Regioselective O-glucuronidation of deoxynivalenol

Abweichender Titel laut Übersetzung der Verfasserin/des VerfassersDeoxynivalenol (DON) ist ein Mycotoxin (Pilzgift), das von verschiedenen Gattungen der Fusarienfamilie gebildet wird. Aufgrund der Stabilität des Toxins und dessen Teratogenität ist das Interesse an diesem Mycotoxin in den letzten Jahren erheblich gestiegen. Vor allem im Bereich der Agrarbiotechnologie wird versucht, durch neue Technologien die verursachten Schäden zu verringern. DON ist mittlerweile gut untersucht, und Grenzwerte sowie Messmethoden für dessen Nachweis vorhanden. Allerdings können auch die beim Stoffwechsel entstehenden Konjugate mit Glucuronsäure (zur Steigerung der Wasserlöslichkeit) eine Wirkung auf den Wirtsorganismus haben. Darüber hinaus können die gebildeten Glycokonjugate ausgeschieden und an der Oberfläche angereichert werden, wobei diese nicht durch herkömmliche Tests erfasst werden. Aus diesem Grund ist es von großem Interesse, diese Substanzen quantifizieren und deren Wirkung abschätzen zu können.Das hauptsächliche Ziel der vorliegenden Arbeit ist die Synthese der beiden in der Natur auftretenden Glucuronide von DON, DON-3-beta-D-glucuronid bzw. DON-15-beta-D-glucuronid. Anders als eine enzymatische Reaktion, die im Rahmen des Stoffwechsels auftritt, stellt eine synthetische Glucuronidierungsreaktion eine wesentlich schwierigere Aufgabe dar, da es abhängig von der Art des Zielmoleküls eine Vielzahl an verschiedenen Reaktionsmöglichkeiten gibt. Da DON kaum verfügbar ist und dessen Anschaffung üblicherweise mit immensen Kosten verbunden ist, war es notwendig die Methoden an geeigneten Modellverbindungen zu testen. Dazu wurde eine Reihe von einfachen Modellen synthetisiert und verschiedenen Glucuronidierungsmethoden unterzogen. Die dabei gewonnen Erkenntnisse wurden anschließen bei der Gucuronidierung von DON eingesetzt, wobei beide geschützten Glucuronide in guten Ausbeuten erhalten wurden. Eine Isolierung von DON-15-glucuronid gelang nach der Abspaltung der Schutzgruppen aufgrund einer Weiterreaktion des Endproduktes nicht, jedoch konnte DON-3-beta-D-glucuronid stereoselektiv und in zufriedenstellenden Ausbeuten isoliert werden.9

Stereoselective Luche Reduction of Deoxynivalenol and Three of Its Acetylated Derivatives at C8

The trichothecene mycotoxin deoxynivalenol (DON) is a well known and common contaminant in food and feed. Acetylated derivatives and other biosynthetic precursors can occur together with the main toxin. A key biosynthetic step towards DON involves an oxidation of the 8-OH group of 7,8-dihydroxycalonectrin. Since analytical standards for the intermediates are not available and these intermediates are therefore rarely studied, we aimed for a synthetic method to invert this reaction, making a series of calonectrin-derived precursors accessible. We did this by developing an efficient protocol for stereoselective Luche reduction at C8. This method was used to access 3,7,8,15-tetrahydroxyscirpene, 3-deacetyl-7,8-dihydroxycalonectrin, 15-deacetyl-7,8-dihydroxycalonectrin and 7,8-dihydroxycalonectrin, which were characterized using several NMR techniques. Beside the development of a method which could basically be used for all type B trichothecenes, we opened a synthetic route towards different acetylated calonectrins

Pentahydroxyscirpene—Producing Strains, Formation In Planta, and Natural Occurrence

Trichothecenes are a class of structurally diverse mycotoxins with more than 200 naturally occurring compounds. Previously, a new compound, pentahydroxyscirpene (PHS), was reported as a byproduct of a nivalenol producing Fusarium strain, IFA189. PHS contains a hydroxy group at C-8 instead of the keto group of type B trichothecenes. In this work, we demonstrate that IFA189 belongs to the species Fusarium kyushuense using molecular tools. Production of PHS in vitro was also observed for several isolates of other Fusarium species producing nivalenol. Furthermore, we report the formation of 4-acetyl-PHS by F. kyushuense on inoculated rice. Wheat ears of the variety Remus were infected with IFA189 and the in planta production of PHS was confirmed. Natural occurrence of PHS was verified in barley samples from the Czech Republic using a liquid chromatographic-tandem mass spectrometric method validated for this purpose. Toxicity of PHS to wheat ribosomes was evaluated with a coupled in vitro transcription and translation assay, which showed that PHS inhibits protein biosynthesis slightly less than nivalenol and deoxynivalenol

Metabolism of Deoxynivalenol and Deepoxy-Deoxynivalenol in Broiler Chickens, Pullets, Roosters and Turkeys

Recently, deoxynivalenol-3-sulfate (DON-3-sulfate) was proposed as a major DON metabolite in poultry. In the present work, the first LC-MS/MS based method for determination of DON-3-sulfate, deepoxy-DON-3-sulfate (DOM-3-sulfate), DON, DOM, DON sulfonates 1, 2, 3, and DOM sulfonate 2 in excreta samples of chickens and turkeys was developed and validated. To this end, DOM-3-sulfate was chemically synthesized and characterized by NMR and LC-HR-MS/MS measurements. Application of the method to excreta and chyme samples of four feeding trials with turkeys, chickens, pullets, and roosters confirmed DON-3-sulfate as the major DON metabolite in all poultry species studied. Analogously to DON-3-sulfate, DOM-3-sulfate was formed after oral administration of DOM both in turkeys and in chickens. In addition, pullets and roosters metabolized DON into DOM-3-sulfate. In vitro transcription/translation assays revealed DOM-3-sulfate to be 2000 times less toxic on the ribosome than DON. Biological recoveries of DON and DOM orally administered to broiler chickens, turkeys, and pullets were 74%–106% (chickens), 51%–72% (roosters), and 131%–151% (pullets). In pullets, DON-3-sulfate concentrations increased from jejunum chyme samples to excreta samples by a factor of 60. This result, put into context with earlier studies, indicates fast and efficient absorption of DON between crop and jejunum, conversion to DON-3-sulfate in intestinal mucosa, liver, and possibly kidney, and rapid elimination into excreta via bile and urine

Immunoassay and amperometric biosensor approaches for the detection of deltamethrin in seawater

The study of an enzyme-linked immunosorbent assay (ELISA) and an amperometric biosensor for the detection of the pyrethroid deltamethrin in seawater is reported. The preparation of specific polyclonal antibodies is addressed using two immunizing haptens based on deltamethrin and cypermethrin compounds, with a spacer arm placed at the cyano residue in the pyrethroid structure. Different conjugates based on bovine serum albumin and aminodextran are prepared depending on the lipophilic profile of the competitor haptens studied. A reproducible and sensitive indirect competitive ELISA is developed, reaching a limit of detection of 1.2 ± 0.04 μg L−1 and an IC50 value of 21.4 ± 0.3 μg L−1 (both n = 3). For validation of the assays described, artificial seawater samples fortified with deltamethrin are analyzed. For the ELISA assay, these accuracy studies reported a slope of 0.904. An amperometric immunosensor is developed using the same immunoreagents and achieving a comparable detectability in terms of LOD of 4.7 μg L−1, measuring seawater without any pretreatment. These results suggest that both techniques can be used as rapid and simple analytical methods for deltamethrin quantification in seawater samples, which are great candidates for initial environmental screening programs. © 2018, Springer-Verlag GmbH Germany, part of Springer Nature.Funding information This work has been funded by SEA-on-a-CHIP project (FP7-OCEAN-2013, no 614168). The Nb4D group (formerly Applied Molecular Receptors group, AMRg) is a consolidated research group (Grup de Recerca) of the Generalitat de Catalunya and has support from the Departament d’Universitats, Recerca i Societat de la Informació de la Generalitat de Catalunya (expedient: 2014 SGR 1484). CIBER-BBN is an initiative funded by the Spanish National Plan for Scientific and Technical Research and Innovation 2013–2016; Iniciativa Ingenio 2010, Consolider Program, and CIBER Actions are financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. The ICTS BNANOBIOSIS,^ and particularly the Custom Antibody Service (CAbS, IQAC-CSIC, CIBER-BBN), is acknowledged for the assistance and support related to the immunoreagents used in this work.Peer reviewe

The Metabolic Fate of Deoxynivalenol and Its Acetylated Derivatives in a Wheat Suspension Culture: Identification and Detection of DON-15-O-Glucoside, 15-Acetyl-DON-3-O-Glucoside and 15-Acetyl-DON-3-Sulfate

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

Assessment of mycotoxin occurrence in feed samples from the Belgian veal calf industry and the influence of roughage provision

INTRODUCTION: The Belgian white veal industry specializes in raising predominantly male surplus dairy calves to white veal meat. This is obtained by intensive feeding of low-iron diets, historically dominated by liquid milk replacer, containing vegetable protein sources. Consequently, veal calves only had very limited ruminal development and function. Only recently, the share of roughage and concentrates has expanded for welfare and feed-technical reasons, leading to more mature rumen development. Both milk replacer and solid feeds contain grain products, concomitant with possible mycotoxin contamination. Mycotoxins are naturally occurring secondary fungal metabolites that can be produced in crops and other feed commodities both pre- and post-harvest. It is suggested that an optimal rumen functioning is responsible for the biotransformation and detoxification of several Fusarium mycotoxins, including deoxynivalenol (DON), making ruminants less sensitive to these toxins. In the first part of this study, we aimed to evaluate the mycotoxin contamination of feed samples in the Belgian veal industry. Subsequently, we investigated the impact of ruminal development on the biotransformation of DON and its acetylated derivatives, 3- and 15-acetyl-DON (3- and 15-ADON), in calves using a comparative toxicokinetic approach. METHODS: Survey: 45 feed samples were collected from 15 different veal farms. On each farm three to six samples per feed component, respectively milk replacer, roughage (straw or corn silage) and concentrate feed, were collected and subsequently pooled per feed component. Samples were analysed by validated multi-mycotoxin (UHP)LC-MS/MS methods (Monbaliu et al., 2010; Van Pamel et al., 2014). Toxicokinetic study: two ruminating and two non-ruminating male calves each received respectively a bolus of DON (120 µg/kg bodyweight (BW)), 3-ADON (25 µg/kg BW), and 15-ADON (50 µg/kg BW) by intravenous (IV) injection or per os (PO) in a cross-over design, respecting a wash-out period of 96h. Concentrations were based on average feed intake and maximum contamination levels of the feed based on the survey. Following mycotoxin bolus administration, blood and urine was collected at different time points post administration. DON, 3-ADON, 15-ADON as well their metabolites, namely de-epoxy-DON, DON-3-glucuronide, DON-3-sulfate, DON-15-sulfate, 3-ADON-15-sulfate, 15-ADON-3-sulfate and DON-3,15-di-sulfate, in plasma and urine samples were analyzed by LC-MS/MS. RESULTS: Survey: About 13% of the milk replacer samples were contaminated with fumonisin B1 and FB2, with an average contamination level of respectively 32 ± 7 and 13 ± 1 µg/kg. No other mycotoxins were found in these samples. However, all roughage and concentrate feed samples were contaminated with at least one mycotoxin. DON was most prevalent, contaminating 80% of the roughage samples and all 15 concentrate samples. Also the DON acetylated derivatives 3- and 15-ADON were present in 40% of the roughage and concentrate samples. It remains to be determined whether 3- and 15-ADON contribute to the total DON contamination, e.g. by possible in vivo hydrolysis upon ingestion. Besides DON, also enniatin B was highly prevalent (73% of the samples). Toxicokinetic study: Results will be presented at the conference. DISCUSSION: The survey demonstrates a multi-mycotoxin contamination of the feed for veal calves, mainly present in roughage and concentrate feed. The results of the toxicokinetic study will determine the balance between mycotoxin exposure and ruminal biotransformation of the toxins in veal calves. The increased exposure of veal calves to mycotoxins, by the provision of large amounts of solid feed, can negatively affect veal performance, gastro-intestinal health and susceptibility for infectious diseases. However, the provision of large amounts of solid feed stimulates ruminal development, which can make the veal calves less sensitive to mycotoxins. REFERENCES Monbaliu S. , Van Poucke C., Detavernier C., Dumoulin F., Van De Velde M., Schoeters E., Van Dyck S., Averkieva O., Van Peteghem C., De Saeger S. (2010) Occurrence of mycotoxins in feed as analyzed by a multi-mycotoxin LC-MS/MS method, J. Agric. Food Cem. 58, 66-71. Van Pamel E., Antonissen G., Valgaeren B., Croubels S.,Daeseleire E. (2014) A multi-mycotoxin UHPLC-MS/MS method for the detection, quantification and identification of mycotoxins in milk replacer, 36th Mycotoxin Workshop, Göttingen, June 16-18