1a. Objectives (from AD-416):
Assays for detecting low molecular weight toxins rely upon components that can interact with the toxins and facilitate toxin isolation or detection. Because of this, the goal of developing assays with better performance characteristics (such as sensitivity, speed, and robustness) requires the development of improved materials that bind the toxins. To meet this goal, we propose four objectives: Objective 1: Develop novel biologically-based materials that bind foodborne toxicants; Objective 2: Design and develop synthetic-based materials for detection of agricultural and food-related toxins; Objective 3: Develop computational methods for foodborne toxins that enable new strategies to reduce exposure; Objective 4: Develop detection strategies for emerging toxins and toxins of commercial importance.
1b. Approach (from AD-416):
Toxins produced by fungi, mycotoxins, can cause devastating economic effects by affecting the safety and marketability of grain, and by causing disease in livestock. Diagnosis of health problems caused by mycotoxins is often difficult because while certain of the toxins can cause acute disease, most cause sub-acute or chronic effects that are more difficult to discern. For these reasons, attempts are made to detect mycotoxins at the many stages from crop production to finished product. As a result, a vital part of mycotoxin control is the availability of rapid, accurate, sensitive, and cost effective methods for toxin detection and quantitation. Fortunately, many such methods exist and are commercially available. With a few exceptions, most of these methods rely, in some fashion, on components that bind the toxins. Ideally, the binding components are highly specific for the toxins of interest and are capable of performing under their expected conditions of use. As analytical technologies progress, assays are required to perform under increasingly demanding conditions, requiring advances in their component materials. We propose to apply the expertise of the investigators in synthetic chemistry, antibody development, immunoassay and sensor development, and instrumental assay methodology, toward the development of the next generation of toxin binding materials, such materials being the fundamental basis of improved analytical technologies for these toxins. By improving technologies for detecting natural toxins, this project will have a direct impact on the ability of producers, processors, and regulatory agencies to improve monitoring programs for natural toxins, and thereby improve the safety of the U.S. food supply.
3. Progress Report:
T-2 toxin is a mycotoxin produced by several species of fungi that commonly infest grains. T-2 is acutely toxic and has been found at low levels in crops such as wheat, corn, barley, oats, and rye and has also been found in human food and animal feeds. Sensitive detection of T-2 toxin is somewhat limited because it does not fluoresce and does not have a strong ultraviolet or visible absorption band. The interactions of cyclodextrins with T-2 toxin and several other trichothecene toxins were investigated. Computer models were developed that explain experimentally observed toxin-cyclodextrin interactions. Knowledge of the molecular recognition of trichothecenes with cyclodextrins was gained and will be useful for the future development of related materials that can bind and remove trichothecenes as part of improved methods for their detection. A method for detecting metabolites of T-2 toxin was developed, based upon a sensitive analytical tool, ultra-high performance liquid chromatography (UHPLC). Detection of these metabolites is important because metabolites such as the glucose-derivatives of T-2 toxin (T-2-glucosides) evade the techniques normally used for toxin detection, yet may retain the ability to be converted back into toxins following consumption by animals. This work has been conducted in collaboration with scientists from two other projects, 3620-42000-042-00D and 3620-4200-043-00D, where the procedures for producing large amounts of the toxin-glucosides are being developed. The availability of larger amounts of the metabolites will enable more widespread adoption of methods for their detection.
1. A rapid, reusable, sensor for detecting the mycotoxin deoxynivalenol in wheat. Deoxynivalenol (DON) is a toxin produced by certain species of fungi that can infest wheat, barley, and corn. It results in substantial losses to the quality and value of grain worldwide. Due to the toxicity of DON and the desire to protect human and animal health, the U.S. conducts extensive monitoring of commodities both for domestic use and export. Agricultural Research Service (ARS) scientists in the Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Peoria, IL, investigated ways to improve upon a biosensor that they developed last year uses a novel analytical technology, biolayer interferometry (BLI). ARS scientists discovered that the signal from the sensor could be substantially increased through the use of an antibody labeled with colloidal gold, allowing the assays to be conducted more rapidly. The technique was successfully used to measure DON in samples of naturally contaminated wheat, with results that agreed well, but which were obtained more quickly than with a reference method. The result is a rapid and reusable sensor that can be used by industrial, academic, or government laboratories to monitor contamination of wheat and assist in the protection of the human food supply.
2. Development of a protein that binds to the mycotoxin deoxynivalenol. Deoxynivalenol (DON) is a toxin produced by certain species of fungi that can infest wheat, barley, and corn. It results in substantial losses to the quality and value of grain worldwide. Due to the toxicity of DON and the desire to protect human and animal health the U.S. conducts extensive monitoring of commodities both for domestic use and export. Agricultural Research Service (ARS) scientists in the Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Peoria, IL a new material for selectively binding a protein known as an scFv that was based upon a toxin binding material previously developed by this research team. An advantage of the material is that it can be produced in bacterial systems, which may lower production costs. The results suggest that despite the smaller size of the scFv, it retained much of the binding capacity of the original protein. This material, the details of its method of production, and the details of its composition, may be of use to scientists charged with developing the next generation of toxin detection kits.
3. A structural model to improve detection of citrinin and related toxins. Citrinin is a toxin produced by several fungal species that frequently contaminate agricultural commodities. This mycotoxin is associated with kidney disease in livestock and humans. To improve detection of citrinin, a state-of-the-art computational study of the three dimensional structures of this toxin was performed by ARS scientists in the Bacterial Foodborne Pathogens and Mycology Research Unit (BFP), National Center for Agricultural Utilization Research (NCAUR), Peoria, IL, in collaboration with scientists at Bradley University, Peoria, IL. Key features of the structure and properties of citrinin were identified. These results will aid researchers in the design of materials for citrinin detection and provide insight into the methods appropriate for studying related natural products. The results will be useful to food safety scientists seeking improved methods for detection of citrinin.
4. Development of a new material to absorb ochratoxin A from liquids such as wine. Ochratoxin A (OTA) is a toxin produced by certain fungi that contaminate and reduce the value of agricultural commodities. In an effort to reduce exposure to OTA, ARS scientists in the Bacterial Foodborne Pathogens and Mycology Research Unit (BFP), National Center for Agricultural Utilization Research (NCAUR), Peoria, IL, investigated the ability of a nanoporous carbohydrate-polymer material to reduce levels of OTA in water and wine. The interaction between OTA and the material was investigated using experimental and computational techniques. It was discovered that the material is capable of reducing OTA levels in water and wine, through a complex binding mechanism. In addition, these results provide important information on the application of this class of polymers to aid in the detection of toxins and will assist in more broad uses of cyclodextrin polymer materials. This research provides scientists with a new type of material and strategies to selectively reduce exposure to ochratoxin A.
5. Discovery of “masked” metabolites of T-2 toxin and HT-2 toxin. T-2 toxin is produced by a variety of fungi that commonly infest grains. T-2 is acutely toxic and has been found at low levels in crops such as wheat, corn, barley, oats, and rye, and has also been found in human food and animal feeds. Fungi and plants have evolved mechanisms to reduce toxicity of small molecules such as T-2 toxin through metabolism to forms that are less hazardous or that can be excreted. ARS scientists in the Bacterial Foodborne Pathogens and Mycology Research Unit (BFP), National Center for Agricultural Utilization Research (NCAUR), Peoria, IL, discovered the production of novel metabolites of T-2 toxin: the gluocoside derivatives of T-2 and HT-2. Such metabolites may be important because the modified versions of the toxins may evade the techniques normally used for toxin analysis, yet may retain the ability to be converted back into toxins following consumption by animals. This discovery provides the developers of analytical methods for toxin detection assays insights into which compounds are important enough to include in monitoring programs to assure the safety of grain products.
Appell, M.D., Jackson, M.A. 2012. Sorption of Ochratoxin A from aqueous solutions using beta-cyclodextrin-polyurethane polymer. Toxins. 4:98-109. DOI: 10.3390/toxinx4020098.
Appell, M.D., Moravec, D., Bosma, W.B. 2012. Quantum chemical study of the structure and properties of citrinin. Journal of Molecular Structure (Theochem). 38(4):284-292.
Shephard, G.S., Berthiller, F., Burdaspal, P.A., Crews, C., Jonker, M.A., Krska, R., Macdonald, S., Malone, R.J., Maragos, C.M., Sabino, M., Solfrizzo, M., Van Egmond, H.P., Whitaker, T.B. 2012. Developments in mycotoxin analysis: an update for 2010 - 2011. World Mycotoxin Journal. 5(1):3-30.
Maragos, C.M. 2012. Signal amplification using colloidal gold in a biolayer interferometry-based immunosensor for the mycotoxin deoxynivalenol. Journal of Food Additives & Contaminants. 29(7):1108-1117.
Maragos, C.M., Li, L., Chen, D. 2012. Production and characterization of a single chain variable fragment (scFv) for the mycotoxin deoxynivalenol. Food and Agricultural Immunology. 23(1):51-67.
Busman, M., Poling, S.M., Maragos, C.M. 2011. Observation of T-2 and HT-2 glucosides from Fusarium sporotrichioides by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Toxins. 3(12):1554-1568.