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:
Citrinin is a toxin produced by several molds with the potential to contaminate agricultural commodities. Synthetic receptors were developed to help detect citrinin. By using computer aided design, ARS scientists Peoria, Illinois, developed a synthetic material with binding sites capable of selectively isolating citrinin. The material is suitable for application in a new detection method for this toxin in corn. Many countries have problems with the naturally occurring toxin deoxynivalenol (DON, also known as “vomitoxin”). DON is commonly found in wheat, barley, and corn. To improve test kits used for monitoring of the toxin, ARS scientists are pursuing enhancements to the toxin-binding components of the kits. Many such kits are based upon monoclonal antibodies that are produced in large amounts using experimental animals. To reduce the reliance upon animals, antibody fragments (single chain fragment variable: scFv) can be produced in cell culture systems. We have improved the expression (production) of a DON scFv in cell culture systems, and modeled the interaction of the DON with the scFv. Zearalenone (ZEA) is an estrogenic compound produced by certain fungi that commonly infest important cereal crops, including corn and wheat. Binding of ZEA to proteins in the blood, such as albumin, is one factor that could influence the distribution and elimination of ZEA. To provide insights into how ZEA might be distributed within the body following exposure, researchers from Southwest University (Chongqing, China) and ARS scientists investigated the interaction between ZEA and bovine serum albumin (BSA). Results indicated that ZEA binds to BSA with strong affinity, suggesting that serum albumins are likely involved in the distribution of this mycotoxin throughout the body following exposure. This information helps to explain how ZEA exposure is related to toxicity and will be helpful in efforts to mitigate the effects of this toxin. T-2 toxin, which is produced by a variety of fungi during the infestation of growing corn kernels, is acutely toxic to many animals. A technique was developed by ARS scientists to determine levels of T-2 toxin in corn. With the direct analysis in real time mass spectrometry (DART-MS) technique, sample extracts applied to paper surfaces underwent ionization under ambient conditions and the resulting ions were detected by mass spectroscopy. The procedure achieved the rapid and sensitive detection of T-2 toxin from corn samples without the extensive sample preparation or the chromatographic separation normally required. The method permits the convenient analysis of T-2 toxin from corn with greater selectivity than commonly used screening methods. This work provides a rapid, sensitive, and convenient analytical tool to grain processors seeking to assure the safety of corn based foods.
1. A novel method to detect aflatoxin M1 in milk. Cows that ingest aflatoxin B1, a highly carcinogenic mycotoxin commonly found in cattle feed, can metabolize it to another carcinogen, aflatoxin M1 (AFM1), which is excreted in their milk. A technique was developed by ARS scientists in Peoria, Illinois, to determine AFM1 in cow’s milk. The technique combines mass spectrometry with an ambient ionization method that allows sampling from paper surfaces. The procedure, known as direct analysis in real time-mass spectrometry (DART-MS), allows the rapid and sensitive detection of AFM1 from milk samples without extensive sample preparation or the time consuming chromatographic separation that is often used. This work provides a rapid, sensitive and convenient analytical tool to food processors seeking to assure the safety of milk based products.
2. A method for routine measurement of patulin in fruit products. Patulin is a mycotoxin commonly found in certain fruits and fruit products, in particular, apple juice and apple products. Recently very high levels of patulin were found in samples of fruit leathers from Iran. To determine whether patulin was present in dehydrated apple products (apple leathers), two techniques were developed by ARS scientists in Peoria, Illinois. A technique called ultra performance liquid chromatography-photodiode array (UPLC-PDA) was used to determine the fate of patulin during the preparation of home-made apple leathers and to screen samples of commercial fruit leathers. Patulin levels increased during the home-style preparation of the fruit leathers, due to concentration during drying. Of the 36 commercial products tested, 14 were above the limit of detection (3.5 ppb), 9 were above the limit of quantification (12 ppb), and one was above 50 ppb. These results demonstrated that patulin can be concentrated during the preparation of fruit leathers. However, despite this, the levels found in commercial products were relatively low, implying that the commercial process was adequate for controlling the carry-over of patulin into the final product.
3. Structure-activity relationships among trichothecene mycotoxins. Trichothecenes are toxins produced by certain molds, and are of concern due to their potential contamination of agricultural commodities and foods. ARS scientists in Peoria, Illinois, identified important properties of deoxynivalenol, nivalenol, T-2 toxin, and 32 other related trichothecenes using computational chemistry methods that help explain trichothecene toxicity, biosynthesis, and detection. In addition, molecular properties distinguishing type B from type A trichothecenes were identified and provide information on their mechanism of toxicity. Several computer generated structure-activity models from this study possess excellent descriptive ability and serve as inexpensive tools to evaluate biosynthesis and potential trichothecene toxicity and false-positive detection outcomes. The results of this study are helpful to food safety scientists, toxicologists, and regulators concerned with trichothecene exposure.
4. Characterization of “masked” T-2 glucosides. T-2 toxin is produced by a variety of fungi during the infestation of growing corn kernels and is acutely toxic to many animals. Certain fungi, and the plants that they infest, can convert T-2 toxin into less toxic forms by making sugar derivatives (glucosides). Because the derivatives are not detected with commonly used techniques, they are termed “masked” toxins. The masked toxins are of concern because of the possibility that they may be converted back to the original toxin during digestion or food processing. Development of improved methods for T-2 toxin and its derivatives has been hindered by the limited availability of the toxin-glucosides. ARS scientists in Peoria, Illinois, developed an efficient way to produce a T-2 toxin-glucoside. Having this material permitted evaluation of its toxicity and the evaluation of the extent to which it can be detected by commonly available methods. A commercial test kit for T-2 toxin was ineffective at detecting the T2-glucoside, whereas an immunoassay based upon an antibody directed against the T2-glucoside (also developed by this group, see the FY2014 report) was able to do so. The developed antibody will be a useful tool for the effective diversion of contaminated commodities from the human food supply.
Appell, M., Bosma, W.B. 2015. Assessment of the electronic structure and properties of trichothecene toxins using density functional theory. Journal of Hazardous Materials. 288:113-123.
Busman, M., Bobell, J.R., Maragos, C.M. 2014. Determination of the aflatoxin M1 (AFM1) from milk by direct analysis in real time - mass spectrometry (DART-MS). Food Control. 47(2015):592-598.
Berthiller, F., Brera, C., Crews, C., Iha, M.H., Krska, R., Lattanzio, V.M.T., MacDonald, S., Malone, R.J., Maragos, C.M., Solfrizzo, M., Stroka, J., Whitaker, T.B. 2015. Developments in mycotoxin analysis: an update for 2013 – 2014. World Mycotoxin Journal. 8(1):5-36.
Maragos, C.M., Busman, M., Ma, L., Bobell, J.R. 2015. Quantification of patulin in fruit leathers by ultra-high-performance liquid chromatography-photodiode array (UPLC-PDA). Journal of Food Additives & Contaminants. 32(7):1164-1174.
McCormick, S.P., Kato, T., Maragos, C.M., Busman, M., Lattanzio, V.M.T., Galaverna, G., Dall-Asta, C., Crich, D., Price, N.P.J., Kurtzman, C.P. 2014. Anomericity of T-2 toxin-glucosides; masked mycotoxins in cereal crops. Journal of Agricultural and Food Chemistry. 63(2):731-738.
Biswas, A., Appell, M., Liu, Z., Cheng, H.N. 2015. Microwave-assisted synthesis of cyclodextrin polyurethanes. Carbohydrate Polymers. 133:74-79. doi: 10.1016/j.carbpol.2015.06.044.
Busman, M., Maragos, C.M. 2015. Determination of T-2 and HT-2 toxins from maize by direct analysis in real time mass spectrometry. World Mycotoxin Journal. 8(4):489-497.