonisin levels or toxicity of fumonisin- contaminated cornmeal; using chemical analysis and fumonisin-like activity using an in vitro mechanism-based bioassay
determined that traditional Mayan tortilla production methods reduce total fumonisins, conducted a five year survey of fumonisins in corn grown in Guatemala, and used the combined data to produced an exposure assessment for corn consumers in Guatemala; using a combination of in vivo and in vitro bioassays showed that high levels of fumonisins were present in joala, a home-brewed beer consumed in Africa, made from F. verticillioides contaminated corn but that another potentially important novel genotoxic, DNA adduct forming compound made by the fungus is not transferred to the joala.NB The results showed that the processing methods could reduce fumonisins in foods and that the chemical analysis and the biological activity based on the biomarkers developed in objective 1 was in close agreement and indicated that hidden fumonisins are not produced in significant amounts during cooking or frying of cornmeal, nor are they produced during traditional household processes for producing
tortillas. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Unit scientists have been requested to share their expertise in numerous national and international food safety forums and to serve as members of organizations that shape the international thinking on the regulation of and human risks associated with mycotoxin contaminated foods.NB Examples of activities in 2005 include presentations, participation in workshops, development of position papers, design of research programs and contributions to task forces assembled to address specific food safety issues.NB These activities have occurred at numerous venues including the USDA-ARS 4nd Fungal Genomics, 5th Fumonisin Elimination and 17th Aflatoxin Elimination Workshops, The 44rd Annual Meeting of the
Society of Toxicology, The 39th United States-Japan Joint Panel on Toxic Microorganisms, The Society of Toxicology Task Force on Chemical/Biological Terrorism Resource Registry, The W-1122 Multistate Research Project, the International Agency for Research on Cancer Publication 158 Editorial Board, the Natural Sciences and Engineering Research Council (NSERC), Canada, the 2005 International Life Sciences Institute Annual Meeting, the 2005 Midwest AOAC meeting, the Satellite Workshop to the Gordon Research Conference "Application of Emerging Technologies to Mycotoxin and Phycotoxin Research", the 2005 Gordon Research Conference on Mycotoxins and Phycotoxins, and the "EU-USA Bilateral Workshop on Toxigenic Fungi and Mycotoxins".NB The results of the five year fumonisin survey in Guatemala and a preliminary fumonisin exposure assessment for Guatemala has been developed and the results will be presented to representatives of the Guatemala Ministry of Health, Codex Guatemala and the Consejo
Nacional de Cienca y Technologia (28-31 August 2005). NB We have established a trust fund agreement with the National Food Processor Assoc. to determine the chemical fate of deoxynivalrnol (DON) during the preparation of common wheat-based food products.NB The data will be used for the ongoing reevaluation of DON by Codex.NB Preliminarily, we have found that baking cookies and crackers does not appreciable destroy DON.NB We also found that, in contrast, DON appears to be degraded or otherwise "lost" during the frying of donuts.NB The information was furnished to the National Food Processors and USFDA prior to last Springs meeting of Codex.NB The data ultimately will be used for setting new or confirming current guidance for DON in commodities and foods.NB 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Presentations: Riley, R.T., Torres, O.A., Palencia,
E., Lopez de Pratdesaba, L., Glenn, A.E., ODonnell, K. and Fuentes, M. Ingestion of fumonisins in corn and corn tortillas in Guatemala. Presentation at the 55th Anniversary of the Institute of Nutrition of Central America and Panama session on Folic Acid and Iron in Health and Development. September 8-10, 2004. Guatemala City, Guatemala. Voss, K.A., Gelineau-van Waes, J.B., Riley, R.T., Burns, T.D. andNBBacon, C.W. Reproductive toxicity of fumonisins revisited: Comparing the effects of Fusarium verticillioides culture material in female CD1 and LM/Bc mice. Proceedings of the Annual Meeting of the University of Georgia Center for Food Safety. Atlanta, GA. March 1-2, 2005.NB Voss, K.A.,NBGelineau-van Waes, J.B., Riley, R.T., Burns, T.D. and Bacon, C.W. Maternal and fetal toxicity of fumonisin-producing Fusarium verticillioides culture material in LM/Bc and CD1 mice.NBAnnual Meeting of the United States/Japan Joint Panel on Toxic Microorganisms, United States/Japan Cooperative Program for
the Development and Utilization of Natural Resources, Atlanta, GA. November, 2004.NB Gelineau-van Waes, J.B., Maddox, J., Starr, L., Wilberding, J., Bauer, L. , Voss, K.A. and Riley, R.T. Maternal fumonisin exposure, altered folate transport and increased risk for neural tube defects. AOAC International Midwest Section Final Program, p. 34, Kansas City, KA. May 23-26, 2005. NB Riley, R.T., Torres, O.A., Palencia, E., Lopez de Pratdesaba, L., Glenn, A.E., ODonnell, K. and Fuentes, M. Fumonisins in highland and lowland maize in Guatemala and a preliminary exposure estimate. AOAC International Midwest Section Final Program, pp. 34-35, Kansas City, KA. May 23-26, 2005. NB Constable, P.D., Riley, R.T., Waggoner, A.L., Hsiao, S.H., Foreman, J.H., Tumbleson, M.E.; and. Haschek, W.M. Serum sphingosine1-phosphate and sphinganine-1-phosphate are elevated in horses exposed to Fumonisin B1. AOAC International Midwest Section Final Program, pp. 63-64, Kansas City, KA. May 23-26, 2005. Williams, L.D.,
Glenn, A.E. and Riley, R.T. Accumulation of sphingoid bases and sphingoid base 1-phosphates: A possible mechanism for Fusarium verticillioides corn-seedling disease. Fungal Genomics and Fumonisin Elimination Workshops. October 26-28, 2004. Sacramento, California. Riley, R.T. "Fumonisins, risk for health and commerce." Invited seminar presentation for Consejo Nacional de Ciencia y Technologia (CONCYT) /National Congress of Science and Technology and Instituto de Nutricion de Centro America y Panama (INCAP)/Institute of Nutrition of Central America and Panama, Guatemala City, Guatemala, August 30, 2005. Voss, K.A. "Maternal and fetal toxicity of fumonsin-producing Fusarium verticillioides culture material in LM/BC and CD1 mice". Presentation for the 39th Annual Meeting of the U.S./Japan Joint Panel on Toxic Microorganisms of the U.S./Japan Cooperataive on Development and Utilization of Natural Resources (UJNR). Atlanta, GA, November 9-10, 2004.
Impacts
(N/A)
Publications
- Rentz, S.S., Showker, A.J., Meredith, F.I., Riley, R.T. 2005. Inhibition of sphingolipid biosynthesis decreases phosphorylated ERK2 in LLC-PK1 cells. Food and Chemical Toxicology. 43:123-131.
- Humpf, H., Voss, K.A. 2005. Effects of thermal food-processing on the chemical structure and toxicity of fumonisin mycotoxins. Molecular Nutrition and Food Research. 48:255-269.
- Corton, J., Apte, U., Anderson, S.P., Limaye, P., Yoon, L., Latendreasse, J., Dunn, C., Everitt, J.X., Voss, K.A., Swanson, C., Wong, J.S., Gill, S. S., Chandraratna, R., Kwak, M.K., Kenzler, T.W., Stulnig, T., Steffensen, K.R., Gustafsson, J., Mehendele, H. 2004. Mimetics of caloric restriction include agonists of the lipid-activated nuclar receptors. Journal of Biological Chemistry. 279:46204-46212.
- Gelineau-Van Waes, J., Starr, L., Maddox, J.R., Aleman, F., Voss, K.A., Wilberding, J., Riley, R.T. 2005. Maternal fumonisin exposure and risk for neural tube defects: disruption of sphingolipid metabolism and folate transport in an in vivo mouse model. Birth Defects Part A: Clinical amd Molecular Teratology. 73:487-497.
- He, Q., Riley, R.T., Sharma, R.P. 2005. Myriocin prevents fumonisin B1- induced sphingoid base accumulation in mice liver without ameliorating hepatotoxicity. Food and Chemical Toxicology. 43:969-979.
- Williams, L.D., Glenn, A.E., Bacon, C.W., Smith, M.A., Riley, R.T. 2005. Accumulation of sphingoid bases and sphingoid base 1-phosphates: a possible mechanism for Fusarium verticillioides corn-seedling disease [abstract]. Toxicological Sciences. 84(S1):285.
- Riley, R.T., Showker, A.J., Voss, K.A. 2005. Time- and dose-dependent changes in sphingoid base 1-phosphates in tissues from rats fed diets containing fumonisins [abstract]. Toxicological Sciences. 84(S1):284-285.
- Voss, K.A., Gelineau Van-Waes, J.B., Riley, R.T., Burns, T.D., Bacon, C.W. 2005. Developmental toxicity of diets containing fumonisin B1 to LM/BC and CD1 mice: a comparative study. Toxicological Sciences. 84:(S1):1396.
- Stauber, A.J., Liu, J., Waalkes, M.P., Brown-Borg, H., Voss, K.A., Kopchick, J.J., Corton, J.C. 2005. Constitutive expression of peroxisome proliferator-activated receptor alpha-regulated genes in dwarf mice. Toxicological Sciences. 84(S1):119.
- Corton, J.C., Apte, U., Anderson, S.P., Limaye, P., Yoon, L., Latendresse, J., Everitt, J., Voss, K.A., Kimbrough, C., Wong, J.S., Gill, S.S., Chandraratna, R.A., Kwak, M., Kensler, T.W., Stuling, T.M., Steffensen, K. R., Gustaffson, J., Mehendele, H.M. 2005. Role of PPAR alpha in caloric restriction effects in mouse liver [abstract]. Toxicological Sciences. 84(S1):12.
- Riley, R.T., Pestka, J. 2005. Mycotoxins: Metabolism, mechanisms and biochemical markers. In: Diaz, D., editor. The Mycotoxin Blue Book. Nottingham, UK:Nottingham University Press. p. 279-294.
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Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Overview: Over $100 billion of exported food commodities are susceptible to mycotoxin contamination. It has been estimated that in the United States economic crop loss due to mycotoxins is between $418,000,000 and $1,660,000,000 annually. Animal losses are estimated to be $6,000,000 per year. Human health effects have been documented for several food- borne mycotoxins, including aflatoxins, ergot alkaloids, and trichothecenes. In the United States, it is well established that farm animal diseases are caused by Fusarium mycotoxins that occur in major cereal grains, including corn and wheat. Because Fusarium mycotoxins are known causes of farm animal disease and suspected causes of human disease, their presence can adversely affect the marketability and export of crops and agricultural products.
The current basis for setting international tolerance levels for mycotoxins in agricultural products is science based risk assessment. Failure to accurately assess the hazards associated with mycotoxin exposure can result in over regulation of US exports such as corn, wheat and nuts. For example, a recent study funded by RAND Science and Technology estimates that imposing a 0.5 ppm fumonisin international standard on corn would result in a $170,000,000 loss of US corn exports. Likewise, at the EU standard of 4 ppb of aflatoxins in peanuts, US export losses would be about $120,000,000. Reducing the uncertainty in mycotoxin hazard assessment will reduce the likelihood of the over regulation of US agricultural exports. Objectives: The long-term goal of this project is to provide the basis for identifying and reducing hazards to animals and man related to mycotoxin exposure. The objectives are: a) define the biochemical, molecular and physiological mechanisms by which mycotoxins act
including factors that modify susceptibility to disease; b) predict the consequences of exposure to combinations of mycotoxins and determining the extent to which mycotoxins modify the response of animals or humans to infectious agents; and c) evaluate cooking and processing procedures with the intent to minimize mycotoxin exposure to consumers of commodity- based food products. Approaches: The following approaches are being used to accomplish the objectives: a) identify, evaluate and apply biochemical and molecular mechanisms of action as biomarkers and criteria to evaluate control and decontamination methods, as well as to provide information required for risk analysis; b) identify and evaluate the ability of fumonisin to modify the biological activity of other toxins and infectious agents; c) evaluate the effects of cooking and processing on the distribution and biological activity of fumonisin in corn-based food products. Who will benefit and impact. Relevant products resulting
from accomplishment of the objectives will include: basic and applied knowledge documented by scientific publications, as well as patented processes based on mechanisms of action; diagnostic methods; bioassay methods; analytical techniques; decontamination procedures; therapeutic agents; research tools; and scientifically sound risk assessments by regulatory agencies. The public will benefit from this research through the continued availability of safe food. Expected users of the findings from the proposed studies will include the food and feed industry since one objective will be to determine the fate of mycotoxins during processing. In addition, scientists and regulatory agencies will benefit from the research defining toxicity levels, effects of combined toxins as occur in naturally contaminated crops, and the biochemical mechanisms by which toxic effects are produced. Producers, extension personnel, and consumers will also benefit by learning levels of mycotoxins and
combinations of mycotoxins that produce adverse effects. 2. List the milestones (indicators of progress) from your Project Plan. Milestones covering 5/01 to termination on 5/06 (conduct/complete/document = XXX/YYY/ZZZ) 5/01 5/02 5/03 5/04 5/05 5/06 Determine the mechanism of action of mycotoxins to develop functional biomarkers: Enzyme kinetic XXXXXXXXXXXXXXXYYYZZZZZ Plant disease XXXXXXXXXXXXXXXXYYYYZZZZZ TNF/LPS XXXXXXXXXXXXYYYYYYYZZZZZ Screening XXXXXXXXXXXXXXXXXXXXXXXXXXXYYYYYYZZZZZ Cytokines XXXXXXXXXXXXYYYYYZZZZZZZ Peroxisomes XXXXXXXXXXXXXXXXXXXXXXXYYYYYZZZZZ Determine if fungi and their metabolites modify the biological activity of other toxins and infectious agents: 1.a. AFB1/FB1 XXXXXYYYZZZZ 1.b. Mon/FB1 XXXXXXXXXXXXXYYYYYYYYZZZZZ 1.c. DON/ZER XXXXXXXXXXXXXXXXYYYYYYYYYYZZZZZZ 1.d. FB1/MFO/DNA XXXXXXXXXXXXXYYYYYYZZZZZZZ 2.a. FB1/T. cruzi XXXXXXXXXYYYYYZZZZZ 2.b.
FB1/Receptors XXXXXXYYYYYZZZZ To evaluate control by decontamination methods, and for hazard assessments: FB Tortilla chips XXXXXXXXXXXXXXXXXXYYYYZZZZZZ FM/FB Beer XXXXXXXXXXXXXXXXXXXXXXXXXXYYYYYZZZZZZZZ FB Guatemala XXXXXXXXXXXXXXXXXXXXXXXXXXXXYYYYYZZZZZZZ 3. Milestones: A. Fully or substantially met in FY 2004 and those not fully or substantially met. All milestones for objectives 1.1, 1.3, 1.5, 1.6, 2.1.a, 2.2.a, 2.2.b, 3. 1, and 3.3 are fully or substantially completed including documentation. Objective 1.2 is in progress but has been modified to include the use of corn seedlings and the conduct milestone has been extended to 5/05 and the complete and document milestones have been extended to late 2005. Objective 1.4 is completed but has not been documented. Objective 2.1.b was initiated, however, preliminary results obtained in the conduct milestone indicated that the interaction between moniliformin and fumonisin was minimal and additional studies were deemed to
not be cost effective and the results of the preliminary study were documented only as a published abstract. Objectives 2.1.c and 2.1.d have not been initiated due to the retirement of the principle investigators. The conduct milestone for objective 3.2 has been partially completed and documented but aspects of the conduct milestone that involve collaborators at another Federal Laboratory (National Center for Toxicology Research) may never be completed or will be delayed due to a redirection of resources. B. Milestones to be addressed (FY 2005, 2006, & 2007) and expected accomplishments (impacts/outcomes/results) and revisions. 2005-2006: Under objective 1, studies will be conducted to determine the role of fumonisin-induced ceramide synthase inhibition in corn seedling disease and the genetic basis for resistance/susceptibility. The role of soil type in modulating the plant response to fungal infection will be addressed using the elevation in sphingoid bases and their
1-phosphates as a biomarker of exposure and also for the ability of fumonisins to be translocated to and be biologically active in aerial tissues (leaf and stems). IMPACT: These studies will be of great interest to agronomists and plant pathologist because they will provide a validated biomarker for detecting fumonisin effects on corn seedlings in the field and will resolve the debate as to whether or not fumonisin can enter soil ecosystems and be taken up by plants or cause plant disease. In addition understanding the mechanistic basis for susceptibility will aid in the selection for resistant corn varieties. 2005 to 2007 - Within objective 1 we will continue to explore the mechanistic basis for the organ specific toxicity of fumonisins. Using our newly acquired mass spectrometer we will determine the dose- and time dependent changes in sphingolipid biomarkers of toxicity during feeding studies in laboratory animals. In particular we will determine the kinetics of changes in
sphingoid base 1-phosphates and possibly ceramides in target organs including liver, kidney, and heart and also serum. The success of these additional studies will provide an improved biomarker for assessing the organ specific effects of fumonisin and will also improve the sensitivity of the biomarker in anticipated studies to determine fumonisin effects in human populations where corn consumption is high. IMPACT: An improved biomarker will improve the quality of the international fumonisin risk assessment and will provide useful information for the USFDA and other regulatory agencies. Reducing the uncertainty in the fumonisin risk assessment will reduce the chance of over regulation of US corn exports. 2005 to 2007 Also under objective 1 we will conduct reproductive studies in mice to determine the dose response relationships for fumonisin-induce neural tube defects and in collaboration with scientists from the University of Nebraska we will conduct studies to better understand
the possible environmental, nutritional and genetic factors that might predispose humans to birth defects known as neural tube defects (NTD) in countries were corn consumption is high. There is evidence in parts of the US (Texas), China, southern Africa and Guatemala that people consuming large amounts of corn-based foods have a higher incidence NTDs. These studies will help to answer whether or not fumonisin exposure could contribute to these defects. IMPACT: The findings will be used by risk assessors and regulators worldwide to determine the levels of exposure which might pose a risk to consumers and will protect the US corn industry from overregulation due to flawed hazard identification. 2005 to 2007 Under objective 3 we have begun and will expand investigations to determine the extent to which 'hidden fumonisins' (unknown or bound forms that are not detected by routine chemical analysis) occur in cooked products and evaluate, using well established bioassay methods, their
potential toxicity to animals. There is accumulating evidence that 'hidden fumonisins' occur in foods with the implication that fumonisin amounts in cooked products might be underestimated by routine analytical methods. IMPACT: The studies in progress will show the extent 'hidden' fumonisins might be formed under various cooking conditions and whether the toxicity of the cooked products are underestimated by routine chemical analyses. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2004 : In FY 2004 corn samples (>120) from the 2003 crop were analyzed from highland (> 1700 m) and lowland (<360 m) fields in Guatemala. As part of a five year collaborative study with the Instituto de Nutricion de Centro America y Panama, data from the 2000, 2001, 2002 and 2003 crops (>450 samples) were analyzed statistically and the mean total FB and its frequency of detection in corn from the lowlands were significantly higher than
from the highlands. The incidence of FB positive samples and Fusarium verticillioides infection was significantly greater in the lowland corn. It was also found that the stored corn from the 2002 crop collected from households immediately before harvest of the 2003 crop had significantly more FB positive samples than the corn collected at the time of harvest in 2002. Based on a recall study, the daily intake of total FBs was estimated and the results showed that a significant portion of the population in Guatemala could exceed the maximal tolerable daily FB intake established by the World Health Organization. The work is important because it shows that in Guatemala FB exposure will be greatest just before harvest, that Lowland corn is the source of greater FB than corn from the highlands and provides the basis for future studies to establish in humans the potential health risks from FB exposure. The field work conducted in 2003 was supported by the Pan American Health Organization
and previously supported by the USDA-FAS and the International Life Sciences Institute of North America. B. Other significant accomplishment(s), if any. Methods were developed to isolate and purify a new sphingoid base metabolite in renal cells treated with fumonisin B1. The new sphingoid base has been tentatively identified as deoxysphinganine (mw=285.2) and its role in fumonisin toxicity is unknown. However, the levels in treated cells can become extremely high after only a few hours of exposure suggesting that they could play an important role in fumonisin renal toxicity. Studies were conducted to determine the dose- and time-dependent uptake of fumonisin B1 by corn seedlings and to relate the levels accumulated in root tissue to reduced root development and to the degree of disruption of sphingolipid metabolism. Methods were developed to quantify for the first time the ability of fumonisins in soils to cause changes in sphingoid base 1-phosphates in plant tissues. It was
found that fumonisins caused dose-dependent changes in the levels of free sphingoid bases and their 1-phosphates in root tissues and that the degree of elevation was closely correlated with the levels of fumonisins in root tissue and the effects on root growth. The work is significant because it strongly supports the notion that fumonisins are pathogenicity factors in Fusarium verticillioides induced corn seedling disease. Methods were developed to quantitatively measure sphingoid base 1- phosphates in various tissues using ion trap mass spectrometry. The method has been validated using corn roots, horse serum, pig serum, rat kidney and various cultured cell lines. The results indicate that the elevation of sphingoid base 1-phosphates in animal serum and corn root is an extremely sensitive biomarker for exposure to fumonisins. The use of elevated sphingoid-base 1-phosphates as a serum marker in humans could resolve the question of whether or not fumonisins are capable of
inhibiting ceramide synthase in humans exposed to fumonisins in countries where corn consumption is high. This is important because it will allow regulatory agencies to accurately assess the risk posed to humans by fumonisins in corn-based foods, thus reducing the uncertainty in the current risk assessment and preventing overregulation of the corn industry. The first of a series of studies to determine if fumonisins cause neural tube defects or other teratogenic effects in mice was completed. No teratogenic effects occurred in the fetuses of either the LMBc or CD1 mouse strains when the females were fed diets containing 50 ppm fumonisin B1 beginning 5 weeks before mating. This is important because, despite earlier results to the contrary, fumonisin B1 has recently been proven to cause neural tube defects in LMBc mice under some conditions. That finding and other observations fueling concerns that fumonisins are risk factors for birth defects could potentially impact fumonisin risk
assessments, regulatory guide lines and have an unfavorable affect on commodity marketability. Our findings establish a basis to determine the relevance of the LMBc mouse model for fumonisin risk assessments. C. Significant activities that support special target populations. NONE D. Additional programmatic information The results of mechanistic studies and toxicological evaluations of fumonisins by Unit scientists have been used extensively in the setting of the FDA guidelines and the setting of provisional maximal tolerable daily intakes (PMTDI) by the WHO/FAO Joint Expert Committee on Food Additives. Because the mechanistic and toxicicological databases were sufficiently complete, the level of uncertainty was reduced and the safety factor used to calculate the PMTDI was also low. The PMTDI was requested by the CODEX committee and is used for harmonizing world trade. The data obtained by Unit scientists was also used in the carcinogenesis evaluation by the International Agency
for Research on Cancer. The mechanistic data was especially important because it demonstrated a reasonable mechanism of carcinogenicity that did not involve direct interaction of fumonisin with DNA. There are several areas where additional work is needed in order to fill knowledge gaps in the risk assessments for fumonisins and deoxynivalenol that are the basis for regulatory action. First, it has been proposed that fumonisins could be a risk factor for the high incidence of neural tube defects in humans in areas where corn consumption is high. Second, it has been suggested that fumonisin intake is underestimated because of the presence of 'hidden' fumonisins in foods. Third, gaps in the toxicology database for deoxynivalenol have resulted in proposed action limits that could adversely impact US cereal exports; these include knowledge gaps in our understanding of the mechanism of action, pharmacokinetics, and dose- response studies. For both fumonisins and deoxynivalenol, lack
of understanding of the dose-response relationships and mechanistic relevance in humans could increase the safety factors used in future risk assessments and thus adversely impact US agriculture. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Milestones completed under objective 1 of this project plan addressed National Program Action Plan sections 1.1.1.2, 1.1.2.1, 1.1.7.1, 1.1.7.2, and 1.1.7.3. Specifically, used cultured cells, plant models, and genetically modified mice to determine specific molecular endpoints of fumonisin-induced disruption of sphingolipid biosynthesis and downstream mechanisms of action. Specifically, using cell lines demonstrated that cell-specific responses (growth vs. death) to fumonisins are determined by the relative activity of enzymes in the de novo biosynthetic and turnover pathways for sphingolipids; in corn seedlings fumonisin-induced inhibition of root growth is mediated by disruption
of sphingolipid metabolism; using gene knockout and transgenic mouse strains demonstrated the close correlation between inhibition of sphingolipid metabolism and the incidence and severity of liver toxicity, that alterations in cytokine expression and downstream signaling pathways play important roles in hepatoxicity, that hepatocellular apoptosis and mitosis are not mediated by peroxisomal proliferators activated receptor alpha (PPAR() mediated pathways; developed patented in vitro and in vivo mechanism (ceramide synthase inhibition) based bioassays to use for testing of cooking and processing methods and ability of various fungal strains to produce fumonisin-like activity. The results of these studies were the basis for developing mechanism based bioassays for 'fumonisin like' activity in foods and other matrices and provided critical data for science-based risk assessments worldwide, thereby enabling US and international regulatory decisions to protect consumers while insuring the
continued availability of high quality commodities both for US consumption and export to foreign markets. Milestones completed under objective 2 of this project plan addressed National Program Action Plan sections 1.1.7.2 and 1.1.7.3. Specifically, demonstrated that dietary fumonisin can modulate aflatoxin and N-methyl- N'-nitro-nitrosoguanidine tumorigenicity in long-term feeding studies using rainbow trout and that the fumonisin promotion of aflatoxin carcinogenicity is closely correlated with evidence of disruption of sphingolipid metabolism; that dietary fumonisins modulate host resistance to infectious agents, specifically showed increased resistance of mice to infection by the parasite Trypanosoma cruzi and that the mechanism of resistance was related to increased nitric oxide production and altered sphingolipid biosynthesis; using mice, rats and chicks and cell lines showed that fumonisin could modulate glycosphingolipid biosynthesis in intestinal and other tissues and thus
alter microbial receptor expression in tissues; found that fumonisin inhibition of ceramide biosynthesis protected renal cells exposed to E. coli endotoxins under conditions where the cytokine TNF-alpha was increased. The results from the aflatoxin/fumonisin interaction study shows that efforts to reduce aflatoxin in corn should also carefully consider the effect of management strategies on the fumonisin levels. Similarly, the effectiveness of animal vaccines and the response of farm animals and humans to infectious agents will be affected by consumption of fumonisin contaminated feeds and foods. Milestones completed under objective 3 of this project plan addressed National Program Action Plan sections 1.1.1.2, 1.1.7.1, 1.1.7.3. Specifically, identified the critical step that significantly reduces fumonisin levels in masa and tortilla chips prepared from contaminated corn under commercial conditions; using a mechanism-based in vitro bioassay, showed that chemical analyses
accurately predicted biological activity of the masa and tortilla chips; using a rat feeding bioassay and validated toxicological endpoints, showed that frying and baking did not significantly affect measurable fumonisin levels or toxicity of fumonisin- contaminated cornmeal; using chemical analysis and fumonisin-like activity using an in vitro mechanism-based bioassay determined that traditional Mayan tortilla production methods reduce total fumonisins, conducted a four year survey of fumonisins in corn grown in Guatemala, and used the combined data to produced an exposure assessment for corn consumers in Guatemala; using a combination of in vivo and in vitro bioassays showed that high levels of fumonisins were present in joala, a home-brewed beer consumed in Africa, made from F. verticillioides contaminated corn but that another potentially important novel genotoxic, DNA adduct forming compound made by the fungus is not transferred to the joala. The results showed that the
processing methods could reduce fumonisins in foods and that the chemical analysis and the biological activity based on the biomarkers developed in objective 1 was in close agreement and indicated that 'hidden' fumonisins are not produced in significant amounts during cooking or frying of cornmeal, nor are they produced during traditional household processes for producing tortillas. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Unit scientists have been requested to share their expertise in numerous national and international food safety forums and to serve as members of organizations that shape the international thinking on the regulation of and human risks associated with mycotoxin contaminated foods. Examples of activities in 2004 include
presentations, participation in workshops, development of position papers, design of research programs and contributions to task forces assembled to address specific food safety issues. These activities have occurred at numerous venues including the USDA-ARS 3nd Fungal Genomics, 4th Fumonisin Elimination and 16th Aflatoxin Elimination Workshops, The 43rd Annual Meeting of the Society of Toxicology, The XI United States-Japan Joint Panel on Toxic Microorganisms, the XI International Union of Pure and Applied Chemistry, the 2004 Annual Meeting of the American Phytopathological Society, The Society of Toxicology Task Force on Chemical / Biological Terrorism Resource Registry, The W-1122 Multistate Research Project, the Industry Council for Development, the International Agency for Research on Cancer Publication 158 Editorial Board, and the International Life Sciences Institute of Europe Workshop on Trichothecenes. Methods for the assay of the biological activity of fumonisins in diverse
matrices have been developed in our laboratory and shared with University and Industry scientists and mechanistic studies have served as the basis for four US Patents including one in 2004 - US Patent no. 6,720, 184 issued 3 April, 2004. The results of the four year fumonisin survey in Guatemala has been provided to the Instituto de Nutricion de Centro America y Panama (INCAP) and provides a preliminary fumonisin exposure assessment for Guatemala. The results of the survey and the exposure assessment will be presented at the INCAP Scientific Meeting on Food and Nutrition Security and the Millennium Development Goals to be held September 8-10 in Guatemala City. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Corn fungus linked to fatal birth defects. The Atlanta Journal Constitution. April 2, 2004.
Impacts
(N/A)
Publications
- Williams, L.D., Meredith, F.I., Riley, R.T. 2004. Fumonisin- ortho- phthalaldehyde derivative is stabilized at low temperature. Journal of Chromatography B. 806:311-314.
- Voss, K.A., Riley, R.T., Gelineau-Van Waes, J.B., Bacon, C.W. 2004. Fumonisins: toxicology, emerging issues, and prospects for control and detoxification. In: Yoshizawa, T., editor, New Horizon of Mycotoxicology for Assuring Food Safety, Proceedings International Symposium of Mycotoxicology Kagawa 2003. Takamatsu, Kagawa, Japan. p. 41-48.
- Voss, K.A., Riley, R.T., Norred, W.P., Bacon, C.W., Meredith, F.I. 2004. The effects of cooking on fumonisins: overview and recent studies. Multicrop Aflatoxin and Fumonisin Elimination and Fungal Genomics Workshop- The Peanut Foundation. October 13-15, 2003, Savannah, Georgia. p. 19.
- Riley, R.T., Palencia, E., Torres, O.R., Glenn, A.E., Fuentes, M. 2004. Fumonisins in maize in guatemala and a preliminary estimate of daily intakes. Toxicological Sciences. 78:1023
- Williams, L.D., Voss, K.A., Norred, W.P., Saunders, S., Riley, R.T. 2004. In vitro screening for biological activity associated with fumonisins in nixtamalized foods [abstract]. Toxicological Sciences. 78:1029.
- Voss, K.A., Couch, L.H., Howard, P.C., Keller, N.P., Bacon, C.W. 2004. Toxicological evaluation of joala, a home-brewed beverage, prepared from corn contaminated with fusarium verticillioides culture material. Toxicological Sciences. 78:1029.
- Marasas, W.O., Riley, R.T., Hendricks, K.A., Stevens, V.L., Sadler, T.W., Gelineau-Van Waes, J., Missmer, S.A., Cabrera Valverde, J., Torres, O.L., Gelderblom, W.A., Allegood, J., Martinez De Figueroa, A., Maddox, J., Miller, J., Starr, L., Sullards, M., Roman Trigo, A., Voss, K.A., Wang, E., Merrill, Jr., A.H. 2004. Fumonisins disrupt shpingolipid metabolism, folate transport, and neural tube development in embryo culture and in vivo: a potential risk factor for human neural tube defects among populations consuming fumonisin-contaminated. Journal of Nutrition 134:711- 716.
- Riley, R.T. 2004. Panel discussion: 4th annual fumonisin elimination workshop for multicrop workshop-afllatoxin/fumonisin/fungal genomics.. Multicrop Aflatoxin and Fumonisin Elimination and Fungal Genomics Workshop- The Peanut Foundation. October 12-15, 2003, Savannah, Georgia. p.22-23.
- Voss, K.A., Plattner, R.D., Gelineau-Van Waes, J.B., Riley, R.T., Bacon, C. W. 2003. Current trends in mycotoxin research: deoxynivalenol and fumonisins. National Food Research Institute's International Symposium on Food Safety (Japan). November, 1-5, 2003, Tsukuba, Japan, p. 9-12.
- Voss, K.A., Riley, R.T., Sharma, R.P., Corton, J., Miller, J., Bacon, C.W. 2004. The use of genetically modified mice for studying the in vivo mode of action of fumonisins: studies on tumor necrosis factor alpha and the peroxixome proliferator-activated receptor alpha. Abstracts of Proceedings of US-Japan Cooperative Program on Devlopment and Utililization of Natural Products. November 9-14, 2003. Tokyo, Japan.
- Voss, K.A., Riley, R.T., Gelineau-Van Waes, J.B., Bacon, C.W. 2004. Fumonisins: toxicology, emerging issues, and prospects for detoxification [abstract]. Japanese Association of Mycotoxicology - International Symposium of Mycotoxicology in Kagawa. November 4, 2003, Kagawa, Japan. p. 5.
- Riley, R.T., Torres, O.A., Palencia, E., Glenn, A.E., Fuentes, M. 2004. A survey of fumonisins in maize in the highlands and lowlands of guatemala 2000-2002. Multicrop Aflatoxin and Fumonisin Elimination and Fungal Genomics Workshop-The Peanut Foundation. October 13-15, 2003, Savannah, Georgia. p. 15.
- SHARMA, R.P., HE, Q., JOHNSON, V.J., VOSS, K.A. INCREASED HEPATOTOXICITY OF FUMONISIN B1 IN TUMOR NECROSIS FACTOR A-KNOCKOUT MOUSE INVOLVES MODULATION OF FAS SIGNALING [Abstract]. JOURNAL OF FEDERATION OF AMERICAN SOCIETIES FOR EXPERIMENTAL BIOLOGY. 17:A1040.
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Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? The worldwide economic losses caused by mycotoxins are enormous. Over $100 billion of exported food commodities are susceptible to mycotoxin contamination. It has been estimated that in the United States economic crop loss due to mycotoxins is between $418,000,000 and $1,660,000,000 annually. Animal losses are estimated to be $6,000,000 per year. Human health effects have been documented for several food-borne mycotoxins, including aflatoxins, ergot alkaloids, and trichothecenes. In the United States, it is well established that farm animal diseases are caused by Fusarium mycotoxins that occur in major cereal grains, including corn and wheat. Because Fusarium mycotoxins are known causes of farm animal disease and suspected causes of human disease, their presence can adversely affect the marketability and export of crops and agricultural products. Products resulting from
accomplishment of the objectives will include: basic and applied knowledge documented by scientific publications, as well as patented processes based on mechanisms of action; diagnostic methods; bioassay methods; analytical techniques; decontamination procedures; therapeutic agents; research tools; and scientifically sound risk assessments by regulatory agencies. The public will benefit from this research through the continued availability of safe food. Expected users of the findings from the proposed studies will include the food and feed industry since one objective will be to determine the fate of mycotoxins during processing. In addition, scientists and regulatory agencies will benefit from the research defining toxicity levels, effects of combined toxins as occur in naturally contaminated crops, and the biochemical mechanisms by which toxic effects are produced. Producers, extension personnel, and consumers will also benefit by learning levels of mycotoxins and combinations
of mycotoxins that produce adverse effects. 2. How serious is the problem? Why does it matter? Mycotoxins threaten the health of plants, people and animals, reduce the value of crops and cause losses for farmers, create added costs for processors, and reduce our competitiveness in the export market. In particular, Fusarium mycotoxins are major problems for our most important crops, corn and wheat. Important recent developments have emphasized the seriousness of the mycotoxin problem. Based on studies by the National Toxicology Program and research conducted under this CRIS, final guidelines for allowable levels of fumonisins in various corn-based products have been released by the Food and Drug Administration. In 2001 the World Health Organization Joint Expert Committee on Food Additives (JECFA) recommended a provisional maximum tolerated daily intake (PMTDI) for fumonisins, in 2002 the International Agency for Research on Cancer (IARC) evaluated fumonisin B1 and concluded that it
is possibly carcinogenic to humans, and in 2003 the European Commission set a tolerable daily intake (TDI) for fumonisins. The current FDA guidelines, the JECFA PMTDI, EC TDI, and the IARC evaluation were all science-based and will have little impact on the US consumers and US farm exports. However, pending legislation in the European Union Community proposes to lower the maximum limits (four fold less than the FDA guidelines) for both fumonisin and deoxynivalenol in corn and cereals, based on their belief that the scientific hazard assessment is not complete or unclear (The Precautionary Principle) and at the present time the California Office of Environmental Health Hazard Assessment is issuing a notice of intent to list under Proposition 65, fumonisin B1 as a compound known to cause cancer. Therefore, it is in the best interest of both consumers and farmers to maintain strong support for the science-based risk assessment of these mycotoxins so as to counteract the possible over
regulation of our export crops and to minimize perception of excess risk where the risk is in fact small or non- existent. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? National Program 108, Food Safety (100%): Objective 2.1 of Outcome 2 under the General Goal II of the ARS Strategic Plan, 1997-2002, states that ARS shall "Maintain a safe and secure food and fiber system that meets the Nation's needs now and in the future". Specifically Strategies 2.1.2, Plant Animal Ecosystems Protection and 2.2.1, Plant and Animal Product Safety, indicate that ARS shall improve integrated management systems that contribute to protection of plants, animals and ecosystems against pests, and provide knowledge and means for production, storage, and processing of safe plant and animal products, respectively. 4. What were the most significant accomplishments this past year? A. Studies were conducted to determine if the chemical analysis
of the fumonisin mycotoxins in foods underestimates the toxicity of the contaminated foods due to the presence of "hidden" fumonisins or undetected toxic metabolites. Scientists at the Toxicology and Mycotoxin Research Unit used "household kitchen" cooking methods and, in collaboration with scientists at the Instituto de Nutricion de Centro America y Panama, used traditional methods of household tortilla production to test if cooking produces "hidden" fumonisins or fumonisin metabolites that increase the toxicity of the final products. Cooked corn products were fed to animals or extracts of cooked tortillas were used in cell culture assays to assess their toxic potential based on changes in biomarkers that are correlated with fumonisin toxicity in vitro and in vivo. The results showed that the chemical analysis and the biological activity based on the biomarker was in close agreement and indicated that "hidden" fumonisins are not produced in significant amounts during cooking or
frying of cornmeal, nor are they produced during traditional household processes for producing tortillas. B. Research was done to better characterize the biochemical changes in cells resulting from chronic exposure to the fumonisin mycotoxins. Scientists in the Toxicology and Mycotoxin Research Unit have identified a new metabolite in kidney cells treated with fumonisin B1. The new metabolite has been tentatively identified as a lipid structure, deoxysphinganine, but its role in fumonisin toxicity is unknown. However, the levels in treated cells can become extremely high after only a few hours of exposure suggesting that this metabolite might play a role in fumonisin kidney toxicity. Research studies were conducted to determine the biological availability of the fumonisin mycotoxins in soil and the possible role of fumonisin inhibiting the metabolism of a class of specific lipids in corn seedling disease. Scientists in the Toxicology and Mycotoxin Research Unit found that fumonisins
could be detected in soil in which corn seeds inoculated with the toxic fungus Fusarium verticillioides had been planted, and an analysis of the roots of corn plants showed that the fumonisin had inhibited the metabolism of this specific cellular lipid in the roots indicating that the fumonisin in the soil was biologically available to the roots. Similar results were obtained when seeds were watered with solutions containing fumonisins (in the absence of the fungus) suggesting that the fumonisins altered metabolism of this specific lipid in corn seedling disease. Studies were conducted in pigs to determine the ability of mycotoxin binding agents to prevent fumonisin-induced alterations in serum sphingolipid biomarkers indicative of fumonisin toxicity. Scientists in the Toxicology and Mycotoxin Research Unit in collaboration with scientists from the Universita Cattolica del Sacro Cuore developed a specific chemical procedure to analyze serum samples for specific metabolites of lipid
bases (sphingoid lipids) in serum from pigs fed diets containing binding agents that had been shown to be effective in trials with other mycotoxins. It was found that the binding agents were not effective at reducing fumonisin-induced alterations and showed for the first time that fumonisin induced large elevations in class of metabolites (sphingoid lipids) known to alter cardiovascular function in pigs. The results indicate that elevation of this specific sphingoid metabolite in serum can serve as an indicator substance for increased risk of fumonisin-induced cardiovascular dysfunction in pigs. Determining the toxic mode of action of the fumonisin mycotoxins is important for proper risk assessment. Scientists in the Toxicology and Mycotoxin Research Unit in collaboration with scientists from ToxicoGenomics, Chapel Hill, NC, the CIIT Centers for Health Research, Research Triangle Park, and the University of Georgia conducted two studies to make this determination. One study
showed that fumonisin toxicity in mice does not depend on the presence of specific receptor sites, and the second study showed that cell death in fumonisin treated mice is triggered under some circumstances by a specific cellular signaling molecule. These results will help regulatory scientists determine the best animal models to use for purposes of determining fumonisin risk assessments. C. Significant Activities that Support Special Target Populations D. Progress Report In 2003 research results conducted under this CRIS were used by the European Commission (EC) to set a tolerable daily intake for fumonisins which was in agreement with the earlier provisional maximum tolerable daily intake (PMTDI) for fumonisin B1 recommended by the 56th report of the Joint FAO/WHO Expert Committee on Food Additives (JECFA) . The acceptance of the weight and validity of the data generated under this CRIS by authoritative bodies such as JECFA and the EC reduces the likelihood that US corn exports
and US corn product exports will be unfairly penalized by importing countries. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Unit scientists first proposed the molecular mechanism of action of fumonisins in the early 1990's. Studies since then have provided strong evidence that the proposed hypothesis was correct, and that the toxic effects of fumonisins can be explained by their ability to disrupt sphingolipid metabolism. Sphingolipids are important not only as structural components of cells, but also serve as messengers that regulate the activities of cells, including growth, division, differentiation, apoptosis (programmed cell death), and immune response. Research conducted on this CRIS has had a significant impact on the field of sphingolipid biochemistry as well as introducing novel concepts of how fungal metabolites (of which there may be thousands not yet discovered) interact with plants, animals and
microorganisms. The discovery has also spurred the search for new antifungal agents to treat the increasing health risks from fungal pathogens. Unit scientists conducted definitive toxicological studies and were directly involved in the design and successful completion of the National Toxicology Program chronic toxicity and carcinogenicity studies of fumonisin B1 in rats and mice, as well as teratology studies of the mycotoxin in rabbits and rats. Studies showed that fumonisin B1 is carcinogenic in rodents, provided target organ, dose-response and toxicokinetic data, and also showed that fumonisin is not teratogenic. The data is critical for developing risk assessments and regulatory guidelines for fumonisins in food. Unit scientists are important members of various legislation committees for regulating the levels of fumonisins in US corn and corn products. These include European Union Community Legislation for setting maximum limits for mycotoxins in products for direct consumption
and proposed draft legislation for fumonisins that could have a serious detrimental impact on US exports including corn, asparagus, and figs. The low limits proposed by the EU Community are based on their acceptance of the "precautionary principle" as the basis for regulatory actions. However, the only effective basis for regulatory actions is one based on sound scientific evidence that is being supported strongly by members of the TMRU. This evidence is generated from the research data of TMRU, as well as the expertise of TMRU's scientists, and forms the basis for these important legislative considerations. Failure to be associated as advisor to these groups would otherwise have serious negative impacts on the future economic well being of US export of corn. 6. What do you expect to accomplish, year by year, over the next 3 years? 2004: On-going studies in Guatemala are expected to determine if humans exposed to high levels of dietary fumonisin will show serum or urine elevation
of a class of lipid-like substances referred to as sphingoid bases. Also, this study will determine if intervention by providing high quality corn to households where fumonisin consumption is high can reduce the effects on sphingoid bases and the resultant toxicity. These studies are important because risk assessors need to know if humans respond mechanistically in the same way as laboratory and farm animals to high levels of dietary fumonisins. We will continue the sampling for at least the 2003 crop year through support offered by the Pan American Health Organization and the International Life Science Institute of NA. Our USDA FAS support ended in January of 2003. Our current goal is to obtain additional outside funding to continue the survey. We suspect that the occurrence of fumonisin in Guatemala is very cyclic. 2004-2005: Studies will be done to identify cooking methods and other procedures that successfully reduce the toxicity of foods prepared from corn containing
fumonisins. This is important because it is not yet known that loss of measurable fumonisins in many types of food products actually leads to a reduction in toxicity. One study, to be done in cooperation with University of Nebraska scientists, will determine the extent to which extrusion cooking, which significantly lowers fumonisin levels in cooked products, reduces toxicity. Other experiments will determine how selected cooking methods, including masa preparation, boiling and other methods reduces toxicity in animals and, if so, determine the optimal conditions for fumonisin reduction. 2005: On going studies to determine the environmental fate of fumonisins in soil ecosystems are expected to show that fumonisins can accumulate in some soil types while passing readily through other soil types. The occurrence of fumonisin in soils where corn is grown will be evaluated. This work is important because large amounts of fumonisin can be present in corn debris and farm animal
excrement and it is unknown whether or not this fumonisin is biologically available after it has entered the soil or if it can contaminate ground water (these studies are ongoing) 2006: Studies will be conducted to determine the ability of fumonisins to induce neural tube defects in various mouse strains and under various dosing protocols. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Unit scientists have been requested to share their expertise in numerous national and international food safety forums and to serve as members of organizations that shape the international thinking on the regulation of and human risks associated with mycotoxin contaminated foods. Examples of activities including presentations, participation in workshops, development
of position papers, design of research programs and contributions to task forces assembled to address specific food safety issues. This activities have occurred at numerous venues including the -National USDA-ARS, CDC and other federal workshops dealing with fungal genomics, and fumonisin elimination -The United States-Japan Joint Panel on Toxic Microorganisms, -State university centers for food safety and quality -State universities national and international workshops on fumonisins, toxicity and neural tube defects -National wet and dry millers associations -State and national biological terrorism and homeland security workshop and summit meetings -Numerous national toxicological, plant genetic and plant pathological societies Methods for the assay of the biological activity of fumonisins in diverse matrices have been developed in our laboratory and shared with other scientists and purified mycotoxins we have produced have been provided to numerous collaborators. These include the
University of Georgia, University of Nebraska, Universities of Bologna and Piacenza, Emory University, and the Instituto de Centro America y Panama.Unit scientists have been requested to share their expertise in numerous national and international food safety forums and to serve as members of organizations that shape the international thinking on the regulation of and human risks associated with mycotoxin contaminated foods. Examples of activities including presentations, participation in workshops, development of position papers, design of research programs and contributions to task forces assembled to address specific food safety issues. These activities have occurred at numerous venues including the -National USDA-ARS, CDC and other federal workshops dealing with fungal genomics, and fumonisin elimination -The United States-Japan Joint Panel on Toxic Microorganisms, -State university centers for food safety and quality -State universities national and international workshops on
fumonisins, toxicity and neural tube defects -National wet and dry millers associations -State and national biological terrorism and homeland security workshop and summit meetings -Numerous national toxicological, plant genetic and plant pathological societies Methods for the assay of the biological activity of fumonisins in diverse matrices have been developed in our laboratory and shared with other scientists and purified mycotoxins we have produced have been provided to numerous collaborators. These include the University of Georgia, University of Nebraska, Universities of Bologna and Piacenza, Emory University, and the Instituto de Centro America y Panama. 8. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: This does not replace your peer-reviewed publications listed below). Riley, R.T. Aspects of the fumonisin risk assessment that could impact the current regulatory guidelines and
recommendations by authoritative bodies. Proceedings of 44th Annual Corn Dry Milling Conference, North American Corn Millers Association, Peoria, Ill, June 2003. Riley, R. T. and Voss, K.A. Approaches for evaluating human mycotoxin exposure in the United States through a CDC implemented countrywide survey. Center for Disease Control and Prevention, Atlanta, GA. 2003. Voss, K.A. Stability of Fumonisins in Corn Products (Processing can make a difference). Article about CRIS research on the fate of fumonisin in cooked foods appeared in "At-A-Glance", a newsletter for the food industry published by the University of Georgia Center for Food Safety Newsletter, Fall, 2002.
Impacts
(N/A)
Publications
- RILEY, R.T., PALENCIA, E. TORRES, O., HAGLER, W., MEREDITH, FL, WILLIAMS, L. FATE OF FUMONISIN IN MAIZE DURING NIXTAMALIZATION AND TORTILLA PRODUCTION BY MAYAN COMMUNITIES IN GUATEMALA. Toxicologist. 2003. v.77. Abstract. p.252.
- VOSS, K.A., LAUGHTER, A., ANDERSON, A., DUNN, C., STAUBER, A.J., RILEY, R. T., MILLER, J.D., CORTON, J.C. THE ROLE OF THE PEROXISOME PROLIFERATOR- ACTIVATED RECEPTOR ALPHA IN MODULATING THE EFFECTS OF FUMONISIN B1 IN MOUSE LIVER. Toxicologist. 2003. v.77. Abstract. p.7.
- WILLIAM, L.D., GLENN, A.E., BACON, C.W., SHOWKER, A.J., RILEY, R.T. INHIBITION OF CERAMIDE SYNTHASE IN CORN SEEDLINGS INFECTED WITH FUSARIUM VERTICILLIOIDES OR EXPOSED DIRECTLY TO FUMONISIN B1 IN SOIL. Toxicologist. 2003. v.77. Abstract p.189.
- GELINEAU-VAN WAES, J., RILEY, R.T., VOSS, K.A., BENNETT, G., STARR, L. FUMONISIN INDUCED NEURAL TUBE DEFECTS: DISRUPTION OF MEMBRANE SPHINGOLIPIDS AND FOLATE TRANSPORT. Toxicologist. 2003. v.77. p.171.
- CORTON, J.C., ANDERSON, S.P., STAUBER, A.J., LAUGHTER, A., SWANSON, C., XIAO, S., EVERITT, J., VOSS, K.A. PPAR ALPHA-DEPENDENT ALTERATIONS IN CHEMICAL-INDUCED STRESS AND LONGEVITY CORRELATES WITH INCREASED EXPRESSION OF HEAT SHOCK PROTEINS. Toxicologist. 2003. v.77. Abstract. p.340.
- VOSS, K.A., NORRED, W.P., MEREDITH, F.I., RILEY, R.T., BACON, C.W., SAUNDERS, D.S. EFFECTS OF COOKING ON THE BIOLOGICAL ACTIVITY OF FUMONISINS. TOXICOLOGIST. 2003. v.77. Abstract p.252.
- OWEN, J.R., PLATTNER, R.D., ROTTINGHAUS, G.E., RILEY, R.T., VOSS, K.A. SUBCHRONIC TOXICITY IN RATS FED CULTURE MATERIALS OF FUMONISIN-PRODUCING AND MONILIFORMIN-PRODUCING FUNGAL ISOLATES. Toxicologist. 2003. v.77. p. 253.
- PIVA, A., DIAZ, D.E., CASADEI, G., PAGLIUCA, G., GALVANO, F., SOLFRIZZO, M. , RILEY, R.T., PIVA, G. PERFORMANCE AND BIOCHEMICAL PARAMETERS OF WEANLING PIGS CONSUMING CONTAMINATED DIETS WITH OR WITHOUT THE ADDITION OF ACTIVATED CHARCOAL. Journal of Animal Science. 2003. v.81. Abstract. p.249
- SHARMA, R.P., BHANDARI, N., HE, Q., RILEY, R.T., VOSS, K.A. PARADOXICAL ROLE OF TUMOR NECROSIS FACTOR ALPHA IN FUONISIN-INDUCED HEPATOTOXICITY IN MICE. Toxicology. 2002. v.180. p.221-232.
- OSBORNE, C.D., NOBLET, G., ENONGENE, E.N., BACON, C.W., RILEY, R.T., VOSS, K.A. HOST-RESISTENCE TO TRYPANOSOMA CRUZI INFECTION IS ALTERED IN MICE FED FUSARIUM MONILIFORME (=F. VERTICILLIOIDES) CULTURE MATERIAL. Food and Chemical Toxicology. 2002. v.40. p.133-142.
- ROBENS, J.F., RILEY, R.T. INTRODUCTION: AFLATOXIN/FUMONISIN ELIMIINATION AND FUNGAL GENOMICS WORKSHOPS, PHOENIX, ARIZONA. MYCOPATHOLOGIA. 2002. v. 155. p.1-3.
- RILEY, R.T., ROBENS, J.F., Editors. PROCEEDINGS OF THE 1ST FUNGAL GENOMICS, 2ND FUMONISIN ELIMINATION AND 14TH AFLATOXIN ELIMINATION WORKSHOPS. AFLATOXIN ELIMINATION WORKSHOP PROCEEDINGS. USDA-Agricultural Research Service. Beltsville, MD. 2002. p.1-184.
- RILEY, R.T. THE USE OF CHRONIC TOXICANTS BY TERRORISTS TO DISRUPT THE FOOD SUPPLY. PROCEEDINGS INTERNATIONAL LIFE SCIENCES INSTITUTE SYMPOSIUM. CD- ROM. Washington, DC. 2002.
- RILEY, R.T.,IARC WORKING GROUP ON THE EVALUATION OF CARCINOGENIC RISK TO HUMANS. SOME TRADITIONAL HERBAL MEDICINES, SOME MYCOTOXINS, NAPHTHALENE AND STYRENE. International Agriculture Research Council Press. 2002. LYON, FRANCE. v.82. p.301-366.
- NORRED, W.P., RILEY, R.T., MEREDITH, F.I., POLING, S.M., PLATTNER, R.D. INSTABILITY OF N-ACETYLATED FUMONISIN B1 (FA1) AND THE IMPACT ON INHIBITION OF CERAMIDE SYNTHASE IN RAT LIVER SLICES. MYCOPATHOLOGIA. 2002. v.155. Abstract p.35.
- CARLSON, D.B., WILLIAMS, D.E., SPITSBERGEN, J.M., ROSS, P.F., BACON, C.W., MEREDITH, F.I., RILEY, R.T. FUMONISIN B1 PROMOTES AFLATOXIN B1 AND N- METHYL-N'-NITRO-NITROSOGUANIDINE INITIATED LIVER TUMORS IN RAINBOW TROUT. MYCOPATHOLOGIA. 2002. v.155. Abstract p.38.
- RENTZ, S.S., SHOWKER, A.J., MEREDITH, F.I., RILEY, R.T. INHIBITION OF DE NOVO SPHINGOLIPID BIOSYNTHESIS REDUCES EXPRESSION OF P42 MAP KINASE (ERK2) IN LLC-PK1 CELLS. MYCOPATHOLOGIA. 2002. v.155. Abstract p.39.
- WILLIAMS, L.D., SHOWKER, A.J., BACON, C.W., SMITH, M.A., WYATT, R.D., MEREDITH, F.I., RILEY, R.T. KINETICS AND BINDING OF FUMONISIN IN A MODEL SOIL SYSTEM. MYCOPATHOLOGIA. 2002. v.155. Abstract p.40.
- WILLIAMS, L.D., BACON, C.W., MEREDITH, F.I., FRANZLUEBBERS, A.J., WYATT, R. D., SMITH, M.A., RILEY, R.T. LEACHING AND BINDING OF FUMONISIN IN SOIL MICROCOSMS. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY. 2003. v.51. p.685- 690.
- VOSS, K.A., HOWARD, P.C., RILEY, R.T., SHARMA, R.P., BUCCI, T.J., LORENTZEN, R.J. CARCINOGENICITY AND MECHANISM OF ACTION OF FUMONISIN B1, A MYCOTOXIN PRODUCED BY FUSARIUM MONILIFORME (=F. VERTICILLIOIDES). Cancer Detection and Prevention. 2002. v.10. p.1-9.
- ROBENS, J., RILEY, R.T. Editors. SPECIAL ISSUE - AFLATOXIN/FUMONISIN ELIMINATION WORKSHOPS. Mycopathologia. 2002. v.155. p.1-123.
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Progress 10/01/01 to 09/30/02
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Over $100 billion of exported food commodities are susceptible to mycotoxin contamination. In the U.S. animal losses are $6,000,000 per year and losses in developing countries are much greater. Human health effects have been documented for several food-borne mycotoxins. Because Fusarium mycotoxins cause farm animal disease and are suspected to cause of human disease, their presence adversely affects the marketability and export value of contaminated crops and agricultural products. In export markets, mycotoxin contamination is used for price leverage. The objectives of this proposal are directly related to the National Program Action Plan for Food Safety, which identifies mycotoxin contamination of crops as a food safety concern. Since mycotoxins are naturally occurring, their complete elimination from foods is unlikely. Where elimination is impossible the nature of the risk must be
determined in order to establish acceptable levels of exposure. This is accomplished through hazard assessment and development of risk management strategies. The research strategies used include toxicological assessment of - a) the nature of the risk, b) the levels of contamination that pose minimum risk, c) the mechanisms of action, d) the fate of mycotoxins during processing, and e) applied approaches developed for decontamination. Benefits include: enhanced safety and consumer acceptance of products made from corn, wheat and other commodities; reduction of mycotoxin-related animal disease and performance problems; enhanced competitiveness of U. S. agricultural commodities in the world market place; and valuable information provided to regulatory agencies for developing exposure and hazard assessments and strategies for risk management. Products include scientific publications documenting basic and applied research about the toxicological properties of mycotoxins in foods,
patented processes based on mechanisms of action; diagnostic methods; bioassay methods; analytical techniques; decontamination procedures; therapeutic agents; research tools; and scientifically sound risk assessments by regulatory agencies. The public benefit is the continued availability of safe food and end- users include the food and feed industry, other research organizations, and regulatory agencies. 2. How serious is the problem? Why does it matter? Mycotoxins threaten the health of plants, people and animals, reduce the value of crops and cause losses for farmers, create added costs for processors, and reduce our competitiveness in the export market. In particular, Fusarium mycotoxins are major problems for our most important crops, corn and wheat. Important recent developments have emphasized the seriousness of the mycotoxin problem. Based on studies by the National Toxicology Program and research conducted under this CRIS, final guidelines for allowable levels of
fumonisins in various corn-based products have been released by the Food and Drug Administration. In 2001 the World Health Organization Joint Expert Committee on Food Additives (JECFA) recommended a provisional maximum tolerated daily intake (PMTDI) for fumonisin B1 and in 2002 the International Agency for Research on Cancer (IARC) evaluated fumonisin B1 and concluded that it is possibly carcinogenic to humans. The current FDA guidelines, the JECFA PMTDI, and the IARC evaluation were all science-based and will have little impact on the US consumers and US farm exports. However, pending legislation in the European Union Community proposes to lower the maximum limits (four fold less than the FDA guidelines) for both fumonisin and deoxynivalenol in corn and cereals, based on their belief that the scientific hazard assessment is not complete or unclear (The Precautionary Principle). Therefore, it is in the best interest of both consumers and farmers to maintain strong support for the
science-based risk assessment of these mycotoxins so as to counteract the possible over regulation of our export crops. 3. How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? National Program 108, Food Safety (100%): Objective 2.1 of Outcome 2 under the General Goal II of the ARS Strategic Plan, 1997-2002, states that ARS shall "Maintain a safe and secure food and fiber system that meets the Nation's needs now and in the future". Specifically Strategies 2.1.2, Plant Animal Ecosystems Protection and 2.2.1, Plant and Animal Product Safety, indicate that ARS shall improve integrated management systems that contribute to protection of plants, animals and ecosystems against pests, and provide knowledge and means for production, storage, and processing of safe plant and animal products, respectively. Mycotoxin contamination of crops is one of the major food safety issues addressed in National Program 108. Information gained
through the research of this CRIS contributes directly to the reduction of mycotoxin risks to consumers and helps ensure competitiveness for our commodities in the world market. 4. What was your most significant accomplishment this past year? A. A study was done in order to determine how widespread within the Fusarium genera was the ability to produce the carcinogenic mycotoxin fumonisin B1. Scientists in the Toxicology and Mycotoxin Research Unit in collaboration with Dr. Gretchen Kuldau at Penn State University, evaluated over 100 Fusarium isolates for their ability to produce "fumonisin-like" activity using the USDA patented bioassay method and the bioactivity was compared to levels of various fumonisins determined by chemical analysis. The results showed that while many isolates could produce fumonisins, all the producers came from species previously known to be fumonisin producers. This supports the contention that the ability to produce fumonisins is limited to a few species
and is not widespread within the Fusarium genera, thus reducing the scope of crops potentially contaminated with fumonisins to those infected by only a few Fusarium genera. B. 1. Studies were done to develop a better understanding of the non- genotoxic mechanisms of action of fumonisins to be used in the science- based risk assessment conducted by regulatory agencies for establishing acceptable levels of exposure in potentially contaminated export foods. Scientists in the Toxicology and Mycotoxin Research Unit in collaboration with Dr. Raghu Sharma (University of Georgia), Dr. Chris Corton, (Chemical Industry Institute of Toxicology) and Dr. David Miller (Carleton University) conducted studies of the mechanisms by which fumonisin alters cell growth and increases cell death in liver and kidney cells. It was shown that the mechanisms involve alterations in many biochemical pathways that regulate cell growth and cell death including altered expression of a protein known as mitogen
activated protein kinase and expression of a protein known as tumor necrosis factor alpha; however, expression of the protein known as Peroxisome Proliferator Activated Receptor alpha (PPAR) did not alter fumonisin liver toxicity. The results are important because they provide a better understanding of the mechanisms of fumonisin toxicity and provide plausible explanations for fumonisins liver and kidney toxicity and support the notion that there are thresholds of fumonisin exposure below which toxicity and carcinogenicity will not occur. 2. A study was conducted to determine the environmental fate in soils of fumonisins naturally occurring in corn debris. Scientists in the Toxicology and Mycotoxin Research Unit measured the ability of fumonisin to be released from corn debris by rainwater and to leach through and bind to various soil mixtures. It was found that fumonisins can be released from corn debris by rainwater and that when they enter the soil they will pass through sandy
soils but are tightly bound in more complex soils and then released from the soil by acid conditions indicating that the nature of the binding is ionic. The findings are important because they show that under certain environmental conditions fumonisin that is bound to soil can become biologically available to plants and animals living in the soil and could also enter the groundwater. 3. Studies were done to determine if exposure to fumonisins could alter immune response to infectious agents. Scientists in the Toxicology and Mycotoxin Research Unit in collaboration with Dr. Gayle Noblet and Charissa Dresden-Osborne (Clemson, University) determined the effect of fumonisin-contaminated corn on the resistance of mice to parasite infection (Trypanosoma cruzi was used as the model parasite because it causes similar diseases in man and mice and resistance involves all aspects of the immune system). It was found that resistance to the parasite in the fumonisin-exposed mice was increased
and the increased resistance was correlated with an increased ability of the mice to produce nitric oxide, an important chemical in some immune responses. This work shows that fumonisins can affect immune function in intact animals, that not all outcomes are detrimental, and provides basic information on the possible mechanism of increased resistence that may be useful in developing therapeutic interventions to reduce the adverse impact due to parasite infection. C. None D. In 2001 and 2002, guidelines set by US and international regulatory agencies had a significant positive impact on food safety and security worldwide. Research conducted under this CRIS ensured the accuracy of the final fumonisin risk assessment while maintaining the value of corn exports and security of the corn market. Research conducted under this CRIS ensured that sound scientific principles were used in establishing a) the design (final report issued 2001) for the long-term feeding studies required for
hazard assessment of fumonisin, b) the 2001 USFDA Final Guidance for Industry for fumonisin, c) the 2001 provisional maximum tolerable daily intake (PMTDI) for fumonisin recommended by the Joint FAO/WHO Expert Committee on Food Additives, and d) the non-genotoxic mechanism of action used by the International Agency for Research on Cancer in its 2002 evaluation of the carcinogenic risk of fumonisin to humans. 5. Describe your major accomplishments over the life of the project, including their predicted or actual impact? Unit scientists first proposed the molecular mechanism of action of fumonisins in the early 1990's. Studies since then have provided strong evidence that the proposed hypothesis was correct, and that the toxic effects of fumonisins can be explained by their ability to disrupt sphingolipid metabolism. Sphingolipids are important not only as structural components of cells, but also serve as messengers that regulate the activities of cells, including growth, division,
differentiation, apoptosis (programmed cell death), and immune response. Research conducted on this CRIS has had a significant impact on the field of sphingolipid biochemistry as well as introducing novel concepts of how fungal metabolites (of which there may be thousands not yet discovered) interact with plants, animals and microorganisms. The discovery has also spurred the search for new antifungal agents to treat the increasing health risks from fungal pathogens. Unit scientists conducted definitive toxicological studies and were directly involved in the design and successful completion of the National Toxicology Program chronic toxicity and carcinogenicity studies of fumonisin B1 in rats and mice, as well as teratology studies of the mycotoxin in rabbits and rats. Studies showed that fumonisin B1 is carcinogenic in rodents, provided target organ, dose-response and toxicokinetic data, and also showed that fumonisin is not teratogenic. The data is critical for developing risk
assessments and regulatory guidelines for fumonisins in food. European Union Community Legislation for setting maximum limits for aflatoxins in products for direct consumption and proposed draft legislation for deoxynivalenol and fumonisins could have a serious detrimental impact on US exports including nuts, dried fruit, corn and wheat. For example, if the draft maximum limit for fumonisin and deoxynivalenol are implemented, most, if not all US corn and wheat exports to the EU community will be eliminated. The low limits proposed by the EU Community are based on their acceptance of the "precautionary principle" as the basis for regulatory actions. The only effective counter measure is to promote sound science-based risk assessment. With regards to fumonisins, deoxynivalenol and other mycotoxins, the best way to defend against over regulation of agricultural products is to continue strong support for toxicological research within ARS. Failure to do so will have a serious negative
impact on the future economic well being of US agriculture. 6. What do you expect to accomplish, year by year, over the next 3 years? 2003: In collaboration with University of Georgia and Clemson University researchers, the effects of fumonisins on the immune system will be determined. Fumonisins, through their disruption of sphingolipid biosynthesis, can alter the expression of microbial pathogen and toxin receptors in the gut. Thus the immune response to these agents could be altered by fumonisins, as could the response to vaccines that interact with the same receptors. The research will determine whether or not fumonisins could be involved in microbial diseases or in vaccination failures. 2004: On-going studies in Guatemala are expected to determine if humans exposed to high levels of dietary fumonisin will show elevated free sphingoid bases in serum or urine. Also, this study will determine if intervention by providing high quality corn to households where fumonisin
consumption is high can reduce the effects on sphingoid bases and the resultant toxicity. These studies are important because risk assessors need to know if humans respond mechanistically in the same way as laboratory and farm animals to high levels of dietary fumonisins. Also, validation of the functional biomarker in humans will allow researchers to survey human populations to determine if there are human diseases associated with consumption of corn containing fumonisins. We have surveyed corn grown in the Central Highlands and Pacific Lowlands of Guatemala for 2.5 years and have only found a few samples of corn that contain fumonisin. This is unlike the year 1995 when fumonisin levels in tortillas from the Central Highlands contained very high levels of fumonisins. At this point we have yet to identify any population that is currently exposed to high levels of fumonisin in tortillas. The validation of the biomarker requires that we identify exposed individuals. Our current
goal is to obtain additional outside funding to continue the survey. We suspect that the occurrence of fumonisin in Guatemala is very cyclic. 2005: On going studies to determine the environmental fate of fumonisins in soil ecosystems are expected to show that fumonisins can accumulate in some soil types while passing readily through other soil types. The occurrence of fumonisin in soils where corn is grown will be evaluated and the possible effects of soil-bound fumonisin on plants and animals living in the soil will be assessed. This work is important because large amounts of fumonisin can be present in corn debris and farm animal excrement and it is unknown whether or not this fumonisin is biologically available after it has entered the soil or if it can contaminate ground water. 7. What technologies have been transferred and to whom? When is the technology likely to become available to the end user (industry, farmer other scientist)? What are the constraints, if known, to the
adoption durability of the technology? Unit scientists have been requested to share their expertise in numerous national and international food safety forums and to serve as members of organizations that shape the international thinking on the regulation of and human risks associated with mycotoxin contaminated foods. Examples of technology transfer activities by CRIS members include: a). Organized the USDA-ARS 1st Fungal Genomics, 2nd Fumonisin Elimination and 14th Aflatoxin Elimination Workshops. The workshops provided a forum for government, university and industry scientists to develop strategies for the control, reduction, and elimination of mycotoxins from U.S. commodities. The proceedings were edited and published both as an ARS in house publication and by the peer reviewed international journal Mycopathologia as a special supplemental issue (vol. 155 (1&2) in press 2002). b). Chair and presenters at the Society of Toxicology platform session on the mechanisms of action of
fumonisins. The audience included international leaders in the area of risk assessment of mycotoxins and the abstracts of the session were published in the supplemental issue of the journal Toxicological Sciences in 2002. c). Invited member of the International Agency for Research on Cancer (IARC) Monographs Working Group on the evaluation of carcinogenic risks to humans. The report of the group will be published as the 82nd volume of the IARC monographs in 2003 and will be provided to regulatory agencies worldwide. A summary of the evaluation by the working group is available on the IARC website (www.iarc.fr) d). Invited member and speaker at the The United States-Japan Joint Panel on Toxic Microorganisms. Presented experimental strategies for identifying and evaluating the risks from toxic mycotoxins in foods to Japanese and American scientists involved in food safety research. e). Invited member and presenter at The University of Georgia Center for Food Safety and Quality
Enhancement. Presented experimental strategies for assessing the role of chemical food contaminants on host resistance to infectious agents. Attendees included food safety researchers and food industry representatives. f). Invited Faculty for the USFDA JIFSAN International Workshop on Mycotoxins for the purpose of transferring the technology required to establish mycotoxin surveillance programs in developing countries. The proceedings and the lecture series on video tape and CD was or will be provided to over 50 invited scientists and regulators from developing countries in Africa, South America, Mexico, Central America, China, and other countries (Workshop proceedings in press 2002). g). Invited Expert and presenter at the The International Life Sciences Institute of North America Workshop on Biological and Chemical Agents of Terrorism in Food. The workshop was developed to address specific needs of the major North American food companies and organizations and was described by
the Surgeon General Dr. David Satcher as a model for the nation in terms of responding to future threats. The workshop deliberations were taped, transcribed and published as Proceedings and a consensus statement was developed outlining key research needs to improve our ability to responds to terrorist threats to our food supply. Proceedings published 2002. h). Invited expert and presenter at the Agro-Security Work Conference. The conference was developed to train first responders from various state and local emergency management organizations in Georgia on the risks associated with the potential use of chronic chemical toxicants by terrorist. Proceeding of the conference available at www.agrosecurity.uga. edu/conference.html i). Methods developed in our laboratory and purified mycotoxins we have produced have been provided to numerous collaborators. These include the University of Georgia, Health Canada, University of Nebraska, Auburn University, University of Iowa, Universities of
Bologna and Piacenza, Emory University, and the Instituto de Centro America y Panama. 8. List your most important publications and presentations, and articles written about your work (NOTE: this does not replace your review publications which are listed below) Popular Press: Scientists discover masa-making process lowers toxins, Food and Chemical News, April 8, 2002. ARS and Frito-Lay chip away at fumonisins. ARS News & Information, http://www.ars.usda.gov/is/pr/2002/020401.html, April 1, 2002. Presentations: Norred, W.P., Riley, R.T., Meredith, F.I., Poling, S.M., Plattner, R.D. Instability of N-Acetylated Fumonisin B1 (FA1) and the Impact on Inhibition Of Ceramide Synthase in Rat Liver Slices. Presented at the 1st Fungal Genomics, 2nd Fumonisin Elimination and 14th Aflatoxin Elimination Workshops, October 23-26, 2001, Phoenix, Arizona. Carlson, D.B., Williams, D.E., Spitsbergen, J.M., Ross, P.F., Bacon, C.W. , Meredith, F.I., Riley, R.T.. Fumonisin B1 Promotes Aflatoxin B1 and N-
Methyl-N'-Nitro-Nitrosoguanidine Initiated Liver Tumors in Rainbow Trout. Presented at the 1st Fungal Genomics, 2nd Fumonisin Elimination and 14th Aflatoxin Elimination Workshops, October 23-26, 2001, Phoenix, Arizona. Rentz, S.S., Showker, J.L., Meredith, F.I., Riley, R.T. Inhibition of de novo Sphingolipid Biosynthesis Reduces Expression of p42 MAP Kinase (pERK2) in LLC-PK1 Cells. Presented at the 1st Fungal Genomics, 2nd Fumonisin Elimination and 14th Aflatoxin Elimination Workshops, October 23-26, 2001, Phoenix, Arizona. Williams, L.D., Showker, J.L., Bacon, C.W., Smith, M.A., Wyatt, R.D., Meredith, F.I., Riley, R.T. Kinetics and Binding of Fumonisin in a Model Soil System. Presented at the 1st Fungal Genomics, 2nd Fumonisin Elimination and 14th Aflatoxin Elimination Workshops, October 23-26, 2001, Phoenix, Arizona. Norred, W.P. Overview: Mycotoxin risk analysis in the USA. Presented at the 9th International Symposium on Toxic Microorganisms/United States- Japan Cooperative
Program on Development and Utilization of Natural Resources, Joint Panel on Toxic Microorganisms, March 12-13, 2002, Tokyo, Japan. Voss, K.A. The role of toxicology and related studies in evaluating the rsk of mycotoxins: fumonisin B1 as an example. Presented at the 9th International Symposium on Toxic Microorganisms/United States-Japan Coopeative Program on Development and Utilization of Natural Resources, Joint Panel on Toxic Microorganisms, March 12-13, 2002, Tokyo, Japan.
Impacts
(N/A)
Publications
- Williams, L.D. Kinetics of leaching and binding of fumonisin B1 in soil microcosms. MS Thesis. University of Georgia, Athens. 2002. 54 p. Rentz, S.S. The role of fumonisin B1 and other inhibitors of de novo sphingolipid biosynthesis in the expression of p42 MAP Kinase (pERK2) in LLC-PK1 cells. MS Thesis. The University of Georgia, Athens. 2002. 73 p. Howard, P.C., Eppley, R.M., Stack, M.E., Warbritton, A., Voss, K.A., Lorentzen, R.J., Kovach, R.M., Bucci, T.J. Fumonisin B1 carcinogenicity in a two-year feeding study using F344 rats and B6C3F1 mice. Environmental Health Perspectives. 2001. v.109. p.277-282.
- Howard, P.C., Warbritton, A., Voss, K.A., Lorentzen, R.J., Thurman, J.D., Kovach, R.M., Bucci, T.J. Compensatory regeneration as a mechanism for renal tubule carcinogenesis of fumonisin b1 in the F344/N/Nctr BR rat. Environmental Health Perspectives. 2001. v.109. p.309-314. Norred, W.P., Riley, R.T., Meredith, F.I., Poling, S.M., Plattner, R.D. Instability of N- acetylated fumonisin B1 (FA1) and the impact on inhibition of ceramide synthase in rat liver slices. Food and Chemical Toxicology. 2001. v.39. p. 1071-1078.
- Sharma, R.P., Bhandari, N., He, Q., Riley, R.T., Voss, K.A. Decreased fumonisin hepatoxicity in mice with a targeted deletion of tumor necrosis factor receptor 1. Toxicology. 2001. v.159. p.69-79. He, Q., Riley, R.T., Sharma, R.P. Fumonisin-induced tumor necrosis factor-alpha expression in a porcine kidney cell line is independent of sphingoid base accumulation induced by ceramide synthase inhibition. Toxicology and Applied Pharmacology. 2001. v.174. p.69-77. Haschek, W.M., Voss, K.A., Beasley, V. R. Selected mycotoxins affecting animal and human health. Haschek, W.M., Roussex, C.G., Wallig, M.A., editors. Academic Press, New York, NY. Handbook of Toxicologic Pathology, Volume I. 2002. Chapter 25. p.645-699.
- Enongene, E.N., Sharma, R.P., Bhandari, N., Miller, J.D., Meredith, F.I., Voss, K.A., Riley, R.T. Persistence and reversibility of the elevation in free sphingoid bases induced by fumonisin inhibition of ceramide synthase. Toxicological Sciences. 2002. v.67. p.173-181. He, Q., Riley, R.T., Sharma, R.P. Pharmacological antagonism of fumonisin B1 cytotoxicity in porcine renal epithelial cells (LLC-PK1): A model for reducing fumonisin- induced nephrotoxicity in vivo. Pharmacology and Toxicology. 2002. v.90. p. 268-277. Riley, R.T. Chronic toxicants. Biological and Chemical Agents of Bioterrorism in Food. The Use of Chronic Toxicants to Disrupt the Food Supply. International Life Sciences Institute, Washington, DC. 2002. p.27- 29.
- Norred, W.P. Significance of fumonisins in the global food supply. Significance of Mycotoxins in the Global Food Supply - Symposium Series on Food Microbiology. International Life Sciences Institute, Washington, DC. 2002. p.43-44. Bhandari, N., Enongene, E.N., Riley, R.T., Meredith, F.I., Sharma, R.P. Temporal expression of fumonisin B1-induced tumor necrosis factor-K and interferon gamma in mice. Comparative Biochemistry and Physiology, C. Toxicology and Pharmacology. 2002. v.131. p.113-122. Voss, K.A., Humpf, H.U., Sullards, M.C., Allgood, K., Hartl, M., Riley, R. T., Merrill, A.H. Jr. In vivo formation of ceramide-like N-acylated aminopentols from hydrolyzed fumonisin B1. The Toxicologist. 2002. v.66. p. 6:Abstract No.30.
- Riley, R.T., Yoo, H.-S., Rentz, S.S. Fumonisin B1 (FB) alters ouabain- insensitive but not ouabain-sensitive Rb86 uptake in LLC-PK1 cells without altering membrane permeability. The Toxicologist. 2002. v.66. p.69- 70:Abstract No. 31. Rentz, S.S., Showker, J.L., Meredith, F.I., Riley. R. T. Fumonisin B1 induces decreased expression of P42 MAP kinase in LLC-PK1 cells. The Toxicologist. 2002. v.66. p.6:Abstract No. 32. Williams, L.D., Bacon, C.W., Meredith, F.I., Franzluebbers, A.J., Smith, M.A., Riley, R.T. Kinetics of leaching and binding of fumonisin B1 in a model soil ecosystem. The Toxicologist. 2002. v.66. p.69-70:Abstract No. 338. Howard, P.C., Couch, L.H., Voss, K.A., Lorentzen, R.J.. Buci, T.J. Prediction of the carcinogenesis of fumonisin B1 in male and female F344/N/Nctr rats from urinary and tissue sphingolipid changes. The Toxicologist. 2002. v.66. p. 1492:Abstract No. 305.
- Bolger, M., Coker, R.D., Dinovi, M., Gaylor, D., Gelderblom, M.O., Paster, N., Riley, R.T., Shephard, G., Speijers, J.A. Fumonisins. In, Safety evaluation of certain mycotoxins in food; Food and Agriculture Organization of the United Nations, Paper No. 74. World Health Organization Food Additives. 2001. v.47. p.103-279. Norred, W.P. Toxicokinetics: Fate and distribution studies of fumonisins. Journal Food Additives and Contaminants. 2001. v.18. Abstracts p.200. Riley, R.T., Bacon, C.W., Meredith, F.I., Torres, O., Saena De Tejada, S., Wang, E., Merrill, A.H. Jr. A Summary of ongoing studies in the central highlands of Guatemala. Journal Food Additives and Contaminants. 2001. v.18. Abstracts p.197. Cohen, S.M., Bidlack, W., Drjagan, Y., Goldsworthy, T., Hard, G., Howard, P., Riley, R., Voss, K. Apoptosis and its implications for toxicity, carcinogenicity and risk: fumonisin B1 as an example. Journal Food Additives and Contaminants. 2001. v.18. Abstracts p.208.
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