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NEW HEADINGS: R712 . Protect Food from Contamination by Pathogenic Microorganisms, Parasites, & Naturally Occurring Toxins; S1510 . Corn; F1102 . Mycology; S1549 . Wheat, general/other; F1150 . Toxicology

OBJECTIVES: Determine if Fusarium sp. toxins, alone or in combination, alter the effects of other mycotoxins, increase susceptibility to infectious diseases, or produce chronic toxicity. Determine mechanisms of action, and use them as bioassay tools and as criteria to evaluate control and decontamination methods. Determine effects of processing of Fusarium contaminated corn on mycotoxin content of final products.

APPROACH: Use in vivo and in vitro methods to assess whether Fusarium toxins or other mycotoxins that co-occur naturally contribute to acute or chronic diseases. In vivo studies will include pathology, reproduction, and fate and distribution. In vitro studies will be done with primary cultures and cell lines, tissue slices, or cell fractions. Endpoints will include enzyme release, cell respiration, DNA alterations, and inhibition of protein synthesis. Disruption of sphingolipid metabolism by fumonisins will be studied in depth to help determine how fumonisins contribute to animal diseases, and whether it is related to the reputed non-genotoxic carcinogenicity of fumonisins. The effect of processing of corn on fumonisins, and occurrence of toxins with fumonisin-like activity will be studied by chemical analysis and mechanism-based bioassays.

KEYWORDS: corn; fusarium; mycotoxins; food contamination; food safety; fumonisin; toxicity; wheat; food processing; bioassays; sphingolipids; fusarium moniliforme; toxins; disease susceptibility; mechanism of action; decontamination; chronic diseases; acute infection; lipid metabolism; carcinogenicity; chemical analysis

PROGRESS: 1999/01 TO 1999/09
1. What major problem or issue is being resolved and how are you resolving it? This CRIS addresses a number of problems, the nature of which are highly important from both food safety and economic aspects. Several diseases of farm animals are caused by toxic metabolites produced by Fusarium species associated with major crops, including corn and wheat. These mycotoxins, including fumonisins and trichothecenes, have an adverse impact on the marketability of the crops, especially in export markets where mycotoxin contamination, even at very low levels, is used for price leverage. Fumonisins and other Fusarium toxins also appear to be factors in some human diseases, thus raising concern about the safety of food products manufactured from contaminated commodities. Elimination of the fungi and their mycotoxins from the commodity is the topic of on-going research and is perhaps an ultimate solution. However, there has been little if any success by this method, and the prospects for success are poor. This CRIS attempts to resolve this problem by (1) determining the biochemical and molecular mechanisms of action, and using the knowledge to develop therapeutic agents and bioassay methods for analytical use, diagnostic procedures and decontamination evaluation,(2) determining the effects of processing of Fusarium contaminated corn on mycotoxin content of final products and (3) determining whether Fusarium sp. toxins, alone or in combination, alter the biological activity of other mycotoxins, increase susceptibility to infectious diseases, or produce chronic toxicity. The outcome of this research will be reduction of animal and human health concerns through mycotoxin management that does not depend on elimination of the fungus. Recognition of the high quality of TMRU research in this area ensures USDA representation on panels and committees involved in regulatory activities or in making recommendations to agencies in the U. S. (FDA, EPA), European Union, United Nations (World Health Organization, Food and Agriculture Organization), and other national (Office of Environmental Health Hazard Assessment, Prop. 65) and international organizations (International Life Sciences Institute, International Union for Pure and Applied Chemistry). Another important outcome is that through understanding mechanisms of action, new approaches to control and detection of fungal contamination will be possible. 2. How serious is the problem? Why does it matter? An important development in 1999 heightened food safety concerns in the mycotoxin area. The National Toxicology Program announced the results of its long-term feeding studies on fumonisin B1, the prevalent Fusarium mycotoxin found on corn. The studies, conducted in two species, provided "clear evidence" of the carcinogenicity of fumonisin. This finding presents serious problems for corn producers and processors, and for regulatory agencies that must decide whether or not to impose action limits, and what level of contamination will cause increased risks to consumers. The general public is increasingly concerned about food safety, and press reports of mycotoxins or other naturally occurring toxins in food often contain inaccurate speculation. Competitiveness of our agricultural products in the world marketplace is a vital concern. Criticism by foreign buyers of low-level mycotoxin contamination of U. S. grains and the perceived problems they cause is often used for price leverage. ARS has a mission to provide the technology necessary to insure safe, nutritious food and feed. If a potential hazard cannot be simply and effectively eliminated, ways must be found to reduce the risk to the lowest possible levels. For mycotoxins, this can be done by determining how the toxin works, what levels of contamination pose minimum risk, the fate of mycotoxins during processing of raw commodities, and development of strategies for destruction or inactivation of the toxins. Mechanistic data can be used to develop control strategies that involve increased plant resistance to the growth of the fungus. 3. How does it relate to the National Program(s) and National 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 plans, 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 insure competitiveness for our commodities in the world market. 4. What were the most significant accomplishments this past year? Unit scientists proposed a novel mechanism of carcinogenicity for fumonisin B1. This model is based on TMRU's past research on the role of disrupted sphingolipid metabolism as the mechanism of action for fumonisin induced apoptosis and altered cell proliferation and the recent discovery of the role of inhibition of de novo ceramide biosynthesis in the protection of DNA damaged cells from ceramide mediated cell death. Long term studies (9-month feeding trial) with fusaric acid in the diet of rats demonstrated altered testicular weight:brain weight ratios in males and decreased body weight in females. Furthermore, fusaric acid modified brain neurochemicals associated with reproduction and seasonality in these animals. The results were proportional with the increased concentrations of fusaric acid in the diets, and suggest the long tern ingestion of this mycotoxin may contribute to unexplained Fusarium toxicities in animals. A segment I reproduction study of F, moniliforme culture material showed, aside from slight increases in litter weights at relatively high doses, no significant reproductive effects. Subsequent studies showed fumonisin B1 does not cross the placenta. Teratology studies in rats, mice and rabbits were negative. The amount of measurable fumonisins in masa and fried tortilla chips was found to be considerably less than that of the corn from which these products were made. Relatively low amounts of hydrolyzed fumonisins were measured in the products. It was found that cooking, steeping and rinsing the corn was the most important step in reducing fumonisins and the content of hydrolyzed fumonisin in the products. Identification of those steps that reduce fumonisins will aid in developing cooking/processing methods for removing fumonisins from corn-based products by both industrial and private concerns. Collaborative studies with scientists at Clemson University have shown that T. cruzi infected mice can be used as a model for studying the effects of F. moniliforme on the overall immune system in vivo. Future studies using this system can be used to obtain immunotoxicity data, a current data gap in our understanding of F. moniliforme and fumonisin toxicity. Previous studies, relying on in vitro systems, have focused only on parts of the immune system and do not take into account the multidimensional and integrated immune system found in vivo. 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 directly contributed to the design and successful completion of chronic toxicity and carcinogenicity studies of fumonisin B1 in rats and mice, as well as teratology studies of the mycotoxin in rabbits and rats, which were done by the National Toxicology Program at FDA's National Center for Toxicological Research. Studies showed that fumonisin B1 is carcinogenic in rodents, provided target organ and dose-response data, and also showed that fumonisin is not teratogenic. The data is critical for developing risk assessments and (any) regulatory guidelines for fumonisins in food. 6. What do you expect to accomplish, year by year, over the next 3 years? Continue studies to determine: 1) if dietary inhibitors of glycosphingolipid biosynthesis alter expression of microbial pathogen and toxin receptors in the digestive epithelia of rodents and poultry, 2) why sphingoid bases accumulate in cells exposed to fumonisins, 3) the mechanism by which fumonisin B1 alters the expression of proinflammatory cytokines and the response of genetically modified mice to fumonisins-induced lever and kidney toxicity and immune response, 4) if serine palmitoyltransfersase inhibition will reduce the toxicity of fumonisin in plants, 5) the biological activity of isomers of acetylated fumonisins, 6) if humans exposed to high levels of dietary fumonisin will have elevated free sphingoid bases in serum or urine, 7) how fumonisin-induced changes in ceramide and glycosphingolipid biosynthesis can modify the cellular response to pathogens, microbial toxins, and how these responses can be modified by concurrent alterations in proinflammatory cytokines. The Frito-Lay CRADA is in the process of being extended. The additional work will concentrate on a) confirmation of current findings by developing more selective and sensitive analytical methods, b) identification, quantitation and fate of fumonisin-adducts and byproducts which are formed during the cook/steep, baking and frying steps of the process, c) develop the best possible "mass balance" for following fumonisins through the manufacturing process, and d) finally, determination of the relative toxicities of the raw corn, process intermediates (such as masa) and the final product (tortilla chips). An in vitro bioassay for fumonisin-like activity using tissue cultures will be used to screen fungal extracts for agents that prevent the fumonisin-induced inhibition of ceramide synthase, and which thereby might have utility in counteracting the effects of fumonisin consumption. An in vivo interaction study on whether moniliformin producing fungi have any effect on the toxicity caused by fumonisin-producing F. moniliforme. The study will further explore the possible role of fumonisins in causing cardiac toxicity, as has been suggested by recent work using swine as a model. 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 & durability of the technology product? 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 include Unit scientists who are: invited members of the International Life Science Institute Apoptosis Working Group; invited to organize and chair a symposium on "Chemical Modifiers of Response to Food-Borne Microbial Pathogens"; invited to be FAO/WHO/UNEP Resource Person for drafting the Report of the Third Joint FAO/WHO/UNEP International Conference on Mycotoxins; invited to be a Task Group Member and Scientific Expert for the finalizing the Environmental Health Criteria Fumonisin B1 (document will be used by the International Programme on Chemical Safety, World Health Organization, and the European Community, Joint Expert Committee on Food Additives); invited session chair for "Molecular targets for Toxins", 1999 Gordon Research Conference on Mycotoxins and Phycotoxins; invited speaker on mycotoxins and human disease at the 50th anniversary of the Institute of Nutrition of Central America and Panama; invited speakers and session chairs at the International Fumonisin Toxicology Conference held in Washington, DC; invited to speak at the Xth International IUPAC conference on Mycotoxins and Phycotoxins in Brazil. Highly purified fumonisins and Fusarium culture materials were provided to collaborators in the U. S. and foreign countries, including Japan, China, Germany and France. Also ISP-1, a serine palmitoyltransferase inhibitor, was given to collaborators at University of Georgia, University of Illinois, University of South Carolina, Emory University, and the National Institute of Infectious Diseases, Japan. Negotiations are ongoing with Avanti Polar Lipids for the purpose of developing a CRADA. USDA will provide technical advice and crude culture material, Avanti will isolate and purify ISP-I, and Avanti provide USDA with 200 mg of pure ISP-I to be used in animal feeding and other studies. 8. List your most important non-peer reviewed publications and presentations to non-scientific organizations, and articles written about your work(NOTE: this does not replace your peer reviewed publications which are listed below). Popular Presentations: RILEY, R.T.-- Invited presentaton for the Connecticut Environmental Health Concerns 12th Annual Symposium - Fungal Toxins, Challenges to Agriculture and Food Safety, U. of Connecticut, Storrs, CT, December 15, 1998. RILEY, R.T.--Gordon Research Conference on Phycotoxins and Mycotoxins: Satellite Conference on Toxin Testing at Mt. Desert Island Biological Laboratory, Salisbury Cover, ME, presentation and laboratory demonstration, June 17-19, 1999; Chair for "Molecular Targets for Toxins, Plymouth State College, Plymouth, NH, June 20-25, 1999. RILEY, R.T.--Invited Presentation for the Japanese Association of Mycotoxicology, Tokyo, Japan, January 5-9, 1999. ILSI North America International Conference on the Toxicology of Fumonisin, Arlington, VA, June 28-30, 1999, sponsored by U.S. Food and Drug Administration, National Institute of Environmental Health Sciences, U.S. Department of Agriculture, ILSI North America-Technical Committee on Food Toxicology and Safety Assessment. TMRU scientists were invited to serve as follows: William P. Norred on the Scientific Planning Committee; Presentations - "An Overview of Rodent Toxicities", Kenneth A. Voss; "Sphingolipid Perturbations as Mechanisms for Fumonisin Carcinogenesis", Ronald T. Riley; William P. Norred, Co-Chair of Session III. NTP Bioassay and Mechanisms of Carcinogenesis. Based on the above popular presentations, the following news items were generated: March, 1999 ARS Quarterly Report; June, 1999 Agricultural Research; Green Grass Syndicated Features, in press; 5/99 Big Sky Country Radio interview; 7/99 Topeka, KS TV interview. Popular Publications: Visconti, A., Marasas, W.F.O., Miller, J.D., and Riley, R.T. Fumonisins. Proceedings of the Third International Conference on Mycotoxins, World Health Organization, Food and Agricultural Organization, www.fao.org/WAICENT/FAOINFO/ECONOMIC/ESN/mycoto /mycoto.htm, 1999. Bhandari, N., Riley, R.T., Sharma, R.P. Expression of tumor necrosis factor-alpha in mouse liver after treatment with fumonisin B1. University of Georgia Research Symposium, Science and Technology for a Sustainable Future, Program and Abstracts. March 22, 1999. Marasas, W.F.O., Miller, J.D., Riley, R.T., and Visconti, A. Fumonisins-occurrence, toxicology, metabolism, and risk assessment. In: B. Summerell and L. Burgess, eds, Paul Nelson Memorial Symposium, APS Press, (accepted June, 1999).

PUBLICATIONS: 1999/01 TO 1999/09
1. RILEY, R.T., VOSS, K.A., NORRED, W.P., BACON, C.W., MEREDITH, F.I., and SHARMA, R.P. 1999. Serine palmitoyltransferase inhibition reverses ... kidney of BALB/c mice. Env. Tox. Pharm. 7:109-118.
2. RILEY, R.T. and NORRED, W.P. 1999. Mycotoxin prevention and decontamination: Corn-a case study. Food, Nutrition and Agriculture 23:25-32.
3. RILEY, R.T., NORRED, W.P., and VOSS, K.A. 1999. Fungal inhibitors of sphingolipid biosynthesis: Tools, toxins, and therapeutics. Mycotoxins 49:9-17.
4. MEREDITH, F.I., TORRES, O.R., MERRILL, A.H. Jr., and RILEY, R.T. Fumonisin B1 and hydrolyzed fumonisin B1 levels in nixtamalized maize...Guatemala. J. of Food Prot. (accepted June 22, 1999).
5. RILEY, R.T. and PLATTNER, R.D. Fermentation, partial purification, and use ...from Isaria (=Cordyceps) sinclairii. Methods in Enzymol., Vol. 314. A. Merrill, Jr. and Y. Hannun (eds.). (Accepted April 5, 1999).
6. NORRED, W.P., VOSS, K.A., RILEY, R.T., MEREDITH, F.I., BACON, C.W., and MERRILL, A.H., JR. 1998. Mycotoxins and health hazards: Toxicological ... of fumonisins. J. Toxicol. Sci. 23:160-164.
7. LI, W. 1998. Fumonisin-induced sphingolipid alterations, Hepatotoxicity and Apoptotic cell d eath in regenerating rat liver. Ph.D. Dissertation, University of Georgia, Athens, GA. 191 pp.
8. VOSS, K.A., RILEY, R.T., BACON, C.W., MEREDITH, F.I. and NORRED, W.P. Simultaneous exposure to fusaric acid did not modify the toxicity ... materials. Food Chem. Tox. (accepted June 1, 1999).
9. WANG, E., RILEY, R.T., MEREDITH, F.I. and MERRILL, A.H., Jr. 1998. Fumonisin B1 consumption by rats cause reversible, dose-dependent increases in urinary sphinganine and sphingosine. J. of Nutrition 129:214-220.
10. BAILEY, C.A., FAZZINO, J.J., Jr., ZIEHR, M.S., SATTAR, M., HAQ, A.U., ODVODY, G., and PORTER, J.K. Effects of sorghum ergot on broiler performance. Poultry Sci. (accepted July 20, 1999).
11. DUGYALA, R.R., SHARMA, R.P. and MEREDITH, F.I. 1999. Modification of fumonisin B1 response by endotoxin and tumor necrosis factor alpha. Environmental and Nutritional Interactions 3:33-45.
12. GROVES, F.D., ZHANG, L., CHANGE, Y.-S., ROSS, P.F., CASPER, H., NORRED, W. P., YOU, W.-C. And FRAUMENI, J.R. 1999. Fusarium mycotoxins in corn and corn products...Shangdong Province, China. J. AOAC Internl. 82:657-662.
13. ENONGENE, E.N., SHARMA, R.P., VOSS, K.A., and RILEY, R.T. 1999. Subcutaneous fumonisin administration disrupts sphingolipid...in the digestive epithelia in mice. Toxicol. Sci. 48:54.
14. LI, W., RILEY, R.T., VOSS, K.A., and NORRED, W.P. 1999. Role of proliferation in the in vivo response of rat liver to fumonisin B1. Toxicol. Sci. 48:53.
15. SHARMA, R.P., TSUNODA, M., BHANDARI, N., RILEY, R.T., and VOSS, K.A. 1999. Modification of fumonisin B1 response in mice carrying human tumor necrosis factor alpha transgene. Toxicol. Sci. 48:149.
16. RILEY, R.T., PESTKA, J.J., AND ROTH, R.A. 1999. Chemical modifiers of response to food-borne microbial pathogens. Toxicol. Sci. 48:302.
17. RILEY, R.T., VOSS, K.A., KUMAGAI, S., SUGITA-KONISHI, Y., and SHARMA, R.P. 1999. Xenobiotic-induced alterations in glycosphingolipid receptors...response. Toxicol. Sci. 48:303.

PUBLICATIONS: 1998/01/01 TO 1998/09/30
1. RILEY, R.T., VOSS, K.A., NORRED, W.P., SHARMA, R.P., WANG, E. and MERRILL, Jr., A.H. 1998. Fumonisins: Mechanism of Mycotoxicity. Revue de Medicine Veterniaire 149:617-626.
2. RILEY, R.T. 1998. Mechanistic interaction of mycotoxins: Theoretical considerations. IN: Mycotoxins in Agriculture and Food Safety, SINHA, K.K. and BHATNAGAR, D., Eds., Marcel Dekker, Inc., New York, NY.
3. VOSS, K.A., PLATTNER, R.D., RILEY, R.T., MEREDITH, F.I. and NORRED, W.P. 1998. In vivo effects of fumonisin B1-producing ... cause hepato- and nephrotoxicity in rats. Mycopathologia 141:45-58.
4. VOSS, K.A., RILEY, R.T., BACON, C.W., MEREDITH, F.I. and NORRED, W.P. 1998. Toxicity and sphinganine levels are correlated in rats fed fumonisin B1 (FB1) or hydrolyzer FB1. Env. Toxicol. Pharmacol. 5:101-104.
5. TSUNODA, M., SHARMA R.P. and RILEY, R.T. 1998. Early fumonisin B1 toxicity in relation to disrupted sphingolipid metabolism in male BALB/c Mice. J. Biochem Molecular Toxicol. 12:281-289.
6. DUGUALA, R.R., SHARMA, R.P., TSUNODA, M. and RILEY, R.T. 1998. Tumor necrosis factor-alpha as a contributor in fumonisin B1 toxicity. Journal of Pharmacology and Experimental Therapeutics 285:317-324.
7. NORRED, W.P., PLATTNER, R.D, RILEY,R.T., MALCOM, and VOSS,K.A. 1998. Sphingoid base elevation in precision-cut liver slices by fumonisin analogs: Dependence of the primary amino group. Toxicol. Sci. 42(1S):289.
8. VOSS, K.A. and PORTER, J.K. 1998. Fusaric acid is not hepato- or nephrotoxic when fed to rats: A preliminary study. Toxicological Sciences 42(1S):289.
9. KANATI , A., SHARMA, R.P. and RILEY, R.T. 1998. Fumonisin B1 exposure potentiates the effects of tumor necrosis factor in a sensitive cell line. Toxicological Sciences 42(1S):288.
10. RILEY, R.T., BACON, C.W. and SHARMA, R.P. 1998. Fungal serine palmitoyltransferase inhibitor reverses fumonisin-induced antiproliferative effects in LLC-PK1 cells. Toxicological Sciences 42(1S):288-289.
11. SHARMA, R.P., TSUNODA, M., RILEY, R.T. and VOSS, K.A. 1998. Relationship of fumonisin dose and biochemical effects with cellular apoptosis in mouse liver and kidney. Toxicological Sciences 42(1S):289.

PUBLICATIONS: 1996/05/01 TO 2001/05/01
1. RIMANDO, A.M. and PORTER, J.K. 1997. Fusaric acid increases melatonin (MEL) in serum of weanling rats. J. Toxicol. Environ. Health
2. RILEY, R.T., SHOWKER, J.L., OWENS, D.L. and ROSS, P.F. 1997. Disruption of sphingolipid metabolism...by Fusarium proliferatum culture material containing fumonisin B2 or B3. Environ. Toxicol.
3. RILEY, R.T., LIOTTA, D.C. and MERRILL, JR., A.H. 1996. Sphingolipid analogue biomodulators in foods alter sphingolipid metabolism:...health and disease. Proceedings of the Nutrition Soc.
4. MERRILL, JR., A.H., LIOTTA, D.C. and RILEY, R.T. 1996. Sphingolipids as regulators of cellular growth...behavior. pp. 273-298. IN: Gross, R.W. (ed.), Advances in Lipobiology, Vol. 1. JAI Press, Inc.,
5. MERRILL, JR., A.H., et al. 1997. Sphingolipids - the enigmatic lipid class: Biochemistry, physiology, and pathophysiology. Toxicol. Appl. Pharmacol. 142:208-225.
6. NORRED, W.P., RILEY, R.T., VOSS, K.A. and BACON, C.W. 1996. Fumoninsin-mechanism of action and toxicology: implications for the immune response. La Selezione Veternaiia. Accepted April 23, 1997.
7. NORRED, W.P., PLATTNER, R.D., MEREDITH, F.I. and RILEY, R.T. 1997. Mycotoxin-induced elevation of free sphingoid bases in precision-cut rat liver slices... Toxicol. Appl. Pharmacol. Accepted July 24, 1997.

CRIS SUPPLEMENTARY DATA:     Region: 2   Process Date: 2001/07/11   Progress Update: 2000/05/16