Deoxynivalenol and its masked forms in food and feed

Deoxynivalenol and its masked forms in food and feed

Accepted Manuscript Title: Deoxynivalenol and its masked forms in food and feed Author: Veronika Nagl Gerd Schatzmayr PII: DOI: Reference: S2214-7993...

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Accepted Manuscript Title: Deoxynivalenol and its masked forms in food and feed Author: Veronika Nagl Gerd Schatzmayr PII: DOI: Reference:

S2214-7993(15)00101-0 http://dx.doi.org/doi:10.1016/j.cofs.2015.08.001 COFS 82

To appear in: Received date: Revised date: Accepted date:

7-7-2015 7-8-2015 10-8-2015

Please cite this article as: Nagl, V.,Deoxynivalenol and its masked forms in food and feed, COFS (2015), http://dx.doi.org/10.1016/j.cofs.2015.08.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

DON occurs in more than half of grain samples, often together with other mycotoxins

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Advances in analytical techniques enabled novel insights into the metabolism of DON

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The biomarker approach facilitates an individual DON exposure assessment in humans

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The masked mycotoxin DON-3-Glc is partly cleaved during mammalian digestion

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First feed additive for DON detoxification approved in the EU

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Deoxynivalenol and its masked forms in food and feed Veronika Nagl a and Gerd Schatzmayr a,* Biomin Research Center, Technopark 1, 3430 Tulln, Austria;

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Corresponding author. Tel.: +43 2272 81166 0, [email protected]

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[email protected], [email protected]

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Abstract Deoxynivalenol (DON) is one of the most frequently occurring mycotoxins in cereal crops worldwide. DON poses a risk to human and animal health due to its wide range of adverse effects. Recently, novel insights into the metabolism of DON enabled the use of the

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biomarker approach for human exposure assessment. In certain subpopulations, a considerable proportion of tested individuals were found to exceed the maximum tolerable intake for DON. Since the masked mycotoxin deoxynivalenol-3-β-D-glucoside (DON-3-Glc)

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is partly cleaved during mammalian digestion, liberation of DON might further increase the total mycotoxin burden. However, progress in better understanding detoxification strategies

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along the food and feed chain has been made and the first product for DON detoxification in

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feed was approved in the EU. Keywords

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Trichothecenes, biotransformation, deoxynivalenol-glucuronide, urine, tolerable daily intake Abbreviations DON-3-Glc,

deoxynivalenol-3-β-D-glucoside;

DON,

deoxynivalenol;

DON-3-GlcA,

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deoxynivalenol-3-glucuronide; DON-15-GlcA, deoxynivalenol-15-glucuronide; DON-GlcA,

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deoxynivalenol-glucuronide; DOM-1, de-epoxy-deoxynivalenol; DOM-1-GlcA, de-epoxydeoxynivalenol-glucuronide; EFSA, European Food Safety Authority; GIT, gastrointestinal

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tract; JECFA, Joint FAO/WHO Expert Committee on Food Additives; PMTDI, provisional maximum tolerable daily intake

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Introduction The type-B trichothecene deoxynivalenol (DON), mainly produced by Fusarium

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graminearum and Fusarium culmorum, is a frequent contaminant of cereal crops worldwide.

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DON exhibits its biological activity through binding to the 60S subunit of ribosomes, thus

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inhibiting protein biosynthesis. Furthermore, DON affects the transcription and mRNA

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stability of pro-inflammatory genes. In animals, DON causes symptoms such as emesis,

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anorexia, weight loss and increased susceptibility to infectious diseases. In addition, DON

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affects intestinal integrity and alters the local gut immune response [1]. In humans, DON is

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associated with episodes of gastroenteritis [2]. Due to its wide range of adverse health effects,

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DON has a significant economic impact and is of major concern for public health. As a

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consequence, many countries have established maximum limits or guidance values for DON

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in food and feed. Masked forms of DON, deriving from conjugation reactions in the course of

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phase II metabolism in plants, have not been implemented in these regulations due to a lack

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of toxicological data. Most prominently, deoxynivalenol-3-β-D-glucoside (DON-3-Glc)

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might be cleaved in the mammalian digestive tract, thus increasing the total DON burden of

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an individual [3].

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In this review we summarize the latest surveys on the occurrence of DON and DON-3-

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Glc in food and feed and on human DON exposure. In addition, we highlight recent findings

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regarding the metabolism and toxicological relevance of these toxins and present an overview

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on current DON detoxification strategies.

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Occurrence in cereal grains, feed and food Several cereal grains, e.g. wheat, barley, and maize, as well as their by-products, can be

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affected by DON [4]. In a survey on the worldwide occurrence of mycotoxins in feedstuffs

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DON was found in 72% to 79% of corn samples sourced in North America and Central

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Europe, while the proportion of DON-positive corn samples ranged from 17% to 47% in

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South America and Southern Europe. The average contamination levels in raw materials and

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compound feeds varied from 794 to 1,304 µg/kg [5]. By contrast, average DON

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concentrations in grain milling products, bread and rolls, breakfast cereals and pasta deriving

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from 21 EU member states and Norway were 104, 70, 66 and 89 µg/kg, respectively. In total,

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less than 1% of 16,958 samples exceeded the respective EU maximum limits for foods [6].

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In comparison, reports on the occurrence of DON-3-Glc are rare. Still, survey data

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show that more than half of cereals are contaminated with DON-3-Glc at mean concentration

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levels of 85 µg/kg [7]. The relative proportion of DON-3-Glc to DON in cereals was

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proposed to be 20% on average, but varies depending on factors such as genotype or season

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[3]. During food processing, e.g. beer brewing, this proportion can increase further, resulting

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in DON-3-Glc concentrations exceeding those of DON in the final product [7]. In processed

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foods, the average contamination levels are moderate, reaching 6.9 µg/L in beer, 34 µg/kg in

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bread, and 13 µg/kg in breakfast cereals [8,9]. However, detected maximum values of up to

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844 µg/kg DON-3-Glc [10] support the case of including masked mycotoxin in routine

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analysis.

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Besides DON-3-Glc, the presence of other mycotoxins needs to be considered. Co-

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contamination by multiple mycotoxins appears to be the rule rather than the exception, with

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individual samples containing up to 69 fungal metabolites [11]. So far, the toxicological

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interactions between mycotoxins have been poorly investigated. Recent findings indicate

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synergistic effects between DON and other type-B trichothecenes on intestinal cells,

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especially at low concentrations [12]. Hence, the risk posed by mycotoxin co-occurrence

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needs to be carefully assessed and subsequently considered in regulations regarding

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mycotoxins in food and feed.

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Mammalian metabolism

In recent years, advances in analytical techniques enabled new insights into the

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metabolism of DON. In principle, three metabolic pathways are described for DON in

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mammals.

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First, ingested DON can be biotransformed by intestinal or ruminal microbes to de-

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epoxy-deoxynivalenol (DOM-1; [1]). While this represents an important metabolic pathway

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in ruminants, the microbial detoxification activity in pigs and humans is moderate [13-15].

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Second, absorbed DON is conjugated to glucuronic acid, resulting in the formation of

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deoxynivalenol-glucuronide (DON-GlcA). The glucuronidation intensity differs considerably

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among species, most likely due to species-dependent variations in the activity of uridine-

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diphosphoglucuronyltransferases [16]. Deoxynivalenol-15-glucuronide (DON-15-GlcA) is

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the major DON metabolite in human urine [17]. In contrast, deoxynivalenol-3-glucuronide

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(DON-3-GlcA) was identified as the predominant isoform in rats [18], while the DON-3-

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GlcA/DON-15-GlcA ratio underlies individual variations in pigs [19]. The formation of

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further DON-GlcA isomers has only minor relevance in vivo [18,20]. Similarly, absorbed 5 Page 5 of 19

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DOM-1 can be metabolized to de-expoxy-deoxynivalenol-glucuronide (DOM-1-GlcA, [15]).

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However, the presence and structures of different DOM-1-GlcA isoforms have not been

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clarified. Third, sulfonation was long thought to have negligible significance in vivo. In 2014,

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Wan et al. [21] presented sulfonation as an important detoxification route for DON in

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chickens. Shortly thereafter, Schwartz-Zimmermann et al. [22] successfully identified three

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different DON-sulfonate and two DOM-1-sulfonate conjugates in excreta of DON exposed

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rats. These findings may facilitate a deeper understanding of species-specific variations in the

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susceptibility to DON.

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The classical approach to assess human DON exposure is based on a combination of

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contamination and consumption data [23]. Besides the inclusion/exclusion of acetylated and

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masked forms of DON, factors such as inhomogeneous distribution of mycotoxins in

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foodstuffs and limitations of dietary questionnaires can have an impact on the accuracy of

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intake estimates. The biomarker approach enables a more individual and comprehensive

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exposure assessment. For establishment of an exposure biomarker, a correlation between the

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biological measure and the quantity of ingested xenobiotic is crucial. The sum of urinary

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DON+DON-GlcA is regarded as validated biomarker for human DON intake [24].

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Consequently, the number of epidemiological investigations employing biomarker

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methods is constantly increasing. Table 1 presents an overview on DON biomarker surveys

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published since 2013. Differences between listed studies in terms of analytical methodology

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(e.g.

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measurement of urinary creatinine) or calculations regarding the provisional maximum

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tolerable daily intake (PMTDI; 1 µg/kg b.w.; [25]) have to be considered for comparison of

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results. Observed interregional variations in DON excretion may result from differences in

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the nutritional habits or the quality of consumed foodstuffs [26]. Furthermore, urinary DON

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levels can vary considerably from year to year, most likely due to annual fluctuations in

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Fusarium prevalence [14]. Children represent one of the most exposed population groups, as

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demonstrated by approximately 2.5-fold increased urinary DON concentrations in children

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compared to adults in Norway and the United Kingdom [27]. Up to 58% of children were

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found to exceed the PMTDI in certain Tanzanian villages [28]. An exceptionally high urinary

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total DON concentration (1,238 ng/mL), corresponding to a DON intake of 3,300% of the

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detection/quantification

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direct/indirect

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DON-GlcA,

PMTDI, was determined in a pregnant woman from Croatia [20]. 6 Page 6 of 19

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Similarly, major efforts have been made to establish DON biomarkers in farm animals.

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However, factors such as age or sampling time greatly influence the levels of DON and its

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metabolites in blood, thus impairing the use of biomarkers under practical conditions [13].

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Biological activity of masked deoxynivalenol Plants can defend themselves against DON by modification of the chemical structure of

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the mycotoxin. Most prominently, enzymatic conjugation to glucose leads to the formation of

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the masked mycotoxin DON-3-Glc [3]. Despite its frequent occurrence, the toxicological

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relevance of DON-3-Glc is largely unknown.

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DON-3-Glc does not induce ribotoxic stress response in contrast to DON, most likely

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due to its inability to bind the peptidyl transferase center of the ribosome [29]. However,

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there are concerns that DON-3-Glc is cleaved during mammalian digestion, thus contributing

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to the overall toxicity of DON [25]. Although DON-3-Glc was found to be resistant to acidic

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and enzymatic conditions in vitro, liberation of DON was observed after incubation with

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intestinal bacteria or fecal slurry [30]. Accordantly, in vivo reports showed that DON-3-Glc

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itself is minimally bioavailable, but hydrolyzed to DON after oral administration in rats

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[18,31] and pigs [19]. Since intravenously administered DON-3-Glc was quite stable, authors

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assumed that cleavage of the masked mycotoxin occurred predominantly in distal parts of the

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gastrointestinal tract (GIT). The absorption of DON-3-Glc and its metabolites from the GIT

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was limited, indicating a reduced toxicological relevance of DON-3-Glc compared to DON

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[19]. Also, DON-3-Glc does not exhibit intestinal toxicity, as it neither a) impairs the cell

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viability, or the barrier function in intestinal cells nor b) alters the gene expression of pro-

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inflammatory cytokines in intestinal explants [29]. The latter is consistent with Wu et al.

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[32], who showed that splenic mRNA expression of cytokines and chemokines is largely

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unaffected by DON-3-Glc. Yet, DON-3-Glc induced anorexia in mice and emesis in minks.

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These effects were less pronounced compared to DON and may result to some extent from

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liberation of the parent toxin [33].

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DON-3-Glc has not been considered in mycotoxin regulations due to the lack of further

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toxicological data. In particular, effects arising from chronic DON-3-Glc exposure are

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completely unknown. Challenges regarding compound synthesis and purification have to be

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met before those issues can be addressed properly. The same applies to the investigation of

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other masked forms of DON, such as glutathione-related metabolites and sulfate-conjugates,

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which have been identified recently in artificially infected plants [34,35].

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Detoxification and decontamination procedures The prevention of fungal growth and formation of mycotoxins in the field and during

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storage is of utmost importance. Measures to reduce DON production in the field, including

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proper crop rotation, soil tillage, use of fungicides, pesticides or antagonists and application

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of appropriate harvesting procedures are not always feasible and remain challenging [36].

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Therefore, decontamination procedures are necessary to secure the supply of safe raw

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materials for food and feed industries. Physical and chemical procedures, microorganisms

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and enzymes have been postulated to reduce DON contamination [37].

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Sieving, dehulling and density segregation are the most important physical methods as

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damaged grain kernels are more prone to fungal infection and infected kernel have a lower

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density. Furthermore, processes such as milling, cleaning, baking or boiling were reported to

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reduce the DON content up to 77%, 74%, 19%, 71%, and 41%, respectively (summarized in

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[38]). However, these results seem to be quite questionable, as most of respective studies

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were conducted in the 1980s when analytical methods were not advanced and calibrants for

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DON degradation products were unavailable. Recently, Vidal et al. [39] observed significant

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reduction of DON during baking. The reduction was more prominent at higher initial toxin

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concentrations and at higher temperatures. Besides trace levels of DON-3-Glc, no other

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breakdown or conjugated products were determined, which is crucial to assess the efficacy of

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this treatment.

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The use of adsorbents, often referred to as mycotoxin binders or enterosorbents, is

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discussed critically for efficacy against DON. These agents are supposed to bind the

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mycotoxin in the GIT of animals and remove it via feces, thus prohibiting its systemic

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absorption. So far, no adsorbent has been approved by the EC for DON reduction, possibly

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because of the inefficiency of different materials, such as clay-based minerals and yeast cell

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wall derivatives, to selectively bind DON. For example, Murugesan et al. tested 27

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commercially available feed additives claiming to bind mycotoxins and assessed DON

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absorption rates of less than 25% for all products [40]. Recently, specific activated carbon

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was found to prevent the absorption of DON in the GIT of pigs [41]. Although this approach

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is promising, it only can be used for short-term treatments because activated carbon is a non-

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specific binder that also sequesters essential nutrients.

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Chemical detoxification 8 Page 8 of 19

Methods for chemical detoxification of DON include alkalization (e.g. ammonia,

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sodium or calcium hydroxide), oxidation (e.g. ozone, sodium hypochlorite or chlorine) and

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reduction (e.g. sodium bisulfite and sodium metabisulfite) [37]. So far, chemical

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detoxification has no practical relevance as such treatments are often costly, lack efficiency

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or produce deleterious side effects on the food products. The most promising technique might

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be the application of sulfur reagents. Paulick et al. observed complete DON reduction in

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maize after eight days of treatment with sodium sulfite [42]. The structure, formation pattern

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and toxicity of resulting DON-sulfonate-conjugates has been elucidated, revealing a

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reduction of toxicity by a factor of at least 29 compared to DON [43]. Nonetheless, this

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approach cannot be used for detoxification of DON contaminated food or feed due to

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regulatory restrictions. Recently, partial DON degradation was proposed at very low pH in

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aqueous solution [44]. However, characterization of reaction products and relevance of these

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findings (e.g. DON degradation at acidic pH in stomach) remains to be clarified.

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DON can be metabolized by microorganisms in various ways such as de-epoxydation,

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acetylation, oxidation, epimerization, glycosylation and mineralization (Figure 1; [45]). The

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GIT harbors microorganisms with detoxification activities. Recently, an anaerobic bacterium

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previously isolated from rumen fluid was positively assessed by EFSA for its efficacy and

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safety as a DON detoxifying feed additive for swine [46] and subsequently approved by the

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European Commission [47]. This bacterium was designated a member of a genus novus of

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family Coriobacteriaceae (strain no. 11798) and given the code BBSH 797. In addition,

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Devosia and Nocardioides strains capable of detoxifying DON to 3-keto-4-deoxynivalenol or

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3-epi-deoxynivaleonol were isolated from soils [48,49]. However, many of the bacteria

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described in the literature lack proof of DON metabolization, with no convincing data

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available showing that the reduction of the toxin occurs due to cleavage, transformation or

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mineralization and not from reversible surface binding to cell walls. Criteria for

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microorganisms to be used for practical applications were summarized by Murugesan et al.

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[40].

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Compared to microorganisms, purified DON detoxifying enzymes could be used more

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widely in food and feed processing industries, such as bakery, starch or biofuel production.

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The structure of DON may explain the lack of commercially available enzyme for DON

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detoxification. All detoxification mechanisms described so far are reductions, oxidations, or

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combinations thereof which might require impracticable multi-enzyme complexes. The only 9 Page 9 of 19

recombinant bacterial enzyme for DON metabolization was published recently by Ito et al.

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[50]. Other enzymes, which catalyze the formation of DON-conjugates, derive from plants

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(glucosidases) or fungi (acetyltransferases) [51]. Their application seems to be limited since

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DON conjugates can be split in the GIT of mammals by endogenous microflora or enzymes.

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Conclusions

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DON is a frequent contaminant in feed and food and its exposure exceeds the tolerable

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daily intake in certain subpopulations. Assessment of multi-mycotoxin exposure will become

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more important in future since toxic effects of DON might be potentiated by the presence of

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other mycotoxins. For example, the masked mycotoxin DON-3-Glc is cleaved in the

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digestive tract of pigs and rats and might also increase the total DON burden of humans.

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Studies on the toxicological interaction of DON and other mycotoxins are needed to facilitate

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proper risk assessment and development of specific counteracting strategies. Currently, there

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is still no detoxification method available which can be used for various food and feed

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ingredients and processing methods. Most of the physical and chemical methods are not

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technically feasible or lack a clear understanding of the metabolites formed, which can be

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even more toxic than the parent compound. In contrast, specific detoxification actions are

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realized by certain microorganisms and enzymes. The latter represent a promising perspective

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for DON detoxification as appropriate enzyme modification may allow a broad application in

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the food and feed industry.

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Acknowledgments

The authors express their gratitude to Ryan Hines for proofreading the manuscript.

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224 References

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1. Maresca M: From the Gut to the Brain: Journey and Pathophysiological Effects of the Food-Associated Trichothecene Mycotoxin Deoxynivalenol. Toxins 2013, 5:784820. 2. Pestka JJ: Deoxynivalenol: Mechanisms of action, human exposure, and toxicological relevance. Archives of Toxicology 2010, 84:663-679. 3. Berthiller F, Crews C, Dall'Asta C, Saeger SD, Haesaert G, Karlovsky P, Oswald IP, Seefelder W, Speijers G, Stroka J: Masked mycotoxins: A review. Molecular nutrition & food research 2013, 57:165-186. 4. Wang Z, Wu Q, Kuča K, Dohnal V, Tian Z: Deoxynivalenol: signaling pathways and human exposure risk assessment—an update. Archives of Toxicology 2014, 88:1915-1928. 5. Rodrigues I, Naehrer K: A three-year survey on the worldwide occurrence of mycotoxins in feedstuffs and feed. Toxins 2012, 4:663-675. 6. European Food Safety Authority (EFSA): Deoxynivalenol in food and feed: occurrence and exposure. EFSA Journal 2013, 11:3379. 7. Broekaert N, Devreese M, De Baere S, De Backer P, Croubels S: Modified Fusarium mycotoxins unmasked: From occurrence in cereals to animal and human excretion. Food and Chemical Toxicology 2015, 80:17-31. 8. De Boevre M, Jacxsens L, Lachat C, Eeckhout M, Di Mavungu JD, Audenaert K, Maene P, Haesaert G, Kolsteren P, De Meulenaer B: Human exposure to mycotoxins and their masked forms through cereal-based foods in Belgium. Toxicology Letters 2013, 218:281-292. 9. Varga E, Malachova A, Schwartz H, Krska R, Berthiller F: Survey of deoxynivalenol and its conjugates deoxynivalenol-3-glucoside and 3-acetyl-deoxynivalenol in 374 beer samples. Food Additives & Contaminants: Part A 2013, 30:137-146. 10. Wang W, Ma J, Yu C, Lin X, jiang h, Shao B, Li F: Simultaneous Determination of Masked Deoxynivalenol and Some Important Type B Trichothecenes in Chinese Corn Kernels and Corn-Based Products by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry. Journal of Agricultural and Food Chemistry 2012, 60:11638-11646. 11. Streit E, Schwab C, Sulyok M, Naehrer K, Krska R, Schatzmayr G: Multi-mycotoxin screening reveals the occurrence of 139 different secondary metabolites in feed and feed ingredients. Toxins 2013, 5:504-523. 12. Alassane-Kpembi I, Puel O, Oswald IP: Toxicological interactions between the mycotoxins deoxynivalenol, nivalenol and their acetylated derivatives in intestinal epithelial cells. Archives of Toxicology 2014:1-10. *13. Dänicke S, Brezina U: Kinetics and metabolism of the Fusarium toxin deoxynivalenol in farm animals: Consequences for diagnosis of exposure and intoxication and carry over. Food and Chemical Toxicology 2013, 60:58-75. In this review, current limitations concerning the use of DON biomarkers in farm animals are highlighted. Among others, factors such as sampling time point, feed intake and age of the animal influence the levels of DON and its metabolites in blood. 14. Gratz SW, Richardson AJ, Duncan G, Holtrop G: Annual variation of dietary deoxynivalenol exposure during years of different Fusarium prevalence: A pilot biomonitoring study. Food Additives & Contaminants: Part A 2014, 31:1579-1585.

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15. Huybrechts B, Martins J, Debongnie P, Uhlig S, Callebaut A: Fast and sensitive LC– MS/MS method measuring human mycotoxin exposure using biomarkers in urine. Archives of Toxicology 2014:1-13. *16. Maul R, Warth B, Schebb NH, Krska R, Koch M, Sulyok M: In vitro glucuronidation kinetics of deoxynivalenol by human and animal microsomes and recombinant human UGT enzymes. Archives of Toxicology 2014, 89:949-960. In this in vitro study, authors demonstrate species-specific variations in the glucuronidation activity of uridine-diphosphoglucuronyltransferases, which is presumably an important reason for the differences in the susceptibility to DON among species. In addition, the formation of DON-GlcA isomers was associated with the specific expression of human UGT isoforms. 17. Warth B, Sulyok M, Berthiller F, Schuhmacher R, Krska R: New Insights into the Human Metabolism of the Fusarium Mycotoxins Deoxynivalenol and Zearalenone. Toxicology Letters 2013, 220:80-94. 18. Nagl V, Schwartz H, Krska R, Moll WD, Knasmüller S, Ritzmann M, Adam G, Berthiller F: Metabolism of the masked mycotoxin deoxynivalenol-3-glucoside in rats. Toxicology Letters 2012, 229:88-94. *19. Nagl V, Woechtl B, Schwartz-Zimmermann HE, Hennig-Pauka I, Moll W-D, Adam G, Berthiller F: Metabolism of the masked mycotoxin deoxynivalenol-3-glucoside in pigs. Toxicology letters 2014, 229:190-197. This study presents the first in vivo data on the fate of DON-3-Glc in pigs. Since there are certain similarities in metabolism of DON between pigs and humans, findings are of special relevance for risk assessment. Ingested DON-3-Glc was nearly completely hydrolyzed in pigs, but only partially absorbed. 20. Šarkanj B, Warth B, Uhlig S, Abia WA, Sulyok M, Klapec T, Krska R, Banjari I: Urinary analysis reveals high deoxynivalenol exposure in pregnant women from Croatia. Food and Chemical Toxicology 2013, 62:231-237. 21. Wan D, Huang L, Pan Y, Wu Q, Chen D, Tao Y, Wang X, Liu Z, Li J, Wang L: Metabolism, Distribution and Excretion of Deoxynivalenol with Combined Techniques of Radio-tracing, HPLC-IT-TOF/MS and Online Radiometric Detection. Journal of Agricultural and Food Chemistry 2013, 62:288-296. *22. Schwartz-Zimmermann HE, Hametner C, Nagl V, Slavik V, Moll W-D, Berthiller F: Deoxynivalenol (DON) sulfonates as major DON metabolites in rats: from identification to biomarker method development, validation and application. Analytical and bioanalytical chemistry 2014:1-14. Using state-of-the-art analytical techniques, novel sulfonate conjugates of DON, DOM-1 and DON-3-Glc were identified in excreta of rats, thus highlighting the importance of this metabolic pathway in rodents. 23. Marin S, Ramos A, Cano-Sancho G, Sanchis V: Mycotoxins: occurrence, toxicology, and exposure assessment. Food and Chemical Toxicology 2013, 60:218-237. *24. Turner PC, Flannery B, Isitt C, Ali M, Pestka J: The role of biomarkers in evaluating human health concerns from fungal contaminants in food. Nutrition research reviews 2012, 25:162-179. Authors provide a comprehensive overview on the current knowledge of mycotoxin biomakers in humans. Special focus is on the development and validation of biomarkers for DON and fumonisin B1 expsoure. In addition, adverse health effects of aflatoxin B1, DON and fumonsin B1 are discussed. 25. Joint FAO/WHO Expert Committee on Food Additives (JECFA): Evaluation of certain contaminants in food: seventy-second report of the Joint FAO/WHO Expert Committee on Food Additives. WHO Technical Report Series 2011, 959.

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26. Gerding J, Ali N, Schwartzbord J, Cramer B, Brown DL, Degen GH, Humpf H-U: A comparative study of the human urinary mycotoxin excretion patterns in Bangladesh, Germany, and Haiti using a rapid and sensitive LC-MS/MS approach. Mycotoxin Research 2015:1-10. 27. Brera C, de Santis B, Debegnach F, Miano B, Moretti G, Lanzone A, Del Sordo G, Buonsenso D, Chiaretti A, Hardie L, et al.: Experimental study of deoxynivalenol biomarkers in urine. EFSA supporting publication 2015:EN-818 2015:136. *28. Srey C, Kimanya ME, Routledge MN, Shirima CP, Gong YY: Deoxynivalenol exposure assessment in young children in Tanzania. Molecular Nutrition & Food Research 2014, 58:1574-1580. Besides survey data on DON expsoure in Tanzania, the excretion rate of DON was assessed for the first time in children. Urinary DON excretion accounted for 74% of ingested DON (which is quite similar to results previously in adults). 29. Pierron A & Mimoun S, Murate L, Lippi Y, Loiseau N, Bracarense A-PFL, Liaubet L, Schatmayr G, Berthiller F, Moll W-D, Oswald IP: Intestinal toxicity of the masked mycotoxin Deoxynivalenol-3-β-D-glucoside. In press. 30. De Boevre M, Graniczkowska K, De Saeger S: Metabolism of modified mycotoxins studied through invitro and invivo models: An overview. Toxicology Letters 2015, 233:24-28. 31. Veršilovskis A, Geys J, Huybrechts B, Goossens E, De Saeger S, Callebaut A: Simultaneous determination of masked forms of deoxynivalenol and zearalenone after oral dosing in rats by LC-MS/MS. World Mycotoxin Journal 2012, 5:303-318. 32. Wu W, He K, Zhou H-R, Berthiller F, Adam G, Sugita-Konishi Y, Watanabe M, Krantis A, Durst T, Zhang H: Effects of oral exposure to naturally-occurring and synthetic deoxynivalenol congeners on proinflammatory cytokine and chemokine mRNA expression in the mouse. Toxicology and applied pharmacology 2014, 278:107-115. *33. Wu W, Zhou HR, Bursian SJ, Pan X, Link JE, Berthiller F, Adam G, Krantis A, Durst T, Pestka JJ: Comparison of anorectic and emetic potencies of deoxynivalenol (vomitoxin) to the plant metabolite deoxynivalenol-3-glucoside and synthetic deoxynivalenol derivatives EN139528 and EN139544. Toxicological sciences : an official journal of the Society of Toxicology 2014, 142:167-181. Results of this study, demonstrating a reduced emetic response of DON-3-Glc in comparison to DON, contribute essentially to the risk assessment of this masked mycotoxin. 34. Kluger B, Bueschl C, Lemmens M, Berthiller F, Häubl G, Jaunecker G, Adam G, Krska R, Schuhmacher R: Stable isotopic labelling-assisted untargeted metabolic profiling reveals novel conjugates of the mycotoxin deoxynivalenol in wheat. Analytical and bioanalytical chemistry 2013, 405:5031-5036. 35. Warth B, Fruhmann P, Wiesenberger G, Kluger B, Sarkanj B, Lemmens M, Hametner C, Fröhlich J, Adam G, Krska R: Deoxynivalenol-sulfates: identification and quantification of novel conjugated (masked) mycotoxins in wheat. Analytical and bioanalytical chemistry 2014:1-7. *36. Miller JD, Schaafsma AW, Bhatnagar D, Bondy G, Carbone I, Harris LJ, Harrison G, Munkvold GP, Oswald IP, Pestka JJ, et al.: Mycotoxins that affect the North American agri-food sector: State of the art and directions for the future. World Mycotoxin Journal 2014, 7:63-82. Summary of a conference giving direction to future research in the area of prevention and decontamination of mycotoxins and regulatory perspectives.

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37. He J, Zhou T, Young JC, Boland GJ, Scott PM: Chemical and biological transformations for detoxification of trichothecene mycotoxins in human and animal food chains: a review. Trends in Food Science and Technology 2010, 21:6776. *38. Kaushik G: Effect of Processing on Mycotoxin Content in Grains. Critical Reviews in Food Science and Nutrition 2015, 55:1672–1683. Very recent summary of DON reductions during thermal treatment, especially in the bakery. The weaknesses of analytical methods and toxicity studies of breakdown products are indirectly shown. 39. Vidal A, Sanchis V, Ramos AJ, Marín S: Thermal stability and kinetics of degradation of deoxynivalenol, deoxynivalenol conjugates and ochratoxin A during baking of wheat bakery products. Food Chemistry 2015, 178:276-286. 40. Murugesan GR, Ledoux DR, Naehrer K, Berthiller F, Applegate TJ, Grenier B, Phillips TD, Schatzmayr G: Prevalence and effects of mycotoxins on poultry health and performance, and recent development in mycotoxin counteracting strategies. Poultry Science 2015, 94:1298-1315. 41. Devreese M, Antonissen G, Backer PD, Croubels S: Efficacy of Active Carbon towards the Absorption of Deoxynivalenol in Pigs. Toxins 2014, 2014:6:2998-3004. 42. Paulick M, Rempe I, Kersten S, Schatzmayr D, Elisabeth H, Schwartz-Zimmermann, Dänicke S: Effects of Increasing Concentrations of Sodium Sulfite on Deoxynivalenol and Deoxynivalenol Sulfonate Concentrations of Maize Kernels and Maize Meal Preserved at Various Moisture Content. Toxins 2015, 2015:7:791-811. 43. Schwartz-Zimmermann H, Wiesenberger G, Unbekannt C, Hessenberger S, Schatzmayr D, Unbekannt FB: Reaction of (conjugated) deoxynivalenol with sulphur reagents-novel metabolites, toxicity and application. World Mycotoxin Journal 2014, 7:187-197. 44. Mishra S, Dixit S, Dwivedi PD, Pandey HP, Das M: Influence of temperature and pH on the degradation of deoxynivalenol (DON) in aqueous medium: comparative cytotoxicity of DON and degraded product. Food Additives & Contaminants: Part A 2014, 31:121–131. *45. Hassan YI, Watts C, Li XZ, Zhou T: A Novel Peptide-Binding Motifs Inference Approach to Understand Deoxynivalenol Molecular Toxicity. Toxins (Basel) 2015, 7:1989-2005. Recent summary on the modifications of the DON structure leading to less or nontoxic metabolites. 46. European Food Safety Authority (EFSA): Scientific Opinion on the safety and efficacy of micro-organism DSM 11798 when used as a technological feed additive for pigs. EFSA Journal 2013, 11:3203. 47. European Commission (EC): Commission implementing regulation (EU) No 1016/2013 of 23 October 2013 concerning the authoristation of a preparation of a micro-organism strain DSM 11798 of the Coriobacteriacae family as a feed additive for pigs. Official Journal of the European Union 2013:L282/236. 48. Ikunaga Y, Sato I, Grond S, Numaziri N, Yoshida S, Yamaya H, Hiradate S, Hasegawa M, Toshima H, Koitabashi M, et al.: Nocardioides sp. strain WSN05-2, isolated from a wheat field, degrades deoxynivalenol, producing the novel intermediate 3epi-deoxynivalenol. Applied Microbiology and Biotechnology 2011, 89:419-427. 49. Sato I, Ito M, Ishizaka M, Ikunaga Y, Sato Y, Yoshida S, Koitabashi M, Tsushima S: Thirteen novel deoxynivalenol-degrading bacteria are classified within two

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genera with distinct degradation mechanisms. Federation of European Microbiological Societies 2011, 327:110-117. *50. Ito M, Sato I, Ishizaka M, Yoshida S-i, Koitabashi M, Yoshida S, Tsushimad S: A bacterial cytochrome P450 system catabolizing the Fusarium toxin deoxynivalenol. Appl. Environ. Microbiol. 2013 79:1619-1628. In this study, the first recombinant enzyme for DON detoxification is described. 51. McCormick SP: Microbial Detoxification of Mycotoxins. Journal of Chemical Ecology 2013, 39:907-918. 52. Ediage EN, Di Mavungu JD, Song S, Sioen I, De Saeger S: Multimycotoxin analysis in urines to assess infant exposure: A case study in Cameroon. Environment international 2013, 57:50-59. 53. Wallin S, Hardie L, Kotova N, Lemming EW, Nälsén C, Ridefelt P, Turner P, White K, Olsen M: Biomonitoring study of deoxynivalenol exposure and association with typical cereal consumption in Swedish adults. World Mycotoxin Journal 2013, 6:439-448. 54. Abia WA, Warth B, Sulyok M, Krska R, Tchana A, Njobeh PB, Turner PC, Kouanfack C, Eyongetah M, Dutton M: Bio-monitoring of mycotoxin exposure in Cameroon using a urinary multi-biomarker approach. Food and Chemical Toxicology 2013, 62:927-934. 55. Shephard GS, Burger H-M, Gambacorta L, Gong YY, Krska R, Rheeder JP, Solfrizzo M, Srey C, Sulyok M, Visconti A: Multiple mycotoxin exposure determined by urinary biomarkers in rural subsistence farmers in the former Transkei, South Africa. Food and Chemical Toxicology 2013, 62:217-225. 56. Solfrizzo M, Gambacorta L, Visconti A: Assessment of multi-mycotoxin exposure in Southern Italy by urinary multi-biomarker determination. Toxins 2014, 6:523538. 57. Warth B, Petchkongkaew A, Sulyok M, Krska R: Utilising an LC-MS/MS-based multibiomarker approach to assess mycotoxin exposure in the Bangkok metropolitan area and surrounding provinces. Food Additives & Contaminants: Part A 2014, 31:2040-2046. 58. Rodríguez-Carrasco Y, Moltó JC, Mañes J, Berrada H: Exposure assessment approach through mycotoxin/creatinine ratio evaluation in urine by GC–MS/MS. Food and Chemical Toxicology 2014, 72:69-75. 59. Kouadio JH, Lattanzio VM, Ouattara D, Kouakou B, Visconti A: Assessment of Mycotoxin Exposure in Côte d’ivoire (Ivory Coast) Through Multi-Biomarker Analysis and Possible Correlation with Food Consumption Patterns. Toxicology international 2014, 21:248.

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Figure captions

458 Figure 1. Modification of deoxynivalenol in microorganisms, fungi, plants and mammals: major types and reaction sites (adapted from Hassan et al. [45]).

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Figure

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Table

Country

Population groupa

Cameroon

us

Table 1. Surveys employing the biomarker approach to assess human deoxynivalenol exposure (since 2013) % Positive

Total urinary DON (mean, ng/mg crea)b

% Exceeding PMTDIc

Reference

Children

240

17

(2.22 ng/mL)

-

[52]

Sweden

Adults

326

90

3.5d

1

[53]

Cameroon

Male and female adults (HIV sero-positive, HIV sero-negative)

175

37-43e

6.04-10.1e

-

[54]

Croatia

Pregnant woman

South Africa

Female adults

Italy

Tanzania

ep te

d

M

an

Total number of subjects

97.5

93.7

48

[20]

53

55f-100e

11.3-20.4e

-

[55]

Males and females

52

96

(11.9 ng/mL)

6

[56]

Children

166

51-80e

(1.1-5.7 ng/mL)e

10-29e

[28]

Ac c

40

Page 18 of 19

ip t Male and female adults

15

100

7.2-19.7e

Thailand

Adolescents, adults

60

11.6f

us

cr

United Kingdom

Belgium

Male and female adults

29

100

Spain

Children, young adults, adults

54

Ivory Coast

Males and females

Bangladesh, Germany, Haiti

Adults, children

Italy, Norway, United Kingdom

Children, adolescents, adults, elderly, vegetarians, pregnant women

[14]

0

[57]

(59.0 ng/mL)

-

[15]

14.8-32.9e

8.1

[58]

M

an

4.8f

Ac c

ep te

d

68.5

0-13e

99

21

(0.01-5.2 ng/mLd,e)

-

[59]

287

0-54e,f

Not detected-20.0e,f

0-6e

[26]

635

76-99e

8.29-31.4e

-

[27]

a

As defined by authors; b If creatinine (crea) content was not determined, mean values in ng/mL are listed; c Provisional maximum tolerable daily intake (PMTDI; 1 µg/kg b.w. [25]); d Median values (mean value not available); e Depending on study-specific clustering (HIV-status, lab, sampling time, age, sex or region); f For most frequently detected metabolite

Page 19 of 19