[Show abstract][Hide abstract] ABSTRACT: Natural food contaminants such as mycotoxins are an important problem for human health. Deoxynivalenol (DON) is one of the most common mycotoxins detected in cereals and grains. Its toxicological effects mainly concern the immune system and the gastrointestinal tract. This toxin is a potent ribotoxic stressor leading to MAP kinase activation and inflammatory response. DON frequently co-occurs with its glucosylated form, the masked mycotoxin deoxynivalenol-3-β-D-glucoside (D3G). The toxicity of this later compound remains unknown in mammals. This study aimed to assess the ability of D3G to elicit a ribotoxic stress and to induce intestinal toxicity. The toxicity of D3G and DON (0-10 µM) was studied in vitro, on the human intestinal Caco-2 cell line, and ex vivo, on porcine jejunal explants. First, an in silico analysis revealed that D3G, contrary to DON, was unable to bind to the A-site of the ribosome peptidyl transferase center, the main targets for DON toxicity. Accordingly, D3G did not activate JNK and P38 MAPKs in treated Caco-2 cells and did not alter viability and barrier function on cells, as measured by the trans-epithelial electrical resistance. Treatment of intestinal explants for 4 h with 10 µM DON induced morphological lesions and up-regulated the expression of pro-inflammatory cytokines as measured by qPCR and pan-genomic microarray analysis. By contrast, expression profile of D3G-treated explants was similar to that of controls, and these explants did not show histomorphology alteration. In conclusion, our data demonstrated that glucosylation of DON suppresses its ability to bind to the ribosome and decreases its intestinal toxicity.
Archives of Toxicology 09/2015; DOI:10.1007/s00204-015-1592-8 · 5.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An extensive study of the metabolism of the
type A trichothecene mycotoxins HT-2 toxin and T-2 toxin
in barley using liquid chromatography coupled to high-resolution
mass spectrometry (LC-HRMS) is reported. A
recently developed untargeted approach based on stable
isotopic labelling, LC-Orbitrap-MS analysis with fast polarity
switching and data processing by MetExtract software
was combined with targeted LC-Q-TOF-MS(/MS)
analysis for metabolite structure elucidation and quantification.
In total, 9 HT-2 toxin and 13 T-2 toxin metabolites
plus tentative isomers were detected, which were successfully
annotated by calculation of elemental formulas and
further LC-HRMS/MS measurements as well as partly identified with authentic standards. As a result,
glucosylated forms of the toxins, malonylglucosides, and
acetyl and feruloyl conjugates were elucidated. Additionally,
time courses of metabolite formation and mass balances
were established. For absolute quantification of those compounds
for which standards were available, the method
was validated by determining apparent recovery, signal
suppression, or enhancement and extraction recovery. Most
importantly, T-2 toxin was rapidly metabolised to HT-2
toxin and for both parent toxins HT-2 toxin-3-O-β-glucoside
was identified (confirmed by authentic standard) as
the main metabolite, which reached its maximum already
1 day after toxin treatment.
Analytical and Bioanalytical Chemistry 09/2015; DOI:10.1007/s00216-015-8975-9 · 3.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the metabolic fate of HT-2 toxin (HT2) and T-2 toxin (T2) in wheat (Triticum aestivum L.) an untargeted metabolomics study utilizing stable isotopic labeling and liquid chromatography-high resolution mass spectrometry was performed. In total, 11 HT2 and 12 T2 derived in planta biotransformation products were annotated putatively. In addition to previously reported mono- and di-glucosylated forms of HT2, evidence for the formation of HT2-malonyl-glucoside and feruloyl-T2, as well as acetylation and de-acetylation products in wheat was obtained for the first time. To monitor the kinetics of metabolite formation, a time course experiment was conducted involving the Fusarium head blight susceptible variety Remus and the resistant cultivar CM-82036. Biotransformation reactions were observed already at the earliest tested time point (6 h after treatment) and formed metabolites showed different kinetic profiles. After ripening, less than 15% of the toxins added to the plants were determined to be unmetabolized.
Journal of Agricultural and Food Chemistry 08/2015; 63(35). DOI:10.1021/acs.jafc.5b02697 · 2.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Deoxynivalenol (DON) is a protein synthesis inhibitor produced by the Fusarium species, which frequently contaminates grains used for human or animal consumption. We treated a wheat suspension culture with DON or one of its acetylated derivatives, 3-acetyl-DON (3-ADON), 15-acetyl-DON (15-ADON) and 3,15-diacetyl-DON (3,15-diADON), and monitored the metabolization over a course of 96 h. Supernatant and cell extract samples were analyzed using a tailored LC-MS/MS method for the quantification of DON metabolites. We report the formation of tentatively identified DON-15-O-β-D-glucoside (D15G) and of 15-acetyl-DON-3-sulfate (15-ADON3S) as novel deoxynivalenol metabolites in wheat. Furthermore, we found that the recently identified 15-acetyl-DON-3-O-β-D-glucoside (15-ADON3G) is the major metabolite produced after 15-ADON challenge. 3-ADON treatment led to a higher intracellular content of toxic metabolites after six hours compared to all other treatments. 3-ADON was exclusively metabolized into DON before phase II reactions occurred. In contrast, we found that 15-ADON was directly converted into 15-ADON3G and 15-ADON3S in addition to metabolization into deoxynivalenol-3-O-β-D-glucoside (D3G). This study highlights significant differences in the metabolization of DON and its acetylated derivatives.
[Show abstract][Hide abstract] ABSTRACT: Fusarium head blight (FHB), mainly caused by Fusarium graminearum, is a devastating disease of wheat that results in economic losses worldwide. During infection, F. graminearum produces trichothecene mycotoxins, including deoxynivalenol (DON), that increase fungal virulence and reduces grain quality. Transgenic wheat expressing a barley UDP-glucosyltransferase (HvUGT13248) were developed and evaluated for FHB resistance, DON accumulation and the ability to metabolize DON to the less toxic DON-3-O-glucoside (D3G). Point inoculation tests in the greenhouse showed that transgenic wheat carrying HvUGT13248 exhibited significantly higher resistance to disease spread in the spike (type II resistance) compared to non-transformed controls. Two transgenic events displayed complete suppression of disease spread in the spikes. Expression of HvUGT13248 in transgenic wheat rapidly and efficiently conjugated DON to D3G, suggesting that the enzymatic rate of DON detoxification translates to type II resistance. Under field conditions, FHB severity was variable, nonetheless, transgenic events showed significantly less severe disease phenotypes compared to the non-transformed controls. In addition, a seedling assay demonstrated that the transformed plants had a higher tolerance to DON-inhibited root growth than non-transformed plants. These results demonstrate the utility of detoxifying DON as a FHB control strategy in wheat.
[Show abstract][Hide abstract] ABSTRACT: In the scientific field, there is a progressive awareness about the potential implications of food processing on mycotoxins especially concerning thermal treatments. High temperatures may cause, in fact, transformation or degradation of these compounds. This work is aimed to study the fate of mycotoxins during bakery processing, focusing on deoxynivalenol (DON) and deoxynivalenol-3-glucoside (DON3Glc), along the chain of industrial rusk production. Starting from naturally contaminated bran, we studied how concentrations of DON and DON3Glc are influenced by modifying ingredients and operative conditions. The experiments were performed using statistical Design of Experiment (DoE) schemes to synergistically explore the relationship between mycotoxin reduction and the indicated processing transformation parameters. All samples collected during pilot plant experiments were analyzed with an LC-MS/MS multimycotoxin method. The obtained model shows a good fitting, giving back relevant information in terms of optimization of the industrial production process, in particular suggesting that time and temperature in baking and toasting steps are highly relevant for minimizing mycotoxin level in rusks. A reduction up to 30% for DON and DON3Glc content in the finished product was observed within an acceptable technological range.
[Show abstract][Hide abstract] ABSTRACT: Glycosylation is an important plant defense mechanism and conjugates of Fusarium mycotoxins often co-occur with their parent compounds in cereal-based food and feed. In case of deoxynivalenol (DON), deoxynivalenol-3-O-β-D-glucoside (D3G) is the most important masked mycotoxin. The toxicological significance of D3G is not yet fully understood so that it is crucial to obtain this compound in pure and sufficient quantities for toxicological risk assessment and for use as an analytical standard. The aim of this study was the biochemical characterization of a DON-inactivating UDP-glucosyltransferase from rice (OsUGT79) and to investigate its suitability for preparative D3G synthesis. Apparent Michaelis constants (Km) of recombinant OsUGT79 were 0.23 mM DON and 2.2 mM UDP-glucose. Substrate inhibition occurred at DON concentrations above 2 mM (Ki = 24 mM DON), and UDP strongly inhibited the enzyme. Cu2+ and Zn2+ (1 mM) inhibited the enzyme completely. Sucrose synthase AtSUS1 was employed to regenerate UDP-glucose during the glucosylation reaction. With this approach, optimal conversion rates can be obtained at limited concentrations of the costly co-factor UDP-glucose. D3G can now be synthesized in sufficient quantity and purity. Similar strategies may be of interest to produce β-glucosides of other toxins.
[Show abstract][Hide abstract] ABSTRACT: A critical assessment of three previously published indirect methods based on acidic hydrolysis using superacids for the determination of "free" and "total" deoxynivalenol (DON) was carried out. The modified mycotoxins DON-3-glucoside (D3G), 3-acetyl-DON (3ADON), and 15-acetyl-DON (15ADON) were chosen as model analytes. The initial experiments focused on the stability/degradation of DON under hydrolytic conditions and the ability to release DON from the modified forms. Acidic conditions that were capable of cleaving D3G, 3ADON, and 15ADON to DON were not found, raising doubts over the efficacy of previously published indirect methods for total DON determination. Validation of these indirect methods for wheat, maize, and barley using UHPLC-MS/MS was performed in order to test the accuracy of the generated results. Validation data for DON, D3G, 3ADON, and 15ADON in nonhydrolyzed and hydrolyzed matrices were obtained. Under the tested conditions, DON was not released from D3G, 3ADON, or 15ADON after hydrolysis and thus none of the published methods were able to cleave the modified forms of DON. In addition to acids, alkaline hydrolysis with KOH for an extended time and at elevated temperatures was also tested. 3ADON and 15ADON were cleaved under the alkaline pH caused by the addition of KOH or aqueous K2CO3 to "neutralize" the acidic sample extracts in the published studies. The published additional DON increase after hydrolysis may have been caused by huge differences in matrix effects and the recovery of DON in nonhydrolyzed and hydrolyzed matrices as well as by the alkaline cleavage of 3ADON or 15ADON after the neutralization of hydrolyzed extracts.
[Show abstract][Hide abstract] ABSTRACT: A reliable and sensitive liquid chromatography-tandem mass spectrometric method was developed for the simultaneous quantitative determination in cereals of the Fusarium mycotoxins HT-2 toxin, T-2 toxin, deoxynivalenol, nivalenol and zearalenone, as well as the modified metabolites 3-acetyl-deoxynivalenol, α-zearalenol, β-zearalenol, deoxynivalenol-3-glucoside, HT-2-3-glucoside, nivalenol-3-glucoside, zearalenone-14-glucoside, zearalenone-14-sulphate, zearalenone-16-glucoside, α-zearalenol-14-glucoside and β-zearalenol-14-glucoside. The 'dilute and shoot' approach was used for sample preparation after extraction with acetonitrile:water:acetic acid (79:20:1, v/v/v). Separation was carried out using reversed-phase liquid chromatography, and detection was performed using tandem mass spectrometry in the selected reaction monitoring mode. The method was in-house validated according to performance characteristics, established in Commission Regulation EC No 401/2006 and Commission Decision EC No 657/2002, prior to its application in a nationwide survey for the analysis of barley, oat and wheat samples (n = 95) harvested in Finland during 2013. Deoxynivalenol and its glucosylated form were the most abundant of the analytes, being detected in 93 and 81 % of the samples, respectively. Concentrations of deoxynivalenol were unusually high in 2013, especially in oats, with some cases exceeding the maximum legislative limits for unprocessed oats placed on the market for first-stage processing. All modified mycotoxins analysed were detected, and the natural occurrence of some of these compounds (e.g. zearalenone-16-glucoside and nivalenol-3-glucoside) in barley, oats and/or wheat was documented for the first time.
[Show abstract][Hide abstract] ABSTRACT: Deoxynivalenol (DON) and zearalenone (ZEN) are mycotoxins produced by fungi of the genus Fusarium which frequently contaminate maize and grain cereals. Mycotoxin-contaminated feed endangers animal health and leads to economic losses in animal production. Several mycotoxin elimination strategies, including the use of commercially available DON and ZEN detoxifying agents, have been developed. However, frequently there is no scientific proof of the efficacy of such adsorbents and degrading products. We therefore tested 20 commercially available products claiming to detoxify DON and/or ZEN either by biodegradation (4 products) or a combination of degradation and adsorption (16 products) under aerobic and anaerobic conditions at approx. pH 7. Under the applied conditions, a complete reduction of DON and consequent formation of the known non-toxic metabolite DOM-1 was exclusively observed in samples taken from the anaerobic degradation experiment of one product. For all other products, incubated under aerobic and anaerobic conditions, a maximum DON reduction of 17% after 72 h of incubation was detected. Aerobic and anaerobic incubation of only one tested product resulted in complete ZEN reduction as well as in the formation of the less-toxic metabolites DHZEN and HZEN. With this product, 68-97% of the toxin was metabolised within 3 h. After 24 h, a ZEN reduction ≥ 60% was obtained with four additional products during aerobic incubation only. Six of the 20 investigated products produced α- and/or β-ZEL, which are metabolites showing similar oestrogenic activity compared to ZEN. Aerobic and anaerobic degradation to unknown metabolites with unidentified toxicity was obtained with 10 and 3 products, respectively. The results of our study demonstrate the importance of in vitro experiments to critically screen agents claiming mycotoxin detoxification.
Food Additives and Contaminants - Part A Chemistry, Analysis, Control, Exposure and Risk Assessment 03/2015; 32(6):1-12. DOI:10.1080/19440049.2015.1023741 · 1.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study, a total of nine different biotransformation products of the Fusarium mycotoxin deoxynivalenol (DON) formed in wheat during detoxification of the toxin are characterized by liquid chromatography-high resolution mass spectrometry (LC-HRMS). The detected metabolites suggest that DON is conjugated to endogenous metabolites via two major metabolism routes, namely 1) glucosylation (DON-3-glucoside, DON-di-hexoside, 15-acetyl-DON-3-glucoside, DON-malonylglucoside) and 2) glutathione conjugation (DON-S-glutathione, "DON-2H"-S-glutathione, DON-S-cysteinyl-glycine and DON-S-cysteine). Furthermore, conjugation of DON to a putative sugar alcohol (hexitol) was found. A molar mass balance for the cultivar 'Remus' treated with 1 mg DON revealed that under the test conditions approximately 15% of the added DON were transformed into DON-3-glucoside and another 19% were transformed to the remaining eight biotransformation products or irreversibly bound to the plant matrix. Additionally, metabolite abundance was monitored as a function of time for each DON derivative and was established for six DON treated wheat lines (1 mg/ear) differing in resistance quantitative trait loci (QTL) Fhb1 and/or Qfhs.ifa-5A. All cultivars carrying QTL Fhb1 showed similar metabolism kinetics: Formation of DON-Glc was faster, while DON-GSH production was less efficient compared to cultivars which lacked the resistance QTL Fhb1. Moreover, all wheat lines harboring Fhb1 showed significantly elevated D3G/DON abundance ratios.
PLoS ONE 03/2015; 10(3):e0119656. DOI:10.1371/journal.pone.0119656 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Schimmelpilzgifte zählen zu den wichtigsten Kontaminanten in Lebensmitteln. Zum Stand der Technik beim analytischen Nachweis dieser Toxine wurden in den letzten zehn Jahren hochempfindliche auf LC-MS/(MS) basierende Multianalytmethoden.
Nachrichten aus der Chemie 02/2015; 63(2). DOI:10.1002/nadc.201590053 · 0.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glycosylation plays a central role in plant defense against xenobiotics including mycotoxins. Glucoconjugates of Fusarium toxins such as deoxynivalenol-3-O-β-D-glucoside (DON-3G) often co-occur with their parental toxins in cereal based food and feed. To date, only limited information exists on occurrence of glucosylated mycotoxins and their toxicological relevance. Due to a lack of analytical standards and the requirement of high end analytical instrumentation for their direct determination, hydrolytic cleavage of β-glucosides followed by analysis of the released parental toxins has been proposed as an indirect determination approach. This study compares the abilities of several fungal and recombinant bacterial β-glucosidases to hydrolyze the model analyte DON-3G. Furthermore, substrate specificities of two fungal and two bacterial (Lactobacillus brevis, Bifidobacterium adolescentis) glycoside hydrolase family 3 β-glucosidases were evaluated on a broader range of substrates. The purified recombinant enzyme from B. adolescentis (BaBgl) displayed high flexibility in substrate specificity and exerted the highest hydrolytic activity towards 3-O-β-D-glucosides of the trichothecenes deoxynivalenol (DON), nivalenol and HT-2 toxin. A Km of 5.4 mM and a Vmax of 16 μmol min-1 mg-1 were determined with DON-3G. Due to low product inhibition (DON, glucose) and sufficient activity in several extracts of cereal matrices, this enzyme has the potential to be used for indirect analyses of trichothecene-β-glucosides in cereal samples.
Applied and Environmental Microbiology 01/2015; · 3.67 Impact Factor
[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; 66(1):1-6. DOI:10.3109/09637486.2014.979316 · 1.21 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.