Research Project: Improved Analytical Technologies for Detection of Foodborne Toxins and Their Metabolites

Location: Mycotoxin Prevention and Applied Microbiology Research

2019 Annual Report

Objectives
The goals of this project are to enhance food safety through the development of tools to more effectively monitor for natural toxins, and to reduce exposure to such toxins. The first goal will be addressed by improving methods for toxin detection, in particular methods to detect multiple toxins and their metabolites simultaneously. To meet this goal requires the development of materials with performance characteristics capable of being used in multiplexed assays and materials that can detect toxins that are currently poorly detected (such as the masked mycotoxins). Development of these materials is integrated with a second goal, the removal of toxins from foods, thereby reducing exposure. To meet these goals we have four objectives. Objective 1. Improve detection of foodborne toxins through development of novel technologies based upon biosensor platforms and new component materials. Sub-objective 1.1. Development and evaluation of multiplexed assay platforms. Sub-objective 1.2. Development and evaluation of materials that can function in multiplexed assays. Objective 2. Improve detection of foodborne toxins through development of direct detection technologies based upon novel mass-spectrometric platforms. Sub-objective 2.1. Develop novel ambient ionization mass spectrometric techniques for detecting single or closely related groups of foodborne toxins. Sub-objective 2.2. Expand techniques such that they can detect multiple toxins simultaneously (multiplexed assays). Objective 3. Improve the ability to detect and measure “masked” mycotoxins and biomarkers of mycotoxin exposure in commodities and foods. Sub-objective 3.1. Application of novel mass spectrometric tools to detect masked toxins. Sub-objective 3.2. Development of novel bio-based materials for masked mycotoxin detection. Objective 4. Improve toxin detection methods and reduce exposure through the development and application of synthetic materials. Sub-objective 4.1. Develop synthetic receptor materials using computational methods and materials science/synthetic strategies. Sub-objective 4.2. Characterize and apply synthetic receptor materials in methods to reduce exposure to toxins.

Approach
Food crops are commonly infested with fungi, both in the field and in storage. Certain fungi produce toxins (mycotoxins) that can adversely affect human health and the health of domestic animals. By permitting the timely diversion of contaminated ingredients from the food supply, detection of foodborne toxins can directly improve food safety and the safety of animal feed. Monitoring for the presence of such naturally occurring toxins is widespread and occurs at many of the stages between the producer and the consumer. Increasing the efficiency and improving the accuracy of monitoring results in more appropriate and efficient diversion of contaminated products. The need to monitor for greater numbers of mycotoxins is a trend that will continue, in particular because of recent concern over the so called “masked” mycotoxins. This project seeks to address the need for improved toxin detection by developing rapid, multi-toxin detection methods. Two major approaches will be used: development of advanced biosensor techniques and development of novel chemical methods based upon mass spectrometry. In support of the former, novel biological binding materials will be developed. Certain toxin-binding materials may also have the potential to be used to remove toxins from foods, and this will be approached through the development and application of novel synthetic materials to reduce exposures.

Progress Report
Progress was made on all four objectives and their subobjectives, all of which fall under National Program 108, Component 1, Foodborne Contaminants. This project is directed towards Problem Statement 4, chemical and biological contaminants: detection and characterization methodology, toxicology, and toxinology. The project is part of a larger management unit that deals with the problems associated with fungi that infest plants and the toxic metabolites that such fungi can produce (mycotoxins). Research conducted under Objective 1 of this project uses novel technologies and materials to improve detection of such toxins. Under this objective we made significant progress in developing improved screening assays for a group of fungal toxins known as the trichothecenes. The trichothecenes are produced by fungi that infest oats, wheat, barley, and corn. Their presence causes substantial economic losses worldwide. Among the trichothecenes T-2 toxin has been associated with the condition Alimentary Toxic Aleukia, a hemorrhagic disorder reported in people that consumed moldy grain. As part of efforts to improve monitoring of these toxins, researchers at the Institute of Sciences of Food Production (ISPA) in Bari, Italy, and ARS researchers in Peoria, Illinois, developed a novel method to detect trichothecenes in wheat. The trichothecenes, produced by fungi, are toxic to plants as well as animals. Plants often protect themselves by modifying toxins to which they are exposed. In this case, wheat plants can combine T-2 with a sugar to render it less toxic. There is concern, however, that during digestion the original toxin may be released. As such, methods that detect the modified forms are needed. Objective 3 of this project targets these so-called “masked” or “hidden” toxins. The method that was developed to detect the original toxin (T-2 toxin) was also used to detect modified (glycosylated) forms of this toxin in wheat. The method used a technology, fluorescence polarization immunoassay, that is both rapid and easy to use. Thus, significant progress has been made on a rapid, sensitive, and convenient method that can be used to monitor specific trichothecenes and their modified forms in wheat. Progress was also made on a confirmatory technique (LC-MS/MS) for the analysis of the modified trichothecene toxins. The technique is being applied in a survey of the modified trichothecenes in U.S. oats. Under the same objective, progress was made on the development of a confirmatory method for detecting modified forms of the fumonisins in corn. The fumonisins are a group of mycotoxins that have been implicated in several diseases of humans and domestic animals. Notably, these toxins produce a type of pulmonary edema in pigs, and a brain-wasting disease in horses (equine leukoencephalomalacia). As with the trichothecenes, plants and microbes can modify the fumonisins by attaching a carbohydrate residue (often a sugar) to the toxin, which can “mask” the fumonisins from detection. The presence of fumonisin-carbohydrate complexes in corn was evaluated by a type of confirmatory technique (LC-HRMS). To improve sensitivity and accuracy the method incorporated isotopically labelled standards that were synthesized by ARS researchers in Peoria, Illinois. In collaboration with researchers at the University of Aberdeen in the United Kingdom, these standards are also being used to evaluate the metabolic fate of modified fumonisins and modified trichothecenes in a laboratory-based system that mimics the mammalian gut. Improved methods for detecting mycotoxins based upon mass spectrometry (MS) are a central component of Objective 2 of this project, in particular the development of novel methods using a set of techniques known collectively as ambient ionization. Progress was made using a variant of ambient ionization known as Direct Analysis in Real Time (DART). In order to detect aflatoxin B1 on corn kernels a laser was combined with DART-MS. Responses were compared with those from another variant of ambient ionization MS: Desorption Electrospray Ionization (DESI). Analysis from maize kernels may be a convenient way to introduce large numbers of samples for MS analysis with minimal sample preparation. The field of ambient ionization MS continues to develop rapidly. A recent modification of the DESI-MS technique involves the direct application of the ionization voltage to the substrate surface, replacing the separate electrospray emitter. This modification of the DESI technique, commonly called “paperspray”, was applied to the analysis of mycotoxin mixtures. The paperspray analysis of the mixtures gave good sensitivity in comparison to DART-MS for several of the more water soluble mycotoxins. This application of the paperspray technique to mycotoxins could be complementary to the DART-MS technique, as devices for high throughput paperspray analysis are currently widely available. Objective 4 of the project is the development and application of synthetic materials for improved toxin detection and reduction of toxin exposure. Progress on this objective was made in the characterization and application of magnetic materials to reduce exposure to the mycotoxin fusaric acid. Fusaric acid is produced by several Fusarium species that can contaminate agricultural commodities. Exposure to this toxin is associated with adverse modulation of neurotransmitter levels. To aid in the selective isolation of fusaric acid, magnetic molecularly imprinted polymer materials were synthesized using picolinic acid as the toxin analog for imprinting. The materials are being evaluated for use in improving the reliability of spectroscopy-based analytical methods for the detection of fusaric acid in corn. Raman spectroscopy studies have identified key spectral features important for the selective detection of fusaric acid.

Accomplishments
1. Detection of “masked” fungal toxins in corn. Fumonisins are a group of fungal toxins (mycotoxins) that are found worldwide in corn and other commodities. They cause a variety of diseases in domestic animals and, in humans, have been implicated in esophageal cancer of adults and neural tube defects of newborns. Monitoring of these toxins is conducted in order to divert infested commodities from food and feed supplies. However, such monitoring is complicated by the fact that fumonisins can form derivatives with other food components which can prevent their detection with commonly used screening techniques. These so-called "masked" toxins are an indeterminate hazard. Rapid methods for their detection are desired. ARS scientists in Peoria, Illinois, developed a screening test that detected several of the masked forms of the fumonisin mycotoxins. Furthermore, to confirm the presence of such masked fumonisins in corn, a liquid chromatography high resolution mass spectrometry (LC-HRMS) method was developed for their detection. The LC-HRMS method has facilitated the production of analytical standards, allowing confident identification of such toxins in corn. Each of these tools will be useful for determining the extent to which the masked fumonisins contaminate corn and whether or not the masked forms represent a hazard to human or animal health.

2. Detection of newly discovered toxins enabled by an existing antibody. Deoxynivalenol (DON) is a toxin produced by certain fungi that cause Fusarium Head Blight, a disease of cereal crops that results in substantial economic losses worldwide. DON is both a mycotoxin, capable of causing disease in animals and a compound that facilitates fungal infection of the host cereal crop. Recently researchers in Europe discovered a group of toxins with similarities to DON. These toxins, known as “NX” toxins, are sufficiently similar to DON that they might be detected using the commercial screening tests commonly used within the industry to detect DON. To determine whether such kits might recognize the NX toxins, scientists from the University of Natural Resources and Life Sciences (Vienna, Austria) and an ARS scientist in Peoria, Illinois, investigated six commercially available test kits, and two immunoassays developed by ARS for their ability to detect DON and the NX-toxins. None of the commercial test kits were able to detect the NX-toxins, whereas one of the ARS-developed immunoassays did. This suggests that the NX-toxins are likely avoiding detection when grain is tested with the commercial kits, but that their detection is possible with the appropriate assay. Based upon these results, the ARS-developed antibody may be the most useful for assessing the frequency and distribution, and therefore the potential risk, of the NX toxins.

3. Determination of more reliable detection properties of Alternaria toxins. Alternariol and related compounds are toxins produced by fungi that contaminate cereal grains and fruits. Reliable detection methods are needed to evaluate exposure to these toxins and the extent to which contaminated food and beverages pose health risks. To aid in the development of better detection methods, researchers at the National Taiwan University, Taipai, Taiwan, and an ARS researcher in Peoria, Illinois, applied computational methods to characterize the detection properties of alternariol and related compounds. Modifications to certain chemical groups of the toxins were identified as being able to influence properties related to detection. By providing insights into the properties influencing detection, this research indirectly facilitates the design of improved analytical methods for these toxins, which benefits analysts, regulators, and the food industry.

4. Improved method for detecting ochratoxin A in fruit juice and wine. Ochratoxin A is a toxic natural product that can contaminate a wide variety of foods and beverages, including fruit juices and wine. ARS scientists in Peoria, Illinois, developed a material to selectively isolate ochratoxin A from beverages using computational chemistry and quantitative structure activity relationship techniques. The material was synthesized and found to improve detection accuracy by removing components of samples that interfered with toxin detection. The results of this study are useful to researchers looking for convenient methods to detect ochratoxin A in beverages.

5. A novel method to detect fungal toxins provides insight into their mode of action. Fungi that infest commodities and foods can produce toxins that bind certain metals. The mycotoxins in this group include cyclopiazonic acid, a neurotoxin, tenuazonic acid, which is toxic to a variety of plants and animals, ochratoxin, and citrinin, which are kidney toxins, and kojic acid. These toxins are found in a variety of commodities, foods, and cosmetics. The manner in which they exert their toxic effects is not well understood but in some cases may be related to their ability to bind to metals. ARS scientists in Peoria, Illinois, discovered that fluorescent complexes could be formed between certain of the mycotoxins and two lanthanide metals (terbium and europium). Generation of a fluorescent signal not only facilitated detection of tenuazonic acid and cyclopiazonic acid, it also permitted the development of a model system under which the effects of these two toxins on other, more common, metals could be studied. Results demonstrated that tenuazonic acid bound copper and aluminum very effectively, provided insight into how this compound may exert its toxic effects on plants. These results provide new methods to detect a variety of fungal toxins that can be found in agricultural commodities before they reach the consumer.

6. Novel biosensor for detecting fungal toxins in corn and camembert cheese. Cyclopiazonic acid is a naturally occurring neurotoxin that is produced by certain fungi that can infest a variety of commodities and foods. This toxin can be produced by some of the same fungi that produce the more widely known aflatoxins and the two groups of toxins have been shown to frequently occur together in corn. Cyclopiazonic acid has also been found at significant levels in certain cheeses, such as camembert, that are deliberately molded. ARS scientists in Peoria, Illinois, developed a method to screen for cyclopiazonic acid in corn and in camembert cheese. The method uses a novel biosensor technology known as imaging surface plasmon resonance (iSPR). The assay can be used as a tool to quickly determine whether this toxin is present in corn or in camembert cheese.

Review Publications
Tittlemier, S.A., Cramer, B., Dall'Asta, C., Iha, M.H., Lattanzio, V.M.T., Malone, R.J., Maragos, C., Solfrizzo, M., Stranska-Zachariasova, M., Stroka, J. 2019. Developments in mycotoxin analysis: an update for 2017-2018. World Mycotoxin Journal. 12(1):3-29. https://doi.org/10.3920/WMJ2018.2398.
Hossain, M.Z., Maragos, C.M. 2019. Coordination of mycotoxins with lanthanides in luminescent complexes. Mycotoxin Research. 35(3):279-292. https://doi.org/10.1007/s12550-019-00356-w.
Tu, Y.-S., Tseng, Y.J., Appell, M. 2018. Theoretical investigation of cyromazine tautomerism using density functional theory and Moller–Plesset perturbation theory methods. Molecular Simulation. 44(16):1344-1352. https://doi.org/10.1080/08927022.2018.1506117.
Leslie, J.F., Lattanzio, V., Audenaert, K., Battilani, P., Cary, J.W., Chulze, S.N., De Saeger, S., Gerardino, A., Karlovsky, P., Liao, Y.-C., Maragos, C.M., Meca, G., Medina, A., Moretti, A., Munkvold, G., Mule, G., Njobeh, P., Pecorelli, I., Perrone, G., Pietri, A., Palazzini, J.M., Proctor, R.H., Rahayu, E.S., Ramirez, M.L., Samson, R., Stroka, J., Sulyok, M., Sumarah, M., Waalwijk, C., Zhang, Q., Zhang, H., Logrieco, A.F. 2018. MycoKey round table discussions of future directions in research on chemical detection methods, genetics and biodiversity of mycotoxins. Toxins. 10(3):109. https://doi.org/10.3390/toxins10030109.
Biswas, A., Cheng, H.N., Kim, S., Appell, M.D., Boddu, V.M., Alves, C.R., Furtado, R.F. 2019. Preparation of sorbitol-based polyurethanes and their semiinterpenetrating polymer networks. Journal of Applied Polymer Science. 136:47602. https://doi.org/10.1002/app.47602.
Appell, M., Evans, K.O., Jackson, M.A., Compton, D.L. 2019. Determination of ochratoxin A in grape juice and wine using nanosponge solid phase extraction clean-up and liquid chromatography with fluorescence detection. Journal of Liquid Chromatography and Related Technologies. 41:949-954. https://doi.org/10.1080/10826076.2018.1544148.
Hossain, Z., Busman, M., Maragos, C.M. 2019. Immunoassay utilizing imaging surface plasmon resonance for the detection of cyclopiazonic acid (CPA) in maize and cheese. Analytical and Bioanalytical Chemistry. 411:3543–3552. https://doi.org/10.1007/s00216-019-01835-w.
Maragos, C.M., Sieve, K.K., Busman, M. 2019. Development of antibodies for N-(1-deoxy-D-fructos-1-yl) fumonisin B1 and cross-reaction with modified fumonisins. World Mycotoxin Journal. 11:493-502. https://doi.org/10.3920/WMJ2018.2308.
Nguyen, N.T., Varga, E., Maragos, C., Baumgartner, S., Adam, G., Berthiller, F. 2018. Cross-reactivity of commercial and non-commercial deoxynivalenol-antibodies to emerging trichothecenes and common deoxynivalenol-derivatives. World Mycotoxin Journal. 12:45-53. https://doi.org/10.3920/WMJ2018.2363.
Tu, Y.-S., Tseng, Y.J., Appell, M. 2019. Quantum chemical investigation of the detection properties of alternariol and alternariol monomethyl ether. Structural Chemistry. https://doi.org/10.1007/s11224-019-01302-3.