Heidi Schwartz

Metabolism of the masked mycotoxin deoxynivalenol-3-glucoside in rats

Highlights ► The metabolism of deoxynivalenol-3-glucoside (D3G) in rats was studied. ► Urine and feces were analyzed by a validated LC–MS/MS biomarker method. ► D3G was readily hydrolyzed to deoxynivalenol (DON) during digestion. ► Most D3G was metabolized by the gut microbiota and recovered in feces. ► D3G is of considerably lower toxicological relevance than DON, at least in rats.

The low intestinal and hepatic toxicity of hydrolyzed fumonisin B1 correlates with its inability to alter the metabolism of sphingolipids

Fumonisins are mycotoxins frequently found as natural contaminants in maize, where they are produced by the plant pathogen Fusarium verticillioides. They are toxic to animals and exert their effects through mechanisms involving disruption of sphingolipid metabolism. Fumonisin B₁ (FB₁) is the predominant fumonisin in this family. FB₁ is converted to its hydrolyzed analogs HFB₁, by alkaline cooking (nixtamalization) or through enzymatic degradation. The toxicity of HFB₁ is poorly documented especially at the intestinal level. The objectives of this study were to compare the toxicity of HFB₁ and FB₁ and to assess the ability of these toxins to disrupt sphingolipids biosynthesis. HFB₁ was obtained by a deesterification of FB₁ with a carboxylesterase. Piglets, animals highly sensitive to FB₁, were exposed by gavage for 2 weeks to 2.8 μmol FB₁ or HFB₁/kg body weight/day. FB₁ induced hepatotoxicity as indicated by the lesion score, the level of several biochemical analytes and the expression of inflammatory cytokines. Similarly, FB₁ impaired the morphology of the different segments of the small intestine, reduced villi height and modified intestinal cytokine expression. By contrast, HFB₁ did not trigger hepatotoxicity, did not impair intestinal morphology and slightly modified the intestinal immune response. This low toxicity of HFB₁ correlates with a weak alteration of the sphinganine/sphingosine ratio in the liver and in the plasma. Taken together, these data demonstrate that HFB₁ does not cause intestinal or hepatic toxicity in the sensitive pig model and only slightly disrupts sphingolipids metabolism. This finding suggests that conversion to HFB₁ could be a good strategy to reduce FB₁ exposure.

Survey of deoxynivalenol and its conjugates deoxynivalenol-3-glucoside and 3-acetyl-deoxynivalenol in 374 beer samples

Beer is one of the most popular beverages worldwide. Malted cereal grains are among the basic ingredients and hence mycotoxin contamination might occur. Previous studies reported the presence of the Fusarium mycotoxins deoxynivalenol (DON) and 3-acetyl-deoxynivalenol (3ADON), as well as of the masked mycotoxin deoxynivalenol-3-glucoside (D3G) in beer. In the present survey, 374u2009beer samples from 38u2009countries with a focus on Austrian (156) and German (64) beers were analysed for the presence of D3G, DON and 3ADON. Beers were assigned to the following six categories: pale (217), wheat (46), dark (47), bock (20), nonalcoholic beers (19) and shandies (25). In total, 348 and 289 beers (93 and 77%, respectively) contained D3G and DON at the levels above the limit of detection, whereas 3ADON was not detected in any of the samples. Average concentrations of all beers were 6.9u2009µgu2009L−1 for D3G and 8.4u2009µgu2009L−1 in the case of DON. Nonalcoholic beers and shandies showed the lowest contaminations, 1.5 and 3.2u2009µgu2009L−1 for D3G and 2.7 and 4.4u2009µgu2009L−1 for DON, respectively. In bock beers characterised by a higher gravity, a significant trichothecene load of 14.8u2009µgu2009L−1 D3G and 12.4u2009µgu2009L−1 DON was found. The highest contamination (81u2009µgu2009L−1 D3G, 89u2009µgu2009L−1 DON) was detected in a pale beer from Austria, underlining the importance of this study for food safety. The molar D3G to DON ratio ranged between 0.11 and 1.25 and was 0.56 on average. Concluding, the average contamination of beer is not of toxicological concern for moderate beer drinkers. However, in the case of heavy beer drinkers, beer consumption may considerably contribute to the overall intake of DON, which might even lead to exceeding the maximum tolerable limits established for this Fusarium toxin.

Characterization of Three Deoxynivalenol Sulfonates Formed by Reaction of Deoxynivalenol with Sulfur Reagents

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-HRMS/MS as well as NMR spectroscopy. Compound 1 is characterized by loss of the epoxide group, and 2 by formation of a hemiketal. Compound 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.

Biotransformation approaches to alleviate the effects induced by fusarium mycotoxins in swine.

Mycotoxin mitigation is of major interest as ingestion of mycotoxins results in poor animal health, decreased productivity, as well as substantial economic losses. A feed additive (FA) consisting of a combination of bacteria (Eubacterium BBSH797) and enzyme (fumonisin esterase FumD) was tested in pigs for its ability to neutralize the effects of mono- and co-contaminated diets with deoxynivalenol (DON) and fumonisins (FB) on hematology, biochemistry, tissue morphology, and immune response. Forty-eight animals, allocated into eight groups, received one of eight diets for 35 days: a control diet, a diet contaminated with either DON (3 mg/kg) or FB (6 mg/kg), or both toxins, and the same four diets with FA. Inclusion of FA restored the circulating number of neutrophils of piglets fed the FB and DON + FB diets. Similarly, FA counteracted the minor changes observed on plasma concentrations of albumin and creatinine. In lung, the lesions induced by the ingestion of FB in mono- and co-contaminated diets were no longer observed after addition of FA in these diets. Lesions recorded in the liver of pigs fed either of the contaminated diets with FA were partly reduced, and the increased hepatocyte proliferation was totally neutralized when FA was present in the co-contaminated diet. After 35 days of exposure, the development of the vaccinal response was significantly improved in animals fed diets supplemented with FA, as shown by results of lymphocyte proliferation, cytokine expression in spleen, and the production of specific Ig. Similarly, in jejunum of animals fed diets with FA, occurrence of lesions and upregulation of pro-inflammatory cytokines were much less obvious. The ameliorative effects provided by FA suggest that this approach would be suitable in the control of DON and FB that commonly co-occur in feed.

Yeast cell based feed additives: studies on aflatoxin B1 and zearalenone

Thirty commercially available yeast cell wall products and two reference bentonites were tested for their ability to bind aflatoxin B1 (AFB1) and zearalenone (ZON) in buffer solutions at pH 3 and pH 6.5 as well as in real gastric juice. For most products, the binding efficacy of AFB1 correlated with the ash content, which was between 2.6 and 89%, and constituted the inorganic non-volatile components, such as mineral clays, of the samples. Samples with smectite as the main ash component showed the highest binding efficacy; yet, a correlation with the content of mannanooligosaccharides (MOS) and β-glucans from yeast cell walls was not observed. Products containing >30% ash showed AFB1 adsorption values >90% at least in one of the investigated media whereas most products with <10% ash did not exceed adsorption rates of 20%. In the case of ZON, adsorption efficiency ranged between 10 and 60%. It tended to be lowest for products with MOS and β-glucan contents <10% and greatest for products with MOS and β-glucan contents >50%. However, there was no general correlation between the adsorption of ZON and the concentration of MOS and β-glucans. Different products of one brand sold in different countries were observed to bind AFB1 to different degrees, which was explained by the difference in ash contents and mineral composition. In the case of ZON, differences in adsorption between products of the same brand were less pronounced.

Enzyme characteristics of aminotransferase FumI of Sphingopyxis sp. MTA144 for deamination of hydrolyzed fumonisin B1

Fumonisins are carcinogenic mycotoxins that are frequently found as natural contaminants in maize from warm climate regions around the world. The aminotransferase FumI is encoded as part of a gene cluster of Sphingopyxis sp. MTA144, which enables this bacterial strain to degrade fumonisin B1 and related fumonisins. FumI catalyzes the deamination of the first intermediate of the catabolic pathway, hydrolyzed fumonisin B1. We used a preparation of purified, His-tagged FumI, produced recombinantly in Escherichia coli in soluble form, for enzyme characterization. The structure of the reaction product was studied by NMR and identified as 2-keto hydrolyzed fumonisin B1. Pyruvate was found to be the preferred co-substrate and amino group receptor (KMu2009=u2009490xa0μM at 10xa0μM hydrolyzed fumonisin B1) of FumI, but other α-keto acids were also accepted as co-substrates. Addition of the co-enzyme pyridoxal phosphate to the enzyme preparation enhanced activity, and saturation was already reached at the lowest tested concentration of 10xa0μM. The enzyme showed activity in the range of pHxa06 to 10 with an optimum at pHxa08.5, and in the range of 6°C to 50°C with an optimum at 35°C. The aminotransferase worked best at low salt concentration. FumI activity could be recovered after preincubation at pHxa04.0 or higher, but not lower. The aminotransferase was denatured after preincubation at 60°C for 1xa0h, and the residual activity was also reduced after preincubation at lower temperatures. At optimum conditions, the kinetic parameters KMu2009=u20091.1xa0μM and kcatu2009=u2009104/min were determined with 5xa0mM pyruvate as co-substrate. Based on the enzyme characteristics, a technological application of FumI, in combination with the fumonisin carboxylesterase FumD for hydrolysis of fumonisins, for deamination and detoxification of hydrolyzed fumonisins seems possible, if the enzyme properties are considered.

Enhancement of solubility in Escherichia coli and purification of an aminotransferase from Sphingopyxis sp. MTA144 for deamination of hydrolyzed fumonisin B1

BackgroundFumonisin B1 is a cancerogenic mycotoxin produced by Fusarium verticillioides and other fungi. Sphingopyxis sp. MTA144 can degrade fumonisin B1, and a key enzyme in the catabolic pathway is an aminotransferase which removes the C2-amino group from hydrolyzed fumonisin B1. In order to study this aminotransferase with respect to a possible future application in enzymatic fumonisin detoxification, we attempted expression of the corresponding fumI gene in E. coli and purification of the enzyme. Since the aminotransferase initially accumulated in inclusion bodies, we compared the effects of induction level, host strain, expression temperature, solubility enhancers and a fusion partner on enzyme solubility and activity.ResultsWhen expressed from a T7 promoter at 30°C, the aminotransferase accumulated invariably in inclusion bodies in DE3 lysogens of the E. coli strains BL21, HMS174, Rosetta 2, Origami 2, or Rosetta-gami. Omission of the isopropyl-beta-D-thiogalactopyranoside (IPTG) used for induction caused a reduction of expression level, but no enhancement of solubility. Likewise, protein production but not solubility correlated with the IPTG concentration in E. coli Tuner(DE3). Addition of the solubility enhancers betaine and sorbitol or the co-enzyme pyridoxal phosphate showed no effect. Maltose-binding protein, used as an N-terminal fusion partner, promoted solubility at 30°C or less, but not at 37°C. Low enzyme activity and subsequent aggregation in the course of purification and cleavage indicated that the soluble fusion protein contained incorrectly folded aminotransferase. Expression in E. coli ArcticExpress(DE3), which co-expresses two cold-adapted chaperonins, at 11°C finally resulted in production of appreciable amounts of active enzyme. Since His tag-mediated affinity purification from this strain was hindered by co-elution of chaperonin, two steps of chromatography with optimized imidazole concentration in the binding buffer were performed to obtain 1.45 mg of apparently homogeneous aminotransferase per liter of expression culture.ConclusionsWe found that only reduction of temperature, but not reduction of expression level or fusion to maltose-binding protein helped to produce correctly folded, active aminotransferase FumI in E. coli. Our results may provide a starting point for soluble expression of related aminotransferases or other aggregation-prone proteins in E. coli.

Development, validation and application of an LC-MS/MS based method for the determination of deoxynivalenol and its conjugates in different types of beer

After water and tea, beer is the third most popular beverage worldwide. Brewed from malted cereal grains, beer is known to be potentially contaminated with mycotoxins. Some studies have shown that not only the Fusarium mycotoxins deoxynivalenol (DON) and 3-acetyl-DON (3-ADON), but also the conjugated mycotoxin deoxynivalenol-3-glucoside (D3G) can be found in beer on a regular basis, albeit usually at low concentrations. The aim of this work was to develop the first triple quadrupole LC-MS/MS based method for the determination of DON, D3G and 3-ADON in beer and to perform an in-house validation. The simple sample preparation includes degassing, precipitation of matrix compounds and reconstitution of the dried-down sample in solvent. Since different kinds of beer exist and method performance parameters will likely differ, we categorised the samples into pale, wheat, dark, bock and non-alcoholic beers, as well as shandies, and validated all six matrices. Although three individual beers for each category were...

Simultaneous preparation of α/β-zearalenol glucosides and glucuronides

An improved and reproducible procedure for the preparation of four different glycosides of the mycotoxins α- and β-zearalenol (α,β-ZEL), both metabolites of the Fusarium toxin zearalenone (ZEN), is reported. These conjugated or masked mycotoxins are formed during phase II metabolism in plants (glucosides) or animals and humans (glucuronides). Improved regioselective Königs-Knorr glucuronidation was applied to ZEN followed by reduction of the keto group of the mycotoxin, leading to α- and β-configuration of ZEL and also to a partial reduction of the glucuronic acid methyl ester to obtain the corresponding glucosides. After deprotection of the sugar moiety, α- and β-zearalenol-14-β,D-glucuronide as well as the corresponding glucosides were isolated at once using preparative HPLC. The reduction step was studied under different reaction conditions to finally develop an optimized and also tunable procedure for the first simultaneous preparation of both, glucosides and glucuronides of a xenobiotic substance in reasonable amounts to be used as reference materials for bioanalytical and toxicological investigations.