Masked Mycotoxins in Food: Formation, Occurrence and Toxicological Relevance / Edition 1 available in Hardcover
Masked Mycotoxins in Food: Formation, Occurrence and Toxicological Relevance / Edition 1
- ISBN-10:
- 1849739722
- ISBN-13:
- 9781849739726
- Pub. Date:
- 11/16/2015
- Publisher:
- RSC
Masked Mycotoxins in Food: Formation, Occurrence and Toxicological Relevance / Edition 1
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Masked Mycotoxins in Food
Formation, Occurrence and Toxicological Relevance
By Chiara Dall'Asta, Franz Berthiller
The Royal Society of Chemistry
Copyright © 2016 The Royal Society of ChemistryAll rights reserved.
ISBN: 978-1-84973-972-6
CHAPTER 1
Introduction to Masked Mycotoxins
FRANZ BERTHILLER, CHRIS M. MARAGOS AND CHIARA DALL'ASTA
1.1 Mycotoxins
Given suitable water activities, moulds can infect almost every agricultural commodity (e.g. cereals, nuts, fruits, etc.) during plant growth and/or after harvest. A variety of these fungi, in particular Aspergillus spp., Penicillium spp. or Fusarium spp., are capable of producing mycotoxins. These substances are low-molecular-weight fungal secondary metabolites that are able to accumulate in food or feed in toxicologically relevant concentrations.1 About 20 years ago, the Food and Agriculture Organization (FAO) of the United Nations estimated that 25% of the world's food crops were significantly contaminated with mycotoxins, leading to an annual loss in the range of 1000 million tons. Recent studies suggest that the percentage of contaminated cereals is much higher: 72%. The difference may be due, in part, to what levels are regarded as contamination, as well as improvements in monitoring.
The potential of mycotoxins to cause harm to human health through dietary exposure has led authorities world-wide to highly regulate these food contaminants. By the end of 2003, about 100 countries had regulations for maximum levels of mycotoxins in various food and feedstuffs. For instance, in the European Union, maximum levels have been set for the mycotoxins aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2), deoxynivalenol (DON), fumonisin B1 (FB1) and B2 (FB2), ochratoxin A (OTA) and patulin (PAT), as well as for zearalenone (ZEN) in foodstuffs. Furthermore, indicative levels have been recommended for T-2 and HT-2 toxins (T2 and HT2) in a variety of cereals and products thereof.
1.2 Masked Mycotoxins
Unaltered mycotoxins are not the only source of mycotoxin exposure for consumers. Plants protect themselves from xenobiotic compounds like mycotoxins by converting them into more polar metabolites (Figure 1.1), which are transported into vacuoles for further storage or are conjugated to biopolymers such as cell wall components. Mycotoxins, which are in contact with highly metabolically active plants in the field, are especially prone to being metabolised. As Fusarium infection usually occurs in the field (in contrast to Aspergillus or Penicillium infections), the Fusarium mycotoxins DON, ZEN, FB1, T2, HT2 and nivalenol (NIV) are the most prominent targets for conjugation.
1.2.1 Terminology
The formed substances are often referred to as masked "mycotoxins". Unfortunately, within the literature there are sufficient inconsistencies in terms that a brief description of how they will be used in this book is needed. The terms "masked", "hidden", "conjugated" and "bound" are frequently used in the literature. Of these, "masked" is the most popular. The term was originally intended to distinguish substances that were the targets during routine analysis from those that, while not targeted analytes, might contribute to the mycotoxin content. In this context, mycotoxin derivatives can arise through a number of mechanisms. They may be precursors, metabolites or degradation products of the "parent" (or free) form of the mycotoxin, or they may have been formed abiotically through chemical reaction of the parent toxin with the matrix (e.g. through food processing). Unfortunately, with this definition, once a masked mycotoxin is a routine target of analysis, it is no longer truly a masked mycotoxin. Despite this contradiction, the definition is widely used because it has merit as an inclusive term for a wide variety of materials that have traditionally been overlooked and that might contribute to toxicity. Masked mycotoxins can be further classified according to how the masked form relates to the parent form; that is, whether the masked form exists as a covalent derivative of the parent toxin or a non-covalent association between the parent toxin and a matrix component. Within the literature, some references describe covalently linked forms as "conjugated", while the non-covalently (e.g. associated) forms have been termed "bound". The latter term is intended to indicate that the parent toxin might be extractable from the non-covalent complex following chemical or enzymatic treatment. This distinction is important, but conjugates, because they contain covalent linkages, are also bound forms of the parent toxin. Perhaps the best description of "hidden" mycotoxins was provided by Dall'Asta et al., where they were classified as non-covalent, associative interactions between a mycotoxin and matrix macroconstituents.
The most recent and comprehensive definition was worked out within the scope of the Committee for Contaminants and other Undesirable Substances in the Food Chain of the German Federal Institute for Risk Assessment (BfR). In this systematic definition, distinctions were made between free mycotoxins, matrix-associated mycotoxins and "modified mycotoxins". Modified mycotoxins were further classified into those that are biologically or chemically modified. Regarding biological modifications, these can be achieved, for example, by plants, animals, fungi or other means. Using the BfR proposal, only plant metabolites of mycotoxins would still be termed masked mycotoxins. This definition was taken up by the Panel on Contaminants in the Food Chain (CONTAM) of the European Food Safety Authority (EFSA) in their recent scientific opinion on modified mycotoxins, as well by the authors of this book.
1.2.2 Historical Perspective
The issue of masked mycotoxins began attracting scientific interest after several mysterious cases of mycotoxicosis during the mid-1980s, in which symptoms in affected animals did not correlate with the low mycotoxin content detected in their feed. Around the same period, the metabolic biotransformation of DON to less toxic derivatives in planta was for the first time hypothesised to occur in field corn inoculated with Fusarium graminearum and in naturally infected winter wheat. It was shown that callus cultures of the Fusarium head blight-resistant wheat cultivar Frontana converted more 14C-labelled DON into uncharacterised products than callus derived from the susceptible wheat cultivar Casavant. Later, the major soluble DON metabolite of plants, deoxynivalenol-3-β-D-glucoside (DON-3-Glc; Figure 1.1), was isolated from DON-treated maize cell suspension cultures. Another decade later, the substance was shown to arise after treatment of Arabidopsis thaliana with DON, until it was for the first time also found in naturally contaminated maize and wheat. It has been shown that, after treatment of plants with DON, wheat produces DON-3-Glc to detoxify this Fusarium graminearum virulence factor. A survey demonstrated that DON-3-Glc concentrations can exceed 1000 µg kg-1 in naturally contaminated wheat and can reach over 70% of the molar DON concentration in maize. DON-3-Glc can also be found in naturally contaminated barley, beer made thereof, breakfast cereals and snacks. The relative proportion of DON-3-Glc to DON in cereals can vary considerably, but on average is in the range of 20%.
ZEN, a Fusarium mycotoxin with high oestrogenic activity, was the next piece to find its place in the masked mycotoxin puzzle, when wheat and maize cell cultures were found to be capable of transforming ZEN into zearalenone-14-β-D-glucopyranoside (ZEN-14-Glc) and other metabolites. The same study indicated that about 90% of the added radiolabelled 14C-ZEN was recovered in soluble form after 3 days. A later paper by the same group reported that, 12 days after treatment, more than 50% of the radio-activity was found in the non-extractable residues. The bioavailability of these bound forms has yet to be determined. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) studies have proven that the model plant Arabidopsis thaliana can rapidly transform ZEN into an array of 17 different compounds (Figure 1.2), including glucosides, malonylglucosides, diglucosides and pentosylhexosides of ZEN and their phase I metabolites α-zearalenol (αZEL) and β-zearalenol (βZEL).
Fumonisin conjugates were long believed to occur only after food processing. However, it was shown that bound fumonisins could also be found in unprocessed maize. The exact chemical nature of these naturally occurring hidden forms is still unknown. Most likely, non-covalent interactions with, for example, starch or proteins occur, rendering the fumonisins difficult to extract.
Generally, very little is known regarding bound mycotoxins. The IUPAC has proposed the following general definition for bound residues: "A xenobiotic bound residue is a residue which is associated with one or more classes of endogenous macromolecules. It cannot be disassociated from the natural macromolecule using exhaustive extraction or digestion without significantly changing the nature of either the exocon or the associated endogenous macromolecules." Depending on the type of linkage to proteins, starch, pectins, hemicellulose, cellulose and lignin, it is conceivable that at least a part of bound mycotoxins could become bioavailable again in the digestive tract of humans and animals. Bound residues are either covalent or non-covalent. Bound residues are usually quantified as the difference of radioactivity compared to the soluble fraction after treatment of plants with the radionuclide-labelled xenobiotics of interest. Depending on the chemical nature of the xenobiotic, very different incorporation rates have been found. Pesticides, for example, showed incorporation rates from just a few to up to 90% of the compound applied to the plant.
1.2.3 Recent Developments
Additional DON conjugates have been identified recently. Oligoglycosides of DON, namely di-, tri- and tetra-glucosides, have been found in beer. The formation of a DON–glutathione conjugate has been shown in vitro, and the occurrence of this compound in cereals was confirmed 3 years later. To do so, a liquid chromatography–high-resolution mass spectrometry (LC-HRMS)-based approach using in vivo stable isotopic labelling, combined with a newly developed software tool37 to extract biological features originating from true metabolites, was employed. Flowering wheat ears were inoculated with a mixture of DON and 13C-labelled DON. In addition to DON-3-Glc, DON–glutathione and its processing products DON-S-cysteine and DON-S-cysteinyl–glycine, as well as DON-malonyl-glucoside, were found. In a continuation of this work, tentative annotation of the remaining biotransformation products was carried out, additionally identifying DON-hexitol (e.g. mannitol), DON-di-hexoside (e.g. glucose), 15-acetyl-DON-3-β-D-glucoside and a DON–glutathione derivative lacking two protons. Most recently, also DON-3-sulphate and DON-15-sulphate have been identified in Fusarium graminearum -inoculated or DON-treated wheat.
Several new masked mycotoxins have also been described during the last few years. Both nivalenol-3-glucoside (NIV-3-Glc) and fusarenon-X-glucoside (FUSX-3-Glc) were found in wheat grain using high-resolution mass spectrometry. Furthermore, several T2 (T2-Glc) and HT2 glucosides (HT2-Glc) were detected in contaminated wheat and oats by LC-MS/MS or LC-HRMS, respectively. The di-glucosides of both T2 and HT2 have also been reported. Glucosides of other type A trichothecenes, namely neosolaniol-glucoside (NEO-Glc) and diacetoxyscirpenol-glucoside (DAS-Glc), were found in maize powder. Most recently, a new ZEN-glucoside, which was isolated from ZEN-treated barley, was found and determined to be ZEN-16-β-D-glucopyranoside (ZEN-16-Glc).
Analytical methods for the determination of masked mycotoxins have been summarised. Moreover, a comprehensive review on the occurrence of masked mycotoxins in food and analytical aspects for their determination, toxicology and impact on stakeholders were published. In brief, there exist at least three analytical strategies to determine masked mycotoxins in food. The first of these are dedicated LC-MS/MS-based methods, which can quantify masked mycotoxins along with their parent forms. Secondly, masked mycotoxins can be detected by immunochemical methods, provided there is cross-reactivity of antibodies towards them. Both LC-MS/MS and immunoassay techniques are strengthened with the availability of appropriate analytical standards of the masked forms. Finally, masked mycotoxins may be hydrolysed to their parent mycotoxins using enzymes or harsh acidic or alkaline conditions. A sum parameter (parent and masked mycotoxins) is derived with the latter two approaches and the masked fraction might be calculated by subtracting the concentration of the parent mycotoxin determined by conventional techniques. Those indirect techniques should be used carefully, however. A recent paper describes the inability of previously published works based on acidic hydrolysis for the determination of masked DON in cereals, none of which were able to liberate DON from its major metabolite DON-3-Glc. Enzymatic cleavage by certain β-glucosidases seems to be far more promising for liberating the parent toxins from DON-3-Glc, NIV-3-Glc or HT2-Glc in cereal matrices. The purified recombinant enzyme from Bifidobacterium adolescentis works rapidly under the given conditions, allowing complete cleavage of DON-3-Glc in cereal extracts within 10 minutes of incubation. The interested reader is referred to Chapters 3 and 4 for far more detailed information on the determination of masked mycotoxins using immunoanalytical and mass spectrometric techniques.
1.2.4 Toxicity of Masked Mycotoxins
In general, intact masked mycotoxins are less potent relatives to their unmodified forms. This is easily understandable, taking into account the severe modifications of the toxins due to conjugation and the fact that masked mycotoxins arise during detoxification reactions of plants. For instance, compared to DON, DON-3-Glc barely binds to the ribosome, resulting in a highly diminished inhibition of protein synthesis — the major mode of action for all trichothecenes. Similarly, ZEN-14-Glc can barely bind to the oestrogen receptor, resulting in a far reduced oestrogenicity compared to its parent toxin ZEN. However, it is assumed that masked mycotoxins can be "reactivated" during mammalian digestion by cleavage of the polar group and liberation of the native toxin. Perhaps because toxicity testing can be very time consuming, the toxic effects caused by masked mycotoxins are only beginning to appear in the literature. Even so, a wealth of information has been gained on DON-3-Glc, especially within the past 3 years. In 2011, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) emphasised the occurrence of DON-3-Glc in cereals and beers, which might contribute to systemic exposure to DON. Besides recommending additional studies to collect occurrence data as well as to investigate the effects of processing on DON-3-Glc, the JECFA also asked for absorption, distribution, metabolism and excretion (ADME) studies on this substance. In the same year, the hydrolytic fate of DON-3-Glc during digestion was assessed using several in vitro assays. DON-3-Glc proved to be stable towards hydrochloric acid and human enzymes, but several lactic acid bacteria showed the ability to partially release DON. Two independent studies verified the results, showing that the release of DON also occurs after incubating DON-3-Glc with human faeces. Nevertheless, in vivo ADME studies were necessary to assess the potential health risk of DON-3-Glc. The fate of orally administered DON-3-Glc was determined in rats and piglets. It was concluded that DON-3-Glc was less bioavailable than DON, but was almost completely hydrolysed in both species. The cleavage took place mostly in the hindgut, where absorption is lower than in the small intestine. Due to differences in anatomy and gut microbiota, the metabolism was species dependent. In addition, the state of digestion and individual differences in gut microbiota can cause differences in the amount of DON released. Again, readers interested in the toxicological aspects of masked mycotoxins are referred to Chapters 6 and 7, where in vitro and in vivo experiments are discussed in far more detail.
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Excerpted from Masked Mycotoxins in Food by Chiara Dall'Asta, Franz Berthiller. Copyright © 2016 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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