[Show abstract][Hide abstract] ABSTRACT: Abstract Food processing may induce thermal degradation of fumonisins in corn via Maillard-type reactions, or alkaline hydrolysis via loss of the two tricarballylic acid moieties. In the former case, N-(1-deoxy-D-fructos-1-yl)-fumonisin B1 (NDF) can be formed, while the latter derivative is called hydrolysed fumonisin B1 (HFB1). The aim of this study was to deepen the knowledge about the gastrointestinal stability of HFB1 and NDF in humans. Due to the lack of standard, NDF was chemically synthesised and cleaned up in high purity to be used for further experiments. While NDF is already partially cleaved (about 41%) during simulated digestion, it remained rather stable towards human colon microflora. In contrast to this, HFB1 is partially metabolised by the colon microflora to unknown compounds after 24 h of fermentation, as seen by a loss of about 22%. Concluding, the cleavage of NDF during digestion as well as the likely metabolisation of HFB1 emphasise the need for animal trials to ascertain their toxicity in vivo.
International Journal of Food Sciences and Nutrition 12/2014; · 1.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The ubiquitous filamentous fungus Fusarium graminearum causes the important disease Fusarium head blight on various species of cereals, leading to contamination of grains with mycotoxins. In a survey of F. graminearum (sensu stricto) on wheat in North America several novel strains were isolated, which produced none of the known trichothecene mycotoxins despite causing normal disease symptoms. In rice cultures a new trichothecene mycotoxin (named NX-2) was characterized by liquid-chromatography-tandem-mass spectrometry. NMR measurements identified NX-2 as 3α-acetoxy-7α,15-dihydroxy-12,13-epoxytrichothec-9-ene. Compared to the well-known 3-acetyl-deoxynivalenol it lacks the keto group at C-8 and hence is a type A trichothecene. Wheat ears inoculated with the isolated strains revealed a ten-fold higher contamination with its deacetylated form, named NX-3, (up to 540 mg kg-1) compared to NX-2. The toxicities of the novel mycotoxins were evaluated utilizing two in vitro translation assays and the alga Chlamydomonas reinhardtii. NX-3 inhibits protein biosynthesis to almost the same extent as the prominent mycotoxin deoxynivalenol, while NX-2 is far less toxic, similar to 3-acetyl-deoxynivalenol. Genetic analysis revealed a different TRI1 allele in the N-isolates which was verified to be responsible for the difference in hydroxylation at C-8.
[Show abstract][Hide abstract] ABSTRACT: Deoxynivalenol (DON) is a trichothecene mycotoxin regularly occurring in cereals. Rats are often used to study toxicokinetics of DON and related compounds, yet only about 30 % of the administered dose is typically recovered. Recently, it was reported that DON is partly metabolised to previously undetected DON- and deepoxy-DON (DOM) sulfonate in rats and tentative structures were proposed. The present work describes the production and characterisation of DON-, DOM- and DON-3-glucoside (D3G) sulfonates of three different series; the development and validation of liquid chromatography tandem mass spectrometry (LC-MS/MS)-based methods for determination of DON, DOM, D3G and their sulfonates in rat faeces and urine; and application of the methods to samples from a DON and D3G feeding trial with rats. In addition to previously produced DON sulfonates (DONS) 1, 2 and 3, D3G sulfonates 1, 2 and 3; and DOM sulfonates (DOMS) 2 and 3 were synthesised, purified and characterised. The developed methods showed apparent recoveries of all investigated compounds between 68 and 151 % in faeces and between 48 and 113 % in urine. The recovery of DON, D3G and their metabolites from faeces and urine of rats (n = 6) administered in a single dose of 2.0 mg/kg b.w. DON or the equimolar amount of D3G was 75 ± 9 % for the DON group and 68 ± 8 % for the D3G group. DON-, DOM- and D3G sulfonates excreted in faeces accounted for 48 and 47 % of the total amount of administered DON and D3G. Urinary excretion of sulfonates was <1 %. In both treatment groups, DONS 2 was the major metabolite 0-24 h after treatment, whereas DOMS 2 was predominant thereafter. The developed methods can also be used for investigation of DON (conjugate) sulfonate formation in other animal species.
Analytical and Bioanalytical Chemistry 10/2014; · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fungal secondary metabolite biosynthesis genes are of major interest due to the pharmacological properties of their products (like mycotoxins and antibiotics). The genome of the plant pathogenic fungus Fusarium graminearum codes for a large number of candidate enzymes involved in secondary metabolite biosynthesis. However, the chemical nature of most enzymatic products of proteins encoded by putative secondary metabolism biosynthetic genes is largely unknown. Based on our analysis we present 67 gene clusters with significant enrichment of predicted secondary metabolism related enzymatic functions. 20 gene clusters with unknown metabolites exhibit strong gene expression correlation in planta and presumably play a role in virulence. Furthermore, the identification of conserved and over-represented putative transcription factor binding sites serves as additional evidence for cluster co-regulation. Orthologous cluster search provided insight into the evolution of secondary metabolism clusters. Some clusters are characteristic for the Fusarium phylum while others show evidence of horizontal gene transfer as orthologs can be found in representatives of the Botrytis or Cochliobolus lineage. The presented candidate clusters provide valuable targets for experimental examination.
PLoS ONE 10/2014; 9(10):e110311. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An LC-MS/MS “dilute and shoot” method for the determination of 295 fungal and bacterial metabolites was optimized and validated according to the guidelines established in the Directorate General for Health and Consumer Affairs of the European Commission (SANCO) document No. 12495/2011. Four different types of food matrices were chosen for validation: apple puree for infants (high water content), hazelnuts (high fat content), maize (high starch and low fat content) and green pepper (difficult or unique matrix). Method accuracy and precision was evaluated using spiked samples in five replicates at two concentration levels. Method trueness was demonstrated through participation in various proficiency tests. Although the method covers a total number of 331 analytes, validation data were acquired only for 295 analytes, either due to the non-availability of analytical standards or due other reasons described in this paper. Concerning the apparent recovery, the percentage of 295 analytes matching the acceptable recovery range of 70-120% lied down by SANCO varied from 21% in green pepper to 74% in apple puree at the highest spiking level. At the levels close to limit of quantification only 20-58% of the analytes fulfilled this criterion. The extent of matrix effects was strongly dependent on the analyte/matrix combination. In general, the lowest matrix effects were observed in apple puree (59% of analytes were not influenced by enhancement/suppression at all at the highest validation level). The highest matrix effects were observed in green pepper, where only 10% of analytes did not suffer from signal suppression/enhancement. The repeatability of the method was acceptable (RSD ≤ 20) for 97% of all analytes in apple puree and hazelnuts, for 95% in maize and for 89% in green pepper. Concerning the trueness of the method, Z-scores were generally between -2 and 2, despite a broad variety of different. Based on these results it can be concluded that quantitative determination of mycotoxins by LC-MS/MS based on a “dilute and shoot” approach is also feasible in case of complex matrices
Journal of Chromatography A 10/2014; · 4.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mycotoxin deoxynivalenol (DON) elicits robust anorectic and emetic effects in several animal species. However, less is known about the potential for naturally-occurring and synthetic congeners of this trichothecene to cause analogous responses. Here we tested the hypothesis that alterations in DON structure found in the plant metabolite deoxynivalenol-3-glucoside (D3G) and two pharmacologically active synthetic DON derivatives, EN139528 and EN139544 differentially impact their potential to evoke food refusal and emesis. In a nocturnal mouse food consumption model, oral administration with DON, D3G, EN139528 or EN139544 at doses from 2.5 to 10 mg/kg BW induced anorectic responses that lasted up to 16, 6, 6 and 3 h, respectively. Anorectic potency rank orders were EN139544>DON>EN139528>D3G from 0 to 0.5 h but DON>D3G>EN139528>EN139544 from 0 to 3 h. Oral exposure to each of the four compounds at a common dose (2.5 mg/kg BW) stimulated plasma elevations of the gut satiety peptides cholecystokinin and to a lesser extent, peptide YY3-36 that corresponded to reduced food consumption. In a mink emesis model, oral administration of increasing doses of the congeners differentially induced emesis, causing marked decreases in latency to emesis with corresponding increases in both duration and number of emetic events. The minimum emetic doses for DON, EN139528, D3G and EN139544 were 0.05, 0.5, 2, and 5 mg/kg BW, respectively. Taken together, the results suggest that while all three DON congeners elicited anorectic responses that mimicked DON over a narrow dose range, they were markedly less potent than the parent mycotoxin at inducing emesis.
[Show abstract][Hide abstract] ABSTRACT: The metabolism of deoxynivalenol-3-glucoside (D3G) was elucidated in pigs. D3G (oral) was nearly completely hydrolyzed in pigs, but only partially absorbed. Two isomers of deoxynivalenol (DON) glucuronides were detected in pig urine. Two times less urinary metabolites were excreted for D3G compared to DON. D3G (i.v.) was almost exclusively excreted in unmetabolized form via urine. Plants can metabolize the Fusarium mycotoxin deoxynivalenol (DON) by forming the masked mycotoxin deoxynivalenol-3-b-D-glucoside (D3G). D3G might be cleaved during digestion, thus increasing the total DON burden of an individual. Due to a lack of in vivo data, D3G has not been included in the various regulatory limits established for DON so far. The aim of our study was to contribute to the risk assessment of D3G by determination of its metabolism in pigs. Four piglets received water, D3G (116 mg/kg b.w.) and the equimolar amount of DON (75 mg/kg b.w.) by gavage on day 1, 5 and 9 of the experiment, respectively. Additionally, 15.5 mg D3G/kg b.w. were administered intravenously on day 13. Urine and feces were collected for 24 h and analyzed for DON, D3G, deoxynivalenol-3-glucuronide (DON-3-GlcA), LOQ, limit of quantification; MS, mass spectrometry; MS/MS, tandem mass spectrometry; PBS, phosphate buffered saline; R A , apparent recovery; R E , recovery of the extraction step; SSE, signal suppression/ enhancement; UHPLC, ultra high performance liquid chromatography.
[Show abstract][Hide abstract] ABSTRACT: As the term "masked mycotoxins" encompasses only conjugated mycotoxins generated by plants and no other possible forms of mycotoxins and their modifications, we hereby propose for all these forms a systematic definition consisting of four hierarchic levels. The highest level differentiates the free and unmodified forms of mycotoxins from those being matrix-associated and from those being modified in their chemical structure. The following lower levels further differentiate, in particular, "modified mycotoxins" into "biologically modified" and "chemically modified" with all variations of metabolites of the former and dividing the latter into "thermally formed" and "non-thermally formed" ones. To harmonize future scientific wording and subsequent legislation, we suggest that the term "modified mycotoxins" should be used in the future and the term "masked mycotoxins" to be kept for the fraction of biologically modified mycotoxins that were conjugated by plants.
[Show abstract][Hide abstract] ABSTRACT: Plant GSK-3/Shaggy-like kinases are key players in brassinosteroid (BR) signalling which impact on plant development and participate in response to wounding, pathogens and salt stress. Bikinin was previously identified in a chemical genetics screen as an inhibitor targeting these kinases. To dissect the structural elements crucial for inhibition of GSK-3/Shaggy-like kinases by bikinin and to isolate more potent compounds we synthesised a number of related substances and tested their inhibitory activity in vitro and in vivo using Arabidopsis thaliana.
[Show abstract][Hide abstract] ABSTRACT: Methylthiodeoxynivalenol (MTD), a novel derivative of the trichothecene mycotoxin deoxynivalenol (DON), was prepared by applying a reliable procedure for the formal Michael addition of methanethiol to the conjugated double bond of DON. Structure elucidation revealed the preferred formation of the hemiketal form of MTD by intramolecular cyclisation between C8 and C15. Computational investigations showed a negative total reaction energy for the hemiketalisation step and its decrease in comparison with theoretical model compounds. Therefore, this structural behaviour seems to be a general characteristic of thia-Michael adducts of type B trichothecenes. MTD was shown to be less inhibitory for a reticulocyte lysate based in vitro translation system than the parent compound DON, which supports the hypothesis that trichothecenes are detoxified through thia-adduct formation during xenobiotic metabolism.
[Show abstract][Hide abstract] ABSTRACT: The foodborne mycotoxin deoxynivalenol (DON) induces a ribotoxic stress response in mononuclear phagocytes that mediate aberrant multi-organ upregulation of TNF-α, interleukins and chemokines in experimental animals. While other DON congeners also exist as food contaminants or pharmacologically-active derivatives, it is not known how these compounds affect expression of these cytokine genes in vivo. To address this gap, we compared in mice the acute effects of oral DON exposure to that of seven relevant congeners on splenic expression of representative cytokine mRNAs after 2 and 6h. Congeners included the 8-ketotrichothecenes 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), fusarenon X (FX), nivalenol (NIV), the plant metabolite DON-3-glucoside (D3G) and two synthetic DON derivatives with novel satiety-inducing properties (EN139528 and EN139544). DON markedly induced transient upregulation of TNF-α IL-1β, IL-6, CXCL-2, CCL-2 and CCL-7 mRNA expression. The two ADONs also evoked mRNA expression of these genes but to a relatively lesser extent. FX induced more persistent responses than the other DON congeners and, compared to DON, was: 1) more potent in inducing IL-1β mRNA, 2) approximately equipotent in the induction of TNF-α and CCL-2 mRNAs, and 3) less potent at upregulating IL-6, CXCL-2, and CCL-2 mRNAs. EN139528's effects were similar to NIV, the least potent 8-ketotrichothecene, while D3G and EN139544 were largely incapable of eliciting cytokine or chemokine mRNA responses. Taken together, the results presented herein provide important new insights into the potential of naturally-occurring and synthetic DON congeners to elicit aberrant mRNA upregulation of cytokines associated with acute and chronic trichothecene toxicity.
Toxicology and Applied Pharmacology 04/2014; · 3.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We report the identification of a barley UDP-glucosyltransferase, HvUGT14077, which is able to convert the estrogenic Fusarium mycotoxin zearalenone into a near equimolar mixture of the known masked mycotoxin zearalenone-14-O-β-glucoside and a new glucose conjugate, zearalenone-16-O-β-glucoside. Biocatalytical production using engineered yeast expressing the HvUGT14077 gene allowed structural elucidation of this compound. The purified zearalenone-16-O-β-glucoside was used as an analytical calibrant in zearalenone metabolization experiments. We confirmed the formation of this new masked mycotoxin in barley seedlings as well as in wheat and Brachypodium distachyon cell suspension cultures. In barley roots, up to 18-fold higher levels of zearalenone-16-O-β-glucoside compared to the known zearalenone-14-O-β-glucoside were found. Incubation of zearalenone-16-O-β-glucoside with human fecal slurry showed that this conjugate can also be hydrolyzed rapidly by intestinal bacteria, converting the glucoside back to the parental mycotoxin. Consequently, it should be considered as an additional masked form of zearalenone with potential relevance for food safety.
Journal of Agricultural and Food Chemistry 01/2014; · 3.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The foodborne mycotoxin deoxynivalenol (DON) induces a ribotoxic stress response in mononuclear phagocytes that mediate aberrant multi-organ upregulation of TNF-α, interleukins and chemokines in experimental animals. While other DON congeners also exist as food contaminants or pharmacologically-active derivatives, it is not known how these compounds affect expression of these cytokine genes in vivo. To address this gap, we compared in mice the acute effects of oral DON exposure to that of seven relevant congeners on splenic expression of representative cytokine mRNAs after 2 and 6 h. Congeners included the 8-ketotrichothecenes 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), fusarenon X (FX), nivalenol (NIV), the plant metabolite DON-3-glucoside (D3G) and two synthetic DON derivatives with novel satiety-inducing properties (EN139528 and EN139544). DON markedly induced transient upregulation of TNF-α IL-1β, IL-6, CXCL-2, CCL-2 and CCL-7 mRNA expression. The two ADONs also evoked mRNA expression of these genes but to a relatively lesser extent. FX induced more persistent responses than the other DON congeners and, compared to DON, was: 1) more potent in inducing IL-1β mRNA, 2) approximately equipotent in the induction of TNF-α and CCL-2 mRNAs, and 3) less potent at upregulating IL-6, CXCL-2, and CCL-2 mRNAs. EN139528’s effects were similar to NIV, the least potent 8-ketotrichothecene, while D3G and EN139544 were largely incapable of eliciting cytokine or chemokine mRNA responses. Taken together, the results presented herein provide important new insights into the potential of naturally-occurring and synthetic DON congeners to elicit aberrant mRNA upregulation of cytokines associated with acute and chronic trichothecene toxicity.
Toxicology and Applied Pharmacology 01/2014; · 3.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pentahydroxyscirpene, a novel trichothecene-type compound, was isolated from Fusarium-inoculated rice. The structure of pentahydroxyscirpene was elucidated by 1D and 2D NMR spectroscopy and X-ray single-crystal diffraction. The conformation in solution was determined by NOESY experiments supported by quantum chemical calculations. In vitro toxicity tests showed that pentahydroxyscirpene inhibits protein synthesis as do other trichothecenes.
Journal of Natural Products 12/2013; · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: After wheat, maize (Zea mays L.) is the second most important cereal crop in Kosovo and a major component of animal feed. The purpose of this study was to analyse the incidence and identity of the Fusarium species isolated from naturally infected maize kernels in Kosovo in 2009 and 2010, as well as the mycotoxin contamination. The disease incidence of Fusarium ear rot (from 0.7% to 40% diseased ears) on maize in Kosovo is high. The most frequently Fusarium spp. identified on maize kernels were Fusarium subglutinans, F. verticillioides/F. proliferatum and F. graminearum. Maize kernel samples were analysed by LC-MS/MS and found to be contaminated with deoxynivalenol (DON), DON-3-glucoside, 3-acetyl-DON, 15-acetyl-DON, zearalenone, zearalenone-14-sulphate, moniliformin, fumonisin B1 and fumonisin B2. This is the first report on the incidence and identification of Fusarium species isolated from naturally infected maize as well as the mycotoxin contamination in Kosovo.
Food Additives and Contaminants: Part B Surveillance 12/2013; 6(4):237-243.
[Show abstract][Hide abstract] ABSTRACT: Reduction of the Fusarium mycotoxin deoxynivalenol (DON) in animal feed by treatment with sodium bisulfite and sodium metabisulfite has been successfully demonstrated in several studies. All of them reported formation of one DON sulfonate of strongly reduced toxicity compared to DON. The starting point of the present work was investigation of different sulfur reagents for reduction of DON. In the course of these experiments, three different DON sulfonates termed DON sulfonate 1 (1), DON sulfonate 2 (2) and DON sulfonate 3 (3) were identified and structurally elucidated by UHPLC-HR-MS/MS as well as NMR spectroscopy. 1 is characterized by loss of the epoxide group and 2 by formation of a hemiketal. 3 is an equilibrating mixture of two isomers, a ketone and a hemiketal. The MS/MS pattern can be used to differentiate the three DON sulfonates, despite their same mass and molecular formula. Investigation of parameters influencing formation and stability of DON sulfonates revealed that rapid formation of 1 and 2 occurs at alkaline pH, whereas at acidic pH, slow formation of 3 takes place, irrespective of the sulfur reagent used. Whereas 1 and 2 are stable across a broad pH range, 3 decomposes to DON, 1, and 2 at alkaline pH. In addition, both 2 and 3 are unstable in solid form. The formation, characterization and stability of three novel DON sulfonates with respect to results from previous studies are discussed, providing insights of relevance for detoxification of DON containing animal feed.
Journal of Agricultural and Food Chemistry 08/2013; · 3.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A combined (triplex) immunoassay for the simultaneous detection of three mycotoxins in grains was developed with superparamagnetic colour-encoded microbeads, in combination with two bead-dedicated flow cytometers. Monoclonal antibodies were coupled to the beads, and the amounts of bound mycotoxins were inversely related to the amounts of bound fluorescent labelled mycotoxins (inhibition immunoassay format). The selected monoclonal antibodies were tested for their target mycotoxins and for cross-reactivity with relevant metabolites and masked mycotoxins. In the triplex format, low levels of cross-interactions between the assays occurred at irrelevant high levels only. All three assays were influenced by the sample matrix of cereal extracts to some extent, and matrix-matched calibrations are recommended for quantitative screening purposes. In a preliminary in-house validation, the triplex assay was found to be reproducible, sensitive and sufficiently accurate for the quantitative screening at ML level. The triplex assay was critically compared to liquid chromatography-tandem mass spectrometry using reference materials and fortified blank material. Results for the quantification of ochratoxin A and zearalenone were in good agreement. However, the fumonisin assay was, due to overestimation, only suitable for qualitative judgements. Both flow cytometer platforms (Luminex 100 and FLEXMAP 3D) performed similar with respect to sensitivity with the advantages of a higher sample throughput and response range of the FLEXMAP 3D and lower cost of the Luminex 100.
Analytical and Bioanalytical Chemistry 06/2013; · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fusarium head blight, caused by Fusarium graminearum, is a devastating disease of wheat. We developed near-isogenic lines (NILs) differing in the two strongest known F. graminearum resistance quantitative trait loci (QTLs), Qfhs.ndsu-3BS (also known as resistance gene Fhb1) and Qfhs.ifa-5A, which are located on the short arm of chromosome 3B and on chromosome 5A, respectively. These NILs showing different levels of resistance were used to identify transcripts that are changed significantly in a QTL-specific manner in response to the pathogen and between mock-inoculated samples. After inoculation with F. graminearum spores, 16 transcripts showed a significantly different response for Fhb1 and 352 for Qfhs.ifa-5A. Notably, we identified a lipid transfer protein which is constitutively at least 50-fold more abundant in plants carrying the resistant allele of Qfhs.ifa-5A. In addition to this candidate gene associated with Qfhs.ifa-5A, we identified a uridine diphosphate (UDP)-glycosyltransferase gene, designated TaUGT12887, exhibiting a positive difference in response to the pathogen in lines harbouring both QTLs relative to lines carrying only the Qfhs.ifa-5A resistance allele, suggesting Fhb1 dependence of this transcript. Yet, this dependence was observed only in the NIL with already higher basal resistance. The complete cDNA of TaUGT12887 was reconstituted from available wheat genomic sequences, and a synthetic recoded gene was expressed in a toxin-sensitive strain of Saccharomyces cerevisiae. This gene conferred deoxynivalenol resistance, albeit much weaker than that observed with the previously characterized barley HvUGT13248.