Hidden toxins : Mycotoxins masked by glucosylation. Mark Busman USDA-ARS Peoria, Illinois

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1 idden toxins : Mycotoxins masked by glucosylation Mark Busman USDA-ARS Peoria, Illinois

2 Fungi in corn Aspergillus Fusarium

3 Mycotoxins in corn Aflatoxins C 3 Tricothecenes Fumonisins C 3 3 C C3 C 3 N 2

4 Mycotoxin effects Aflatoxins: liver toxicity, carcinogenic Fumonisins : carcinogenic, cause birth defects

5 Determination of mycotoxins Biological assays Chemical analysis

6 Problem Why is there often a large discrepancy between biological and chemical toxin analysis? A possible answer idden mycotoxins. Toxins that are masked from conventional chemical analysis.

7 What is a masked mycotoxin molecule? Contains a mycotoxin base Chemically masked Masking can possibly be cleaved to produce the base mycotoxin

8 Masked mycotoxin

9 What does masking do? Detoxification? Interferes with detection of toxin in food / feed materials. Achieves a change in chemical identity Can result in a hiding of toxic behavior

10 What is the nature of the chemical masking? Glucose Glutathione Malonic acid Mediated by plant transferase enzymes

11 Mycotoxin glucosides Zearalenone (1988): DN (1991): Nivalenol (2011): Fusarenon X (2011): T2 toxin (2011): Active area of mycotoxin research

12 Masked mycotoxins: glucosylation glucosylation (glucosidase) glucosylation (glucosyltranferase)

13 Masking of mycotoxins

14 T-2 toxin T-2 toxin

15 T2 glucoside Liquid chromatography Mass spectrometry

16 T2 glucoside ypothesized: Glucoside production Glucoside chromatographic behavior Glucoside mass spectrometric behavior Grow Molecular weight Collision induced dissociation: fragmentation pattern Fusarium sporotrichioides

17 T2 glucoside Parent ion scan: Search for ions with CID behavior analogous to T2 toxin T2 toxin DN-3-glucoside And, molecular weight corresponding to hypothetical toxin glucosides

18 T2-glucoside fragmentation [M+] + ion 100 (a) Relative intensity (%) m/z

19 LC-MS/MS parent ion scan results for F. sporotrichioides culture extracts T T Relative intensity (%) T2 glucoside T2 glucoside

20 T-2 toxin glucoside T-2 toxin glucoside

21 T2 3-glucosides α-glucoside β-glucoside

22 What causes the masking of toxins? Can be produced by fungi Plants Free sugars Plant enzymes Detoxification process for host plant Food processing Fermentation ther processes with plentiful sugar

23 Should we care about hidden toxins? -after all they are hidden Recent research shows that toxin glucosylation might readily be reversed by mammalian digestive systems. Will toxin analysis see the hidden toxins? Maybe. Biological analysis: Probably yes. Chemical analysis: Probably no.

24 Masked toxins and plant breeding Will masked toxins increase in plants bred for fungal / toxin resistance? Will plants bred for higher linoleic and oleic acid content have higher levels of hidden toxins? Recent study showed higher levels of hidden fumonisins in corn varieties bred for higher linoleic and oleic acid content

25 Where do we go from here? Science is still changing rapidly New masked toxins are being discovered Toxicity studies are needed ost plant for fungi is changing Plants are being bred for fungi / toxin resistance Plants are being bred for different nutrient profile Uses for corn are changing ow will the regulatory environment adapt?

26 Should we look to modify toxin analytical methodology to make sure we include the hidden toxins? Do we need to talk in terms of total toxin levels versus free toxin levels ow do we resolve the disagreements between first tier (biological) tests and second tier (chemical) tests Detoxification vs. masking of toxins Toxicology / metabolism studies

27 Status of a few common mycotoxins: zearalenone Estrogenic compound Wide variety of sugar conjugates have been characterized Several surveys of toxin profiles have been published Some toxicity data published

28 Status of a few common mycotoxins: DN / NIV DN-glucoside standard is commonly available, NIV-glucoside is not Several studies regarding metabolic fate of toxin glucosides in animals have been published Cross reactivity of toxin glucosides in antibody-based tests is well characterized

29 Status of a few common mycotoxins: fumonisin A variety of hidden forms known Covalent and non-covalent hidden forms No studies regarding metabolic fate of toxin glucosides in animals have been published In some studies of processed corn (corn flakes, etc.) over 50% of the total FB1 content is in a hidden form

30 Status of a few common mycotoxins: T-2 T-2 and T-2-glucoside standards are not commonly available No studies regarding metabolic fate of toxin glucosides in animals have been published Studies of cross reactivity of toxin glucosides in antibody-based tests are in progress Development of antibodies to toxin glucosides for use in antibody-based tests is in progress

31 Future research Characterize the cross-reactivity of biological based screening methods to masked toxins. Develop antibodies leading to direct biological based screening methods for masked toxins. Characterize the metabolic fate of masked toxins in digestive process. Provide widely available standards for the masked toxins.

32 Acknowledgements NCAUR Peoria: Chris Maragos Thank you for your attention