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Mycotoxin research at the BOKU department IFA-Tulln#

Food analysis – Breeding resistant crops – biomarker research#

Author: Prof. Dr. Rudolf Krska, BOKU Tulln

BOKUs department IFA-Tulln has become one of the world´s leading institution hosting the most cited authors in the area of mycotoxin research in the last decade with special emphasis on novel analytics to study the significance and metabolism of these toxic secondary metabolites in agricultural commodities, foods and humans. In Europe, mycotoxins in cereals and maize are mainly the result of an infestation with Fusarium species of the crop in the field as the causal factors for ear rot of maize, the bleaching and shrinking of wheat kernels and mycotoxin contamination. The Food and Agriculture Organisation (FAO) of the United Nations has estimated that 25% of the world´s food crops are contaminated with mycotoxins leading to an annual loss in the range of 1000 million tons. In fact, recent mycotoxin surveys carried out by IFA´s Center for Analytical Chemistry show a frequency of contamination which is much higher than the figure published by the FAO indicates. In order to protect the consumer, over 100 countries have established regulations for maximum levels of mycotoxins in various food and feedstuffs. Despite huge research investments on mycotoxins, prevention, control and proper exposure assessment remain difficult and agricultural and food industry continue to be vulnerable to problems of contamination. Recognising significant gaps in the current knowledge, the IFA-Tulln scientists around Prof. Rudolf Krska and Prof. Hermann Bürstmayr have been following a highly interdisciplinary approach as demonstrated in the ongoing Special Research Programme FUSARIUM funded by the Austrian Science Fund (FWF) and coordinated by Prof. Gerhard Adam from BOKU/DAGZ.

This approach is

1) based on novel mass spectrometric methods for multi-toxin analysis and stable isotopic labelling-assisted untargeted metabolite profiling

2) to obtain new occurrence data on (masked) mycotoxins in crops, foods and feeds as a basis for proper intake calculations

3) to study the metabolisation of mycotoxins by plants in order to improve the molecular understanding of virulence and resistance mechanisms of fungi and plants, respectivel

4) to study the fate of masked mycotoxins during digestion and to identify novel microbial detoxification strategies to inactivate mycotoxins.

5) and to assess the exposure of humans to mycotoxins through the simultaneous determination of appropriate (multiple) biomarkers in urine.

In the area of novel mass spectrometric methods for multi-toxin analysis and untargeted metabolite profiling, IFA´s research has led to a unique multi-analyte approach in mycotoxin analysis based on liquid chromatography-mass spectrometry (LC-MS/MS) that enabled for the first time the accurate and simultaneous determination 400 mycotoxins and other fungal metabolites which is crucial to elucidate the presence of mycotoxins in agricultural commodities and to study their metabolisation by plants, animals and humans. Within a European Commission funded project, the developed multi-toxin approach has also been key to measuring fungal and bacterial metabolites indoors to facilitate a deeper understanding of the toxicological effects caused by these fungal metabolites and of their possible synergism. In addition, the successful mass spectrometric work included the development of a fast, easy-to-handle and highly accurate isotope dilution mass spectrometric assay. The latter has become feasible through the production of fully isotopically labelled mycotoxins by means of a patented technology. The developed assays are now available to the scientific community and industry as an accurate reference method for the quantification of all regulated mycotoxins in cereal-based food products. In the last few years, the mycotoxin researcher´s work moved from targeted analysis of individual mycotoxins to untargeted metabolite profiling and metabolomics of (ideally) all secondary metabolites that are involved in plant-fungus interactions. This methodology is based on in vivo stable isotopic 13Clabelling and subsequent measurement of biological samples by full scan high-resolution-LC-MS.

The methods for multi-toxin determination have revealed important new occurrence data on mycotoxins in crops, foods and feeds. Many of the mycotoxins found have not been reported before to occur in these commodities, which emphasizes the great variety of mycotoxin co-exposure and the need to take the exposure to multiple mycotoxins into account for proper risk assessment.

The developed LC-MS/MS methods also enabled to provide the first scientific proof of the occurrence of glucose-bound (masked) deoxynivalenol in naturally contaminated wheat and maize, probably as part of the plant´s defense strategy to detoxify this most prevalent Fusarium mycotoxin. As a result, the potential threat to consumer safety from masked mycotoxins has been or will soon be addressed by several institutions, such as ILSI Europe, the European Food Safety Authority (EFSA) and by companies.

The studies performed by the interdisciplinary team of the IFA-Tulln on the metabolisation of mycotoxins by plants have been recognised as ground-breaking new results in a functional genomics context and have led to an improved molecular understanding of virulence and resistance mechanisms of fungi and plants. Indeed, they were able to demonstrate that resistant wheat genotypes have a greater ability to detoxify the major Fusarium mycotoxin deoxynivalenol through glucosylation. The degree of glucosylation even correlates well with the level of Fusarium resistance of the wheat genotype and could be traced back to a specific wheat chromosome locus (QTL) which expresses a glucosyltransferase which is a key for Fusarium head blight resistance in wheat. This finding was the result of the still ongoing successful collaborations between the plant breeders, geneticists and the (bio-)analytical chemists at the BOKU-department IFA-Tulln.

In cooperation with the company Biomin, IFA researcher also paved also the way for new detoxification strategies to reduce the mycotoxin levels in feeding stuffsthrough novel (microbial) deactivation techniques. The team has been able to elucidate the microbial detoxification metabolism of fumonisins and zearalenone by means of metabolite identification using tandem mass spectrometry. These findings have been the basis for new feed additives which have been developed within a research project.

In their efforts to assess the exposure of humans to mycotoxins through the simultaneous determination of appropriate (multiple) biomarkers, IFA´s team developed a novel multi-biomarker LC-MS/MS based method for the determination of 15 mycotoxins and their key metabolites, respectively, in urine. Recently, this biomarker approach has been employed in several human pilot studies. In addition, the developed method enabled the direct quantification of deoxynivalenol glucuronide in humans, for the first time. An example of the relevance of the reported new exposure data is the extent of co-exposure observed in samples from Cameroonian individuals with 63 % of the analysed urine samples (n=175) containing at least one analyte, with a maximum of six mycotoxin biomarkers simultaneously determined in a single individual, a severe co-exposure that had never been reported before. Moreover, a pilot-study carried out in Austria revealed that about 30% the tested individuals (n=27) who provided urine samples, exceeded the tolerable daily intake for deoxynivalenol. Similarly, 47.5% (n=40) of third trimester pregnant women from Eastern Croatia exceeded the provisional maximal tolerable daily intake level for deoxynivalenol, astonishing findings which have also been highly recognised by the scientific community.

Uitlising the power of the advanced multi-biomarker LC-MS/MS method, Prof. Krska and his PhD student Benedikt Warth have also been able to provide new insights into the human metabolism of the Fusarium mycotoxins deoxynivalenol and zearalenone. They conducted the first in vivo case study for these toxins over a period of eight days. Thus, for the first time concrete figures have become available for the excretion pattern of the formed glucuronides throughout a day. Moreover, they were able to provide the first report on the presence of a third DON-glucuronide in human urine and to study the fate of ingested masked forms of deoxynivalenol. These findings contribute to a significantly improved understanding of human in vivo metabolism of mycotoxins and thereby support advanced exposure and risk assessment and the study of the relationship between these mycotoxins and potentially associated chronic diseases in the future.

The developed methods for multi-toxin determination have been leading to new occurrence data on (masked) mycotoxins in crops, foods and feeds as a basis for proper intake calculations and chemical health hazard analysis. Just recently (August 2013), the European Food Safety Authority (EFSA) has received a mandate from the European Commission for a scientific opinion on the risks of masked mycotoxins in food and feed. The results of the recent surveys demonstrating that masked mycotoxins are co-occurring with the native toxins in almost all cases have been the basis for this mandate.

The simultaneous determination of appropriate mycotoxin biomarkers in urine has already led to a better understanding of the human metabolism of major Fusarium mycotoxins and to an improved assessment of exposure of humans to mycotoxins. Ultimately, the multi-biomarker approach could be crucial in the identification of what are some of the most important mycotoxin mysteries: the role of mycotoxins in chronic disease caused by low-dose long-term background exposure through the intake of contaminated food and the toxicological risks posed by combinations of mycotoxins of frequent natural occurrence.

IFA´s highly interdisciplinary approach has been leading to ground-breaking new findings in prevention, determination, metabolism and detoxification of mycotoxins through a great number of scientific and innovative accomplishments. In summary, the implications of the research results to other branches of science are manifold. These achievements are not only of interest to (bio-analytical) chemists but are also of great importance to plant breeders, plant pathologists, toxicologists, epidemiologists, food-, feed- and medical scientists and microbiologists. Consequently, the results are highly relevant to the agro-food industry, the safety of raw food and feed materials and the health of humans and animals. Therefore, scientists of different disciplines and the colleagues from the food and feed industry as well as health science authorities take advantage of IFA´s research.