Philipp Fruhmann

Assessment of human deoxynivalenol exposure using an LC–MS/MS based biomarker method

The Fusarium toxin deoxynivalenol (DON) is one of the most abundant mycotoxins worldwide and poses many adverse health effects to human and animals. Consequently, regulatory limits and a provisional maximum tolerable daily intake (PMTDI) for this important type B-trichothecene were assigned. We conducted a pilot survey to investigate the level of DON exposure in Austrian adults by measurements of DON and its glucuronide conjugates (DON-GlcAs), as biomarkers of exposure, in first morning urine. The average concentration of total DON (free DON+DON-GlcAs) was estimated to be 20.4±2.4 μg L⁻¹ (max. 63 μg L⁻¹). Surprisingly, we found that one third of the volunteers (n=27) exceeded the established PMTDI when consuming regular diet. DON-GlcAs were directly quantified by LC-MS/MS and the results were compared with indirect quantification after enzymatic hydrolysis and confirmed the suitability of the direct method. Moreover, we investigated the in vivo metabolism of DON in humans and were able to determine two closely eluting DON-GlcAs in naturally contaminated urine samples for the first time. In contrast to previous findings we have tentatively identified DON-15-glucuronide as a major DON metabolite in human urine based on the analysis of these samples. About 75% of total glucuronides were derived from this metabolite while DON-3-glucuronide accounted for approximately 25%. The reported new findings clearly demonstrate the great potential of suitable biomarkers to critically assess exposure of humans and animals to DON.

Development and validation of a rapid multi‐biomarker liquid chromatography/tandem mass spectrometry method to assess human exposure to mycotoxins

RATIONALE Mycotoxins regularly occur in food worldwide and pose serious health risks to consumers. Since individuals can be exposed to a variety of these toxic secondary metabolites of fungi at the same time, there is a demand for proper analytical methods to assess human exposure by suitable biomarkers. METHODS This study reports on the development of a liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for the quantitative measurement of 15 mycotoxins and key metabolites in human urine using polarity switching. Deoxynivalenol (DON), DON-3-O-glucuronide, DON-15-O-glucuronide (D15GlcA), de-epoxy DON, nivalenol (NIV), T-2 toxin, HT-2 toxin, zearalenone, zearalenone-14-O-glucuronide, α- and β-zearalenol, fumonisins B(1) and B(2) (FB(1), FB(2)), ochratoxin A (OTA) and aflatoxin M(1) (AFM(1)) were determined without the need for any cleanup using a rapid and simple dilute and shoot approach. RESULTS Validation was performed in the range of 0.005-40 µg L(-1) depending on the analyte and expected urinary concentration levels. Apparent recoveries between 78 and 119% and interday precisions of 2-17% relative standard deviation (RSD) were achieved. The applicability of the method was demonstrated by the analysis of urine samples obtained from Cameroon. In naturally contaminated urine samples up to six biomarkers of exposure (AFM(1), DON, D15GlcA, NIV, FB(1), and OTA) were detected simultaneously. CONCLUSIONS We conclude that the developed LC/MS/MS method is well suited to quantify multiple mycotoxin biomarkers in human urine down to the sub-ppb range within 18 min and without any prior cleanup. The co-occurrence of several mycotoxins in the investigated samples clearly emphasizes the great potential and importance of this method to assess exposure of humans and animals to naturally occurring mycotoxins.

Metabolism of the Fusarium Mycotoxins T-2 Toxin and HT-2 Toxin in Wheat

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 diglucosylated forms of HT2, evidence for the formation of HT2-malonyl-glucoside and feruloyl-T2, as well as acetylation and deacetylation 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.

Synthesis of deoxynivalenol-3-ß-D-O-glucuronide for its use as biomarker for dietary deoxynivalenol exposure

Trichothecene mycotoxins are prevalent toxic secondary metabolic products of several fungal species and pose a serious threat to human and animal health. Deoxynivalenol (DON) is known to undergo ra...

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.

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.

Identification of a novel human deoxynivalenol metabolite enhancing proliferation of intestinal and urinary bladder cells

The mycotoxin deoxynivalenol (DON) is an abundant contaminant of cereal based food and a severe issue for global food safety. We report the discovery of DON-3-sulfate as a novel human metabolite and potential new biomarker of DON exposure. The conjugate was detectable in 70% of urine samples obtained from pregnant women in Croatia. For the measurement of urinary metabolites, a highly sensitive and selective LC-MS/MS method was developed and validated. The method was also used to investigate samples from a duplicate diet survey for studying the toxicokinetics of DON-3-sulfate. To get a preliminary insight into the biological relevance of the newly discovered DON-sulfates, in vitroexperiments were performed. In contrast to DON, sulfate conjugates lacked potency to suppress protein translation. However, surprisingly we found that DON-sulfates enhanced proliferation of human HT-29 colon carcinoma cells, primary human colon epithelial cells (HCEC-1CT) and, to some extent, also T24 bladder cancer cells. A proliferative stimulus, especially in tumorigenic cells raises concern on the potential impact of DON-sulfates on consumer health. Thus, a further characterization of their toxicological relevance should be of high priority.

A barley UDP-glucosyltransferase inactivates nivalenol and provides Fusarium Head Blight resistance in transgenic wheat

Highlight Barley HvUGT13248 catalyzes conversion of nivalenol (NIV) to its non-toxic form 3-O-glucoside, and in transgenic wheat confers resistance to NIV-producing Fusarium graminearum

Isolation and Structure Elucidation of Pentahydroxyscirpene, a Trichothecene Fusarium Mycotoxin

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.

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.