Enhancing the activity of zearalenone lactone hydrolase toward the more toxic α-zearalanol via a single-point mutation.

Zearalenone (ZEN) and its derivatives are estrogenic mycotoxins known to pose significant health threats to humans and animals. Especially, the derivative α-zearalanol (α-ZAL) is over 10 times more toxic than ZEN. Simultaneous degradation of ZEN and its derivatives, especially α-ZAL, using ZEN lactone hydrolases (ZHDs) is a promising solution to eliminate their potential hazards to food safety. However, most available ZHDs exhibit limited activity toward the more toxic α-ZAL compared to ZEN. Here, we identified a broad-substrate spectrum ZHD, named ZHDAY3, from Exophiala aquamarina CBS 119918, which could not only efficiently degrade ZEN but also exhibited 73% relative activity toward α-ZAL. Through rational design, we obtained the ZHDAY3(N153H) mutant, which exhibited the highest specific activity (253.3 ± 4.3 U/mg) reported so far for degrading α-ZAL. Molecular docking, structural comparative analysis, and kinetic analysis collectively suggested that the shorter distance between the side chain of the catalytic residue His242 and the lactone bond of α-ZAL and the increased binding affinity to the substrate were mainly responsible for the improved catalytic activity of ZHDAY3(N153H) mutant. This mechanism was further validated through additional molecular docking of 18 mutants and experimental verification of six mutants.IMPORTANCEThe mycotoxins zearalenone (ZEN) and its derivatives pose a significant threat to food safety. Here, we present a highly promising ZEN lactone hydrolase (ZHD), ZHDAY3, which is capable of efficiently degrading both ZEN and the more toxic derivative α-ZAL. Next, the ZHDAY3(N153H) mutant obtained by single-point mutation exhibited the highest specific activity for degrading α-ZAL reported thus far. We further elucidated the molecular mechanisms underlying the enhanced hydrolytic activity of ZHDAY3(N153H) toward α-ZAL. These findings represent the first investigation on the molecular mechanism of ZHDs against α-ZAL and are expected to provide a significant reference for further rational engineering of ZHDs, which will ultimately contribute to addressing the health risks and food safety issues posed by ZEN-like mycotoxins.

A mechanistic toxicology study to grasp the mechanics of zearalenone estrogenicity: Spotlighting aromatase and the effects of its genetic variability.

Zearalenone (ZEN) is a mycoestrogen produced by Fusarium fungi contaminating cereals and in grain-based products threatening human and animal health due to its endocrine disrupting effects. Germane to the mechanisms of action, ZEN may activate the estrogen receptors and inhibit the estrogens-producing enzyme aromatase (CYP19A1). Both show single nucleotide variants (SNVs) among humans associated with a diverse susceptibility of being activated or inhibited. These variations might modify the endocrine disrupting action of ZEN, requiring dedicated studies to improve its toxicological understanding. This work focused on human aromatase investigating via 3D molecular modelling whether some of the SNVs reported so far (n = 434) may affect the inhibitory potential of ZEN. It has been also calculated the inhibition capability of α-zearalenol, the most prominent and estrogenically potent phase I metabolite of ZEN, toward those aromatase variants with an expected diverse sensitivity of being inhibited by ZEN. The study: i) described SNVs likely associated with a different susceptibility to ZEN and α-zearalenol inhibition - like T310S that is likely more susceptible to inhibition, or D309G and S478F that are possibly inactive variants; ii) proofed the possible existence of inter-individual susceptibility to ZEN; iii) prioritized aromatase variants for future investigations toward a better comprehension of ZEN xenoestrogenicity at an individual level.

Metabolism of Zearalenone in the Rumen of Dairy Cows with and without Application of a Zearalenone-Degrading Enzyme.

The mycotoxin zearalenone (ZEN) is a frequent contaminant of animal feed and is well known for its estrogenic effects in animals. Cattle are considered less sensitive to ZEN than pigs. However, ZEN has previously been shown to be converted to the highly estrogenic metabolite α-zearalenol (α-ZEL) in rumen fluid in vitro. Here, we investigate the metabolism of ZEN in the reticulorumen of dairy cows. To this end, rumen-fistulated non-lactating Holstein Friesian cows (n = 4) received a one-time oral dose of ZEN (5 mg ZEN in 500 g concentrate feed) and the concentrations of ZEN and ZEN metabolites were measured in free rumen liquid from three reticulorumen locations (reticulum, ventral sac and dorsal mat layer) during a 34-h period. In all three locations, α-ZEL was the predominant ZEN metabolite and ß-zearalenol (ß-ZEL) was detected in lower concentrations. ZEN, α-ZEL and ß-ZEL were eliminated from the ventral sac and reticulum within 34 h, yet low concentrations of ZEN and α-ZEL were still detected in the dorsal mat 34 h after ZEN administration. In a second step, we investigated the efficacy of the enzyme zearalenone hydrolase ZenA (EC 3.1.1.-, commercial name ZENzyme®, BIOMIN Holding GmbH, Getzersdorf, Austria) to degrade ZEN to the non-estrogenic metabolite hydrolyzed zearalenone (HZEN) in the reticulorumen in vitro and in vivo. ZenA showed a high ZEN-degrading activity in rumen fluid in vitro. When ZenA was added to ZEN-contaminated concentrate fed to rumen-fistulated cows (n = 4), concentrations of ZEN, α-ZEL and ß-ZEL were significantly reduced in all three reticulorumen compartments compared to administration of ZEN-contaminated concentrate without ZenA. Upon ZenA administration, degradation products HZEN and decarboxylated HZEN were detected in the reticulorumen. In conclusion, endogenous metabolization of ZEN in the reticulorumen increases its estrogenic potency due to the formation of α-ZEL. Our results suggest that application of zearalenone hydrolase ZenA as a feed additive may be a promising strategy to counteract estrogenic effects of ZEN in cattle.

Oxidative stress, glutathione, and gene expression as key indicators in SH-SY5Y cells exposed to zearalenone metabolites and beauvericin.

The co-presence of mycotoxins from fungi of the genus Fusarium is a common fact in raw food and food products, as trace levels of them or their metabolites can be detected, unless safety practices during manufacturing are carried out. Zearalenone (ZEA), its metabolites α-zearalenol (α-ZEL) and ß-zearalenol (ß-ZEL) and, beauvericin (BEA) are co/present in cereals, fruits or their products which is a mixture that consumer are exposed and never evaluated in neuronal cells. In this study the role of oxidative stress and intracellular defense systems was assessed by evaluating reactive oxygen species (ROS) generation and glutathione (GSH) ratio activity in a human neuroblastoma cell line, SH-SY5Y cells, treated individually and combined with α-ZEL, ß-ZEL and BEA. It was further examined the expression of genes involved in cell apoptosis (CASP3, BAX, BCL2) and receptors of (endogenous or exogenous) estrogens (ERß and GPER1), by RT-PCR in those same conditions. These results demonstrated elevated ROS levels in combinations where α-ZEL was involved (2.8- to 8-fold compared to control); however, no significant difference in ROS levels were detected when single mycotoxin was tested. Also, the results revealed a significant increase in GSH/GSSG ratio at all concentrations after 24 h. Expression levels of CASP3 and BAX were up regulated by α-ZEL while CASP3 and BCL2 were down regulated by ß-ZEL, revealing how ZEA´s metabolites can induce the expression of cell apoptosis genes. However, BEA down-regulated the expression of BCL2. Moreover, ß-ZEL + BEA was the only combination treatment which was able to down regulate the levels of cell apoptosis gene expression. Relying to our findings, α-ZEL, ß-ZEL and BEA, induce injury in SH-SY5Y cells elevating oxidative stress levels, disturbing the antioxidant activity role of glutathione system and finally, causing disorder in the expressions and activities of the related apoptotic cell death genes.

Zearalenone and Metabolites in Livers of Turkey Poults and Broiler Chickens Fed with Diets Containing Fusariotoxins.

Zearalenone (ZEN) and metabolites were measured in livers of turkeys and broilers fed a control diet free of mycotoxins, a diet that contained 0.5 mg/kg ZEN (ZEN diet), and a diet that contained 0.5, 5, and 20 mg/kg of ZEN, fumonisins, and deoxynivalenol, respectively (ZENDONFB diet). The feed was individually distributed to male Grade Maker turkeys from the 55th to the 70th day of age and to male Ross chickens from the 1st to the 35th day of age, without any signs of toxicity. Together, the free and conjugated forms of ZEN, α- and ß-zearalenols (ZOLs), zearalanone (ZAN), and α- and ß-zearalanols (ZALs) were measured by UHPLC-MS/MS with [13C18]-ZEN as an internal standard and immunoaffinity clean-up of samples. ZAN and ZALs were not detected. ZEN and ZOLs were mainly found in their conjugated forms. α-ZOL was the most abundant and was found at a mean concentration of 2.23 and 1.56 ng/g in turkeys and chickens, respectively. Consuming the ZENDONFB diet significantly increased the level of total metabolites in the livers of chickens. Furthermore, this increase was more pronounced for the free forms of α-ZOL than for the conjugated forms. An investigation of the presence of ZEN and metabolites in muscle with the methods validated for the liver failed to reveal any traces of these contaminants in this tissue. These results suggest that concomitant dietary exposure to deoxynivalenol (DON) and fumonisins (FB) may alter the metabolism and persistence of ZEN and its metabolites in the liver.

Toxicokinetics of α-zearalenol and its masked form in rats and the comparative biotransformation in liver microsomes from different livestock and humans.

Alpha-zearalenol (α-ZEL) and its masked form α-zearalenol-14 glucoside (α-ZEL-14G) have much higher oestrogenic activity than zearalenone. Owing to very limited toxicokinetic and metabolic data, no reference points could be established for risk assessment. To circumvent it, the toxicokinetic, metabolic profiles, and phenotyping of α-ZEL and α-ZEL-14G were comprehensively investigated in this study. As a result, the plasma concentrations of α-ZEL and α-ZEL-14G were all below LOQ after oral administration, while after iv injection, both could be significantly bio-transformed into various metabolites. A complete hydrolysis of α-ZEL-14G contributed to α-ZEL overall toxicity. Additionally, 31 phase I and 10 phase II metabolites of α-ZEL, and 9 phase I and 5 phase II metabolites were identified for α-ZEL-14G. For α-ZEL, hydroxylation, dehydrogenation, and glucuronidation were the major metabolic pathways, while for α-ZEL-14G, it was deglycosylation, reduction, hydroxylation, and glucuronidation. Significant metabolic differences were observed for α-ZEL and α-ZEL-14G in the liver microsomes of rats, chickens, swine, goats, cows and humans. Phenotyping studies indicated that α-ZEL and α-ZEL-14G were mediated by CYP 3A4, 2C8, and 1A2. Moreover, the deglycosylation of α-ZEL-14G was critically mediated by CES-I and CES-II. The acquired data would provide fundamental perspectives for risk evaluation of mycotoxins and their modified forms.

Evaluation of the epigenetic alterations and gene expression levels of HepG2 cells exposed to zearalenone and α-zearalenol.

Zearalenone, produced by various Fusarium species, is a non-steroidal estrogenic mycotoxin that contaminates cereals, resulting in adverse effects on human health. We investigated the effects of zearalenone and its metabolite alpha zearalenol on epigenetic modifications and its relationship with metabolic pathways in human hepatocellular carcinoma cells following 24 h of exposure. Zearalenone and alpha zearalenol at the concentrations of 1, 10 and 50 µM significantly increased global levels of DNA methylation and global histone modifications (H3K27me3, H3K9me3, H3K9ac). Expression levels of the chromatin modifying enzymes EHMT2, ESCO1, HAT1, KAT2B, PRMT6 and SETD8 were upregulated by 50 µM of zearalenone exposure using PCR arrays, consistent with the results of global histone modifications. Zearalenone and alpha zearalenol also changed expression levels of the AhR, LXRα, PPARα, PPARÉ£, L-fabp, LDLR, Glut2, Akt1 and HK2 genes, which are related to nuclear receptors and metabolic pathways. PPARÉ£, a key regulator of lipid metabolism, was selected from among these genes for further analysis. The PPARÉ£ promoter reduced methylation significantly following zearalenone exposure. Taken together, the epigenetic mechanisms of DNA methylation and histone modifications may be key mechanisms in zearalenone toxicity. Furthermore, effects of zearalenone in metabolic pathways could be mediated by epigenetic modifications.

Identification of a Potent Enzyme for the Detoxification of Zearalenone.

The occurrence of mycotoxin zearalenone (ZEN) and its derivatives has been a severe global threat to food and animals. In addition to the chemical and physical degradation methods, a powerful biocatalyst is urgently required for the detoxification of ZEN. Here, an efficient ZEN-degrading lactonase from Gliocladium roseum, named ZENG, was identified for the first time. The recombinant ZENG exhibited the highest activity at pH 7.0 and 38 °C. In addition, the recombinant enzyme showed a high degrading performance toward ZEN and its toxic derivatives α-zearalenol (α-ZOL) and α-zearalanol (α-ZAL), with the specific activities as 315, 187, and 117 units/mg, respectively. To meet the industrial demands, attempts were also made to enhance the thermostability of ZENG using a structure-based modification. Three double-site mutants, including H134L/S136L, H134F/S136F, and H134I/S134I, in the position between the cap and core catalytic domain of ZENG were designed. Finally, the thermostability of both H134L/S136L and H134F/S136F displayed a significant improvement compared to the wild-type enzyme.

Glucuronidation as a metabolic barrier against zearalenone in rat everted intestine.

Zearalenone (ZON), produced by Fusarium fungi, exhibits estrogenic activity. Livestock can be exposed to ZON orally through contaminating feeds such as cereals, leading to reproductive disorders such as infertility and miscarriage via endocrine system disruption. However, the details of ZON metabolism remain unclear, and the mechanism of its toxicity has not been fully elucidated. In this study, we investigated the kinetics of ZON absorption and metabolism in rat segmented everted intestines. ZON absorption was confirmed in each intestine segment 60 min after application to the mucosal buffer at 10 µM. Approximately half of the absorbed ZON was metabolized to α-zearalenol, which tended to be mainly glucuronidated in intestinal cells. In the proximal intestine, most of the glucuronide metabolized by intestinal cells was excreted to the mucosal side, suggesting that the intestine plays an important role as a first drug metabolism barrier for ZON. However, in the distal intestine, ZON metabolites tended to be transported to the serosal side. Glucuronide transported to the serosal side could be carried via the systemic circulation to the local tissues, where it could be reactivated by deconjugation. These results are important with regard to the mechanism of endocrine disruption caused by ZON.

The Trp183 is essential in lactonohydrolase ZHD detoxifying zearalenone and zearalenols.

Lactonohydrolase ZHD can detoxify oestrogenic mycotoxin zearalenone and zearalenols through hydrolysis and decarboxylation. The detail mechanism, especially the role of Trp183, which interacts with substrate through p-π interaction and one hydrogen bond, is still unknown. The Trp183 mutants abolished activity to ZEN, α-ZOL and ß-ZOL, except that W183F mutant retained about 40% activity against α-ZOL. In two W183F-reactant complex structures the reactants still bind at the active position and it suggested that this p-π interaction takes responsible for the reactants recognization and allocation. Further, the ZHD-productant complex structures showed that the resorcinol ring of hydrolysed α-ZOL and hydrolysed ß-ZOL move a distance of one ring as compare to the resorcinol ring of reactant α-ZOL and ß-ZOL. The same movement also found in comparison of hydrolysed ZEN and ZEN. In the structure of W183F complex with hydrolysed α-ZOL the resorcinol ring of hydrolysed α-ZOL doesn't move as compare to the resorcinol ring of reactant α-ZOL. It suggested the Trp183 coordinated hydrogen bond takes responsible for the movement of the hydrolysed product. These functional and structural results suggested that Trp183 is essential for ZHD detoxifying zearalenone and zearalenols.

Manganese protects wheat from the mycotoxin zearalenone and its derivatives.

Searching for factors that reduce zearalenone (ZEN) toxicity is an important challenge in wheat production, considering that this crop is a basic dietary ingredient. ZEN, absorbed by cells, is metabolized into α-zearalenol and α-zearalanol, and this study focused on the function of manganese ions as potential protectants against the mycotoxins. Stress effects were invoked by an application of 30 µM ZEN and its derivatives. Manganese ions were applied at 100 µM, not stress-inducing concentration. Importance of the biomembrane structures in the absorption of the mycotoxins was demonstrated in in vitro wheat calli and on model membranes. ZEN showed the greatest and α-zearalanol the smallest stressogenic effect manifested as a decrease in the calli growth. This was confirmed by variable increase in antioxidant enzyme activity. Mn ions added to the toxin mixture diminished stressogenic properties of the toxins. Variable decrease in total lipid content and the percentage of phospholipid fraction detected in calli cells exposed to ZEN and its metabolites indicated significance of the membrane structure. An analysis of physicochemical parameters of model membranes build from phosphatidylcholine, a basic lipid in native membranes, and its mixture with the tested toxins made by Langmuir technique and verified by Brewster angle microscopy, confirmed variable contribution of ZEN and its derivatives to the modification of membrane properties. The order of toxicity was as follows: ZEN ≥ α-zearalenol > α-zearalanol. Manganese ions present in the hydrophilic phase interacted with polar lipid groups and reduced the extent of membrane modification caused by the mycotoxins.

Formation of Zearalenone Metabolites in Tempeh Fermentation.

Tempeh is a common food in Indonesia, produced by fungal fermentation of soybeans using Rhizopus sp., as well as Aspergillus oryzae, for inoculation. Analogously, for economic reasons, mixtures of maize and soybeans are used for the production of so-called tempeh-like products. For maize, a contamination with the mycoestrogen zearalenone (ZEN) has been frequently reported. ZEN is a mycotoxin which is known to be metabolized by Rhizopus and Aspergillus species. Consequently, this study focused on the ZEN transformation during tempeh fermentation. Five fungal strains of the genera Rhizopus and Aspergillus, isolated from fresh Indonesian tempeh and authentic Indonesian inocula, were utilized for tempeh manufacturing from a maize/soybean mixture (30:70) at laboratory-scale. Furthermore, comparable tempeh-like products obtained from Indonesian markets were analyzed. Results from the HPLC-MS/MS analyses show that ZEN is intensely transformed into its metabolites α-zearalenol (α-ZEL), ZEN-14-sulfate, α-ZEL-sulfate, ZEN-14-glucoside, and ZEN-16-glucoside in tempeh production. α-ZEL, being significantly more toxic than ZEN, was the main metabolite in most of the Rhizopus incubations, while in Aspergillus oryzae fermentations ZEN-14-sulfate was predominantly formed. Additionally, two of the 14 authentic samples were contaminated with ZEN, α-ZEL and ZEN-14-sulfate, and in two further samples, ZEN and α-ZEL, were determined. Consequently, tempeh fermentation of ZEN-contaminated maize/soybean mixture may lead to toxification of the food item by formation of the reductive ZEN metabolite, α-ZEL, under model as well as authentic conditions.

ERK activation by zeranol has neuroprotective effect in cerebral ischemia reperfusion.

AIMS: Incidence of stroke increases in postmenopausal women with dangerous consequences. In this study we used zeranol to protect ovariectomized (OVX) rats against cerebral I/R damage and our target is to identify the mechanism of its protection, in addition to investigating whether this mechanism inhibits inflammation (by preventing glial cell activation) and apoptosis. MAIN METHODS: First 18 ovariectomized rats were allocated into 3 groups: I/R group, zeranol+ I/R group and U0126, MEK1/2 inhibitor + zeranol+ I/R group. After 24 h reperfusion, protein expression of total extracellular signal-regulated protein kinase (t-ERK1/2), phosphorylated extracellular signal-regulated protein kinase (p-ERK1/2), Bcl-2, and Bax were quantified. Second 36 female rats were allocated into 3 groups: sham group, I/R group (after ovariectomy by 7 weeks, rats exposed to cerebral I/R) and zeranol group (after ovariectomy by 2 weeks, rats received zeranol for 5 weeks). After 24 h of reperfusion, the following parameters were measured; total nitrate/nitrite, interleukin-10, myeloperoxidase, caspase-3, and finally immunohistochemistry analysis of glial fibrillary acidic protein, cyclooxygenase-2 in cortex and hippocampus (CA1) regions were performed. KEY FINDINGS: U-0126 administration reversed the neuroprotective effect induced by zeranol through decreasing ratio of p-ERK1/2:ERK1/2 and Bcl-2/Bax in brain tissue. Activation of ERK signaling pathway by zeranol caused reduction in brain apoptosis and inflammation. SIGNIFICANCE: Zeranol showed protective effect in OVX rats that were exposed to cerebral I/R by activation of ERK signaling pathway which was blocked by U0126. This protective effect in turns led to decrease inflammation and apoptosis.

Insights into In Vivo Absolute Oral Bioavailability, Biotransformation, and Toxicokinetics of Zearalenone, α-Zearalenol, ß-Zearalenol, Zearalenone-14-glucoside, and Zearalenone-14-sulfate in Pigs.

The aim of this study was to determine the toxicokinetic characteristics of ZEN and its modified forms, α-zearalenol (α-ZEL), ß-zearalenol (ß-ZEL), zearalenone-14-glucoside (ZEN14G), and zearalenone-14-sulfate (ZEN14S), including presystemic and systemic hydrolysis in pigs. Crossover pig trials were performed by means of intravenous and oral administration of ZEN and its modified forms. Systemic plasma concentrations of the administered toxins and their metabolites were quantified and further processed via tailor-made compartmental toxicokinetic models. Furthermore, portal plasma was analyzed to unravel the site of hydrolysis, and urine samples were analyzed to determine urinary excretion. Results demonstrate complete presystemic hydrolysis of ZEN14G and ZEN14S to ZEN and high oral bioavailability for all administered compounds, with further extensive first-pass glucuronidation. Conclusively, the modified-ZEN forms α-ZEL, ß-ZEL, ZEN14G, and ZEN14S contribute to overall ZEN systemic toxicity in pigs and should be taken into account for risk assessment.

Rational Reprogramming of O-Methylation Regioselectivity for Combinatorial Biosynthetic Tailoring of Benzenediol Lactone Scaffolds.

O-Methylation modulates the pharmacokinetic and pharmacodynamic (PK/PD) properties of small-molecule natural products, affecting their bioavailability, stability, and binding to targets. Diversity-oriented combinatorial biosynthesis of new chemical entities for drug discovery and optimization of known bioactive scaffolds during drug development both demand efficient O-methyltransferase (OMT) biocatalysts with considerable substrate promiscuity and tunable regioselectivity that can be deployed in a scalable and sustainable manner. Here we demonstrate efficient total biosynthetic and biocatalytic platforms that use a pair of fungal OMTs with orthogonal regiospecificity to produce unnatural O-methylated benzenediol lactone polyketides. We show that rational, structure-guided active-site cavity engineering can reprogram the regioselectivity of these enzymes. We also characterize the interplay of engineered regioselectivity with substrate plasticity. These findings will guide combinatorial biosynthetic tailoring of unnatural products toward the generation of diverse chemical matter for drug discovery and the PK/PD optimization of bioactive scaffolds for drug development.

Theoretical Study on Zearalenol Compounds Binding with Wild Type Zearalenone Hydrolase and V153H Mutant.

Zearalenone hydrolase (ZHD) is the only reported α/ß-hydrolase that can detoxify zearalenone (ZEN). ZHD has demonstrated its potential as a treatment for ZEN contamination that will not result in damage to cereal crops. Recent researches have shown that the V153H mutant ZHD increased the specific activity against α-ZOL, but decreased its specific activity to ß-ZOL. To understand whyV153H mutation showed catalytic specificity for α-ZOL, four molecular dynamics simulations combining with protein network analysis for wild type ZHD α-ZOL, ZHD ß-ZOL, V153H α-ZOL, and V153H ß-ZOL complexes were performed using Gromacs software. Our theoretical results indicated that the V153H mutant could cause a conformational switch at the cap domain (residues Gly161⁻Thr190) to affect the relative position catalytic residue (H242). Protein network analysis illustrated that the V153H mutation enhanced the communication with the whole protein and residues with high betweenness in the four complexes, which were primarily assembled in the cap domain and residues Met241 to Tyr245 regions. In addition, the existence of α-ZOL binding to V153H mutation enlarged the distance from the OAE atom in α-ZOL to the NE2 atom in His242, which prompted the side chain of H242 to the position with catalytic activity, thereby increasing the activity of V153H on the α-ZOL. Furthermore, α-ZOL could easily form a right attack angle and attack distance in the ZHD and α-ZOL complex to guarantee catalytic reaction. The alanine scanning results indicated that modifications of the residues in the cap domain produced significant changes in the binding affinity for α-ZOL and ß-ZOL. Our results may provide useful theoretical evidence for the mechanism underlying the catalytic specificity of ZHD.

Interaction of α- and ß-zearalenols with ß-cyclodextrins.

Zearalenone (ZEN) is a mycotoxin produced by Fusarium fungi. ZEN primarily contaminates different cereals, and exerts a strong xenoestrogenic effect in animals and humans. ZEN is a fluorescent mycotoxin, although molecular interactions and microenvironmental changes significantly modify its spectral properties. During biotransformation, ZEN is converted into α-zearalenol (α-ZOL) and ß-zearalenol (ß-ZOL), the toxic metabolites of ZEN, which mimick the effect of estrogen. Cyclodextrins (CDs) are host molecules, and have been studied extensively; they can form stable complexes with several mycotoxins, including ZEN. However, information is limited regarding the interactions of CDs with ZOLs. Therefore, we studied the interactions of α- and ß-ZOLs with native and six chemically modified ß-CDs by fluorescence spectroscopy. Fluorescence enhancement during complex formation, as well as binding constants, were determined. To understand ZOL-CD interactions better, molecular modeling studies were also carried out. Both mycotoxin derivatives formed the most stable complexes with methylated and sulfobutylated CD-derivatives; however, the CD complexes of α-ZOL were significantly stronger than those of ß-ZOL. The data presented here indicate which of the chemically modified ß-CDs appear more suitable as fluorescence enhancers or as potential mycotoxin binders.

Metabolism of Zearalenone and Its Major Modified Forms in Pigs.

The Fusarium mycotoxin zearalenone (ZEN) can be conjugated with polar molecules, like sugars or sulfates, by plants and fungi. To date, the fate of these modified forms of ZEN has not yet been elucidated in animals. In order to investigate whether ZEN conjugates contribute to the total ZEN exposure of an individual, ZEN (10 µg/kg b.w.) and equimolar amounts of two of its plant metabolites (ZEN-14-O-ß-glucoside, ZEN-16-O-ß-glucoside) and of one fungal metabolite (ZEN-14-sulfate) were orally administered to four pigs as a single bolus using a repeated measures design. The concentrations of ZEN, its modified forms and its mammalian metabolites ZEN-14-glucuronide, α-zearalenol (α-ZEL) and α-ZEL-14-glucuronide in excreta were analyzed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) based methods. The biological recovery of ZEN in urine was 26% ± 10%, the total biological recovery in excreta was 40% ± 8%. Intact ZEN-14-sulfate, ZEN-14-O-ß-glucoside and ZEN-16-O-ß-glucoside were neither detected in urine nor in feces. After ZEN-14-sulfate application, 19% ± 5% of the administered dose was recovered in urine. In feces, no ZEN metabolites were detected. The total biological recoveries of ZEN-14-O-ß-glucoside and ZEN-16-O-ß-glucoside in the form of their metabolites in urine were 19% ± 11% and 13% ± 7%, respectively. The total biological recoveries in urine and feces amounted to 48% ± 7% and 34 ± 3%. An explanation for the low biological recoveries could be extensive metabolization by intestinal bacteria to yet unknown metabolites. In summary, ZEN-14-sulfate, ZEN-14-O-ß-glucoside, and ZEN-16-O-ß-glucoside were completely hydrolyzed in the gastrointestinal tract of swine, thus contributing to the overall toxicity of ZEN.

Reaction of zearalenone and α-zearalenol with allyl isothiocyanate, characterization of reaction products, their bioaccessibility and bioavailability in vitro.

This study investigates the reduction of zearalenone (ZEA) and α-zearalenol (α-ZOL) on a solution model using allyl isothiocyanate (AITC) and also determines the bioaccessibility and bioavailability of the reaction products isolated and identified by MS-LIT. Mycotoxin reductions were dose-dependent, and ZEA levels decreased more than α-ZOL, ranging from 0.2 to 96.9% and 0 to 89.5% respectively, with no difference (p⩽0.05) between pH 4 and 7. Overall, simulated gastric bioaccessibility was higher than duodenal bioaccessibility for both mycotoxins and mycotoxin-AITC conjugates, with duodenal fractions representing ⩾63.5% of the original concentration. Simulated bioavailability of reaction products (α-ZOL/ZEA-AITC) were lower than 42.13%, but significantly higher than the original mycotoxins. The cytotoxicity of α-ZOL and ZEA in Caco-2/TC7 cells was also evaluated, with toxic effects observed at higher levels than 75µM. Further studies should be performed to evaluate the toxicity and estrogenic effect of α-ZOL/ZEA-AITC.

Masked trichothecene and zearalenone mycotoxins withstand digestion and absorption in the upper GI tract but are efficiently hydrolyzed by human gut microbiota in vitro.

SCOPE: Cereal grains are commonly contaminated with Fusarium mycotoxins and their plant-derived masked metabolites. The fate of masked mycotoxins in the human gut is poorly understood. Here we assess the metabolism and transport of glucoside metabolites of common trichothecenes (deoxynivalenol, nivalenol, T-2 toxin) and zearalenone compounds (zearalenone, α- and ß-zearalenol) in the human gut in vitro. METHODS AND RESULTS: Masked mycotoxins were incubated with artificial digestive juices and absorption was assessed in differentiated Caco-2/TC7 cells. Colonic metabolism was studied using fecal batch cultures from five donors and mycotoxins were detected using LC-MS/MS. All masked mycotoxins were stable under upper GI tract conditions and no absorption was observed. Free trichothecenes were absorbed intact whereas free zearalenone compounds were absorbed and metabolized to undetected compounds by Caco-2/TC7 cells. Human gut microbiota efficiently hydrolyzed all masked mycotoxins. Trichothecenes were fully recovered as parent mycotoxins whereas 40-70% of zearalenone compounds were further metabolized to unknown metabolites. CONCLUSION: Our results demonstrate that masked trichothecenes will reach the colon intact to be released as parent mycotoxins by gut microbiota, hence contributing to mycotoxin exposure. Masked zearalenone compounds are metabolized by gut microbiota and epithelial cells and the identity and toxicity of metabolites remain to be determined.