Further insight into the potential toxicity of zearalenone-14-glucoside based on toxicokinetics, tissue distribution, transformation, and excretion in rats.

Bioaccumulation and biotransformation are critical factors that affect the release of easily metabolizable chemicals to cause human toxicity. The glucoside-type modified mycotoxin Zearalenone-14-Glucoside (Z14G) has attracted global attention for its high occurrence in foodstuffs and the potential threat to humans as its high rate of transformation into parent forms. Given the limited toxicokinetics information, this study assessed the absorption, distribution, biotransformation and excretion of Z14G, aiming to define the potential risk of Z14G. The toxicokinetics of Z14G were assessed after intravenous (IV) or oral administration (PO) in SD rats at doses of 10 mg/kg·b.w. In addition, comparative work with the parent mycotoxin ZEN was performed in parallel. The determination of Z14G and its metabolites (ZEN, α-zearalenol, ß-zearalenol, α-zearalanol, ß-zearalanol) proceeded with a sensitive UHPLC-MS/MS method. Our research indicated that Z14G readily disappeared from the blood, and distributed throughout the tissues via transformation into its parent form ZEN, and excreted primarily through urine. More importantly, the metabolite α-ZEL was observed in most analyzed tissue, urine and feces samples. Overall, our findings highlight the importance of biotransformation with regard to Z14G, providing critical insight for the health risk assessment of co-exposure of humans to glucoside-type modified mycotoxins.

Deglucosylation of zearalenone-14-glucoside in animals and human liver leads to underestimation of exposure to zearalenone in humans.

Zearalenone-14-glucoside (ZEN-14G), the modified mycotoxin of zearalenone (ZEN), has attracted considerable attention due to its high potential to be hydrolyzed into ZEN, which would exert toxicity. It has been confirmed that the microflora could metabolize ZEN-14G to ZEN. However, the metabolic profile of ZEN-14G and whether it could be deglucosidated in the liver are unknown. To thoroughly investigate the metabolism of ZEN-14G, in vitro metabolism including phase I and phase II metabolism was studied using liquid chromatography coupled to high-resolution mass spectrometry. Additionally, in vivo metabolism of ZEN-14G was conducted in model animals, rats, by oral administration. As a result, 29 phase I metabolites and 6 phase II metabolites were identified and significant inter-species metabolic differences were observed as well. What is more, ZEN-14G could be considerably deglucosidated into its free form of ZEN after the incubation with animals and human liver microsomes in the absence of NADPH, which was mainly metabolized by human carboxylesterase CES-I and II. Furthermore, results showed that the major metabolic pathways of ZEN-14G were deglucosylation, hydroxylation, hydrogenation and glucuronidation. Although interspecies differences in the biotransformation of ZEN-14G were observed, ZEN, α-ZEL-14G, ß-ZEL-14G, α-ZEL, ZEN-14G-16GlcA and ZEN-14GlcA were the major metabolites of ZEN-14G. Additionally, a larger yield of 6-OH-ZEN-14G and 8-OH-ZEN-14G was also observed in human liver microsomes. The obtained data would be of great importance for the safety assessment of modified mycotoxin, ZEN-14G, and provide another perspective for risk assessment of mycotoxin.

Zearalenone-14-glucoside specifically promotes dysplasia of Gut-Associated Lymphoid Tissue: A natural product for constructing intestinal nodular lymphatic hyperplasia model.

INTRODUCTION: Zearalenone-14-glucoside (Z14G) is a modified mycotoxin that widely contaminates food across the world. Our preliminary experiment showed that Z14G degrades to zearalenone (ZEN) in the intestine exerting toxicity. Notably, oral administration of Z14G in rats induces intestinal nodular lymphatic hyperplasia. OBJECTIVES: To investigate the mechanism of Z14G intestinal toxicity and how it differs from ZEN toxicity. We conducted a precise toxicology study on the intestine of rats exposed to Z14G and ZEN using multi-omics technology. METHODS: Rats were exposed to ZEN (5 mg/kg), Z14G-L (5 mg/kg), Z14G-H (10 mg/kg), and pseudo germ free (PGF)-Z14G-H (10 mg/kg) for 14 days. Histopathological studies were performed on intestines from each group and compared. Metagenomic, metabolomic, and proteomic analyses were performed on rat feces, serum, and intestines, respectively. RESULTS: Histopathological studies showed that Z14G exposure resulted in dysplasia of gut-associated lymphoid tissue (GALT) compared to ZEN exposure. The elimination of gut microbes in the PGF-Z14G-H group alleviated or eliminated Z14G-induced intestinal toxicity and GALT dysplasia. Metagenomic analysis revealed that Z14G exposure significantly promoted the proliferation of Bifidobacterium and Bacteroides compared to ZEN. Metabolomic analysis showed that Z14G exposure significantly reduced bile acid, while proteomic analysis found that Z14G exposure significantly reduced the expression of C-type lectins compared to ZEN. CONCLUSIONS: Our experimental results and previous research suggest that Z14G is hydrolyzed to ZEN by Bifidobacterium and Bacteroides promoting their co-trophic proliferation. This leads to inactivation of lectins by hyperproliferative Bacteroides when ZEN caused intestinal involvement, resulting in abnormal lymphocyte homing and ultimately GALT dysplasia. It is noteworthy that Z14G is a promising model drug to establish rat models of intestinal nodular lymphatic hyperplasia (INLH), which is of great significance for studying the pathogenesis, drug screening and clinical application of INLH.

Development of a reliable UHPLC-MS/MS method for simultaneous determination of zearalenone and zearalenone-14-glucoside in various feed products.

A reliable ultra-high-performance liquid chromatography-tandem mass spectrometry method (UHPLC-MS/MS) was developed for the simultaneous determination of two mycotoxins, that is, zearalenone (ZEN) and zearalenone-14-glucoside (ZEN-14G) in formula feed, concentrated feed, and premixed feed products. An improved sample pretreatment was achieved with the hydrophilic-lipophilic balance (HLB) cartridges efficiently removing the impurities and enriching the target analytes in different feeds. The critical parameters affecting the performance of the solid-phase extraction (SPE) procedure were carefully optimized, and 20% acetonitrile in water as the loading solution, 50% methanol in water as the washing solvent, and 5 ml of methanol as the elution solvent yielded the optimal purification efficiencies. The established method was thoroughly validated in terms of linearity (R 2 ≥ 0.999), sensitivity (limit of quantification in the range of 0.50-5.00 µg kg-1), recovery (89.35 ± 2.67% to 110.93 ± 1.56%), and precision (RSD, 3.00-14.20%), and it was then successfully applied to investigate a total of 60 feed samples. Among them, 50 samples were found to be contaminated with ZEN (an incidence of 83.3%) at levels ranging from 0.63 to 615.24 µg kg-1, whereas 22 samples were contaminated with ZEN-14G (an incidence of 36.7%) in the range of 0.89-15.31 µg kg-1. The developed method proved to be a specific and reliable tool for intensive monitoring of ZEN and ZEN-14G in complex feed matrices.

Zearalenone-14-Glucoside Is Hydrolyzed to Zearalenone by ß-Glucosidase in Extracellular Matrix to Exert Intracellular Toxicity in KGN Cells.

As one of the most important conjugated mycotoxins, zearalenone-14-glucoside (Z14G) has received widespread attention from researchers. Although the metabolism of Z14G in animals has been extensively studied, the intracellular toxicity and metabolic process of Z14G are not fully elucidated. In this study, the cytotoxicity of Z14G to human ovarian granulosa cells (KGN) and the metabolism of Z14G in KGN cells were determined. Furthermore, the experiments of co-administration of ß-glucosidase and pre-administered ß-glucosidase inhibitor (Conduritol B epoxide, CBE) were used to clarify the mechanism of Z14G toxicity release. Finally, the human colon adenocarcinoma cell (Caco-2) metabolism model was used to verify the toxicity release mechanism of Z14G. The results showed that the IC50 of Z14G for KGN cells was 420 µM, and the relative hydrolysis rate of Z14G on ZEN was 35% (25% extracellular and 10% intracellular in KGN cells). The results indicated that Z14G cannot enter cells, and Z14G is only hydrolyzed extracellularly to its prototype zearalenone (ZEN) by ß-glucosidase which can exert toxic effects in cells. In conclusion, this study demonstrated the cytotoxicity of Z14G and clarified the toxicity release mechanism of Z14G. Different from previous findings, our results showed that Z14G cannot enter cells but exerts cytotoxicity through deglycosylation. This study promotes the formulation of a risk assessment and legislation limit for ZEN and its metabolites.

Toxicokinetic Studies in Piglets Reveal Age-Related Differences in Systemic Exposure to Zearalenone, Zearalenone-14-Glucoside, and Zearalenone-14-Sulfate.

Juveniles are considered as one of the most vulnerable population groups concerning mycotoxins and their modified forms. The weaning stage is a particularly vulnerable period in the life of mammals, reflected in intestinal and immune dysfunction. The current study investigated the toxicokinetic (TK) characteristics of zearalenone (ZEN), zearalenone-14-glucoside (ZEN14G), and zearalenone-14-sulfate (ZEN14S) in weaned (4-week-old) piglets, by means of oral and intravenous administration of equimolar doses, i.e., 331, 500, and 415 µg/kg bodyweight, respectively. Plasma and urine were sampled pre- and post-administration and were quantitatively analyzed for ZEN, ZEN14G, ZEN14S, and in vivo metabolites by liquid chromatography-high-resolution mass spectrometry. Tailor-made TK models were elaborated to process data. A statistical comparison of the results was performed with TK data obtained in a previously reported study in pigs of 8 weeks of age. Additionally, porcine plasma protein binding was determined to support TK findings. The TK results for ZEN, ZEN14G, and ZEN14S, obtained in 4- and 8-week-old pigs, revealed significant age-related differences, based on differences in intestinal permeability, body fat content, gastrointestinal transit time, and biotransformation, with a special emphasis on an increased absorbed fraction of ZEN14G, i.e., 94 vs 61% in 4- compared to 8-week-old pigs. Since the growing pig has been reported to be a suitable pediatric animal model for humans concerning TK processes, these results may contribute to refine the risk assessment concerning modified ZEN forms in juvenile animals and humans.

Interactions of zearalanone, α-zearalanol, ß-zearalanol, zearalenone-14-sulfate, and zearalenone-14-glucoside with serum albumin.

The xenoestrogenic mycotoxin zearalenone is a Fusarium-derived food and feed contaminant. In mammals, the reduced (e.g., zearalanone, α-zearalanol, and ß-zearalanol) and conjugated (e.g., zearalenone-14-sulfate) metabolites of zearalenone are formed. Furthermore, filamentous fungi and plants are also able to convert zearalenone to conjugated derivatives, including zearalenone-14-sulfate and zearalenone-14-glucoside, respectively. Serum albumin is the dominant plasma protein in the circulation; it interacts with certain mycotoxins, affecting their toxicokinetics. In a previous investigation, we demonstrated the remarkable species differences regarding the albumin binding of zearalenone and zearalenols. In the current study, the interactions of zearalanone, α-zearalanol, ß-zearalanol, zearalenone-14-sulfate, and zearalenone-14-glucoside with human, bovine, porcine, and rat serum albumins were examined, employing fluorescence spectroscopy and affinity chromatography. Zearalanone, zearalanols, and zearalenone-14-sulfate form stable complexes with albumins tested (K = 9.3 × 103 to 8.5 × 105 L/mol), while the albumin binding of zearalenone-14-glucoside seems to be weak. Zearalenone-14-sulfate formed the most stable complexes with albumins examined. Considerable species differences were observed in the albumin binding of zearalenone metabolites, which may have a role in the interspecies differences regarding the toxicity of zearalenone.

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.

Cyclodextrins Can Entrap Zearalenone-14-Glucoside: Interaction of the Masked Mycotoxin with Cyclodextrins and Cyclodextrin Bead Polymer.

Zearalenone (ZEN) is a Fusarium-derived xenoestrogenic mycotoxin. In plants, zearalenone-14-O-ß-d-glucoside (Z14G) is the major conjugated metabolite of ZEN, and is a masked mycotoxin. Masked mycotoxins are plant-modified derivatives, which are not routinely screened in food and feed samples. Cyclodextrins (CDs) are cyclic oligosaccharides built up from D-glucopyranose units. CDs can form stable host-guest type complexes with lipophilic molecules (e.g., with some mycotoxins). In this study, the interaction of Z14G with native and chemically modified ß- and γ-CDs was examined employing fluorescence spectroscopy and molecular modeling. Furthermore, the removal of Z14G from aqueous solution by insoluble ß-CD bead polymer (BBP) was also tested. Our results demonstrate that Z14G forms the most stable complexes with γ-CDs under acidic and neutral conditions (K ≈ 103 L/mol). Among the CDs tested, randomly methylated γ-CD induced the highest increase in the fluorescence of Z14G (7.1-fold) and formed the most stable complexes with the mycotoxin (K = 2 × 103 L/mol). Furthermore, BBP considerably reduced the Z14G content of aqueous solution. Based on these observations, CD technology seems a promising tool to improve the fluorescence analytical detection of Z14G and to discover new mycotoxin binders which can also remove masked mycotoxins (e.g., Z14G).

Assessing the hydrolytic fate of the masked mycotoxin zearalenone-14-glucoside - A warning light for the need to look at the "maskedome".

Masked mycotoxins are plant metabolites of mycotoxins that contaminate food and feed. They pose health concern as the shortage of toxicological data forces the lack of regulation worldwide. The present work investigated the toxicological relevance of the masked mycotoxin zearalenone-14-glucoside. In vitro, it shows a lower toxicity in respect to the parent compound. However, the major risks related to the consumption of masked mycotoxins depend on the possibility to undergo hydrolysis. Therefore, the hydrolysis and further transformation of zearalenone-14-glucoside in bovine blood and blood components (i.e. plasma, serum and serum albumin) were monitored using LC/MS-MS analysis to gain insights on the possible systemic fate. Hydrolysis was observed in all matrices, and both cell-dependent and -independent contributions were pointed out. Moreover, further metabolism was observed in the whole blood as zearalenol isomers were found. Serum albumin was identified among the active components, and the protein-ligand interaction was investigated via computational analysis. The blood has been pointed out as possible district of reversion and further activation of zearalenone-14-glucoside, and a similar fate cannot be excluded for other masked mycotoxins. Therefore, the systemic hydrolysis should be evaluated beside the absorption, bioavailability and bioaccessibility to deeply understand the toxicity of masked mycotoxins.

Synthesis of Mono- and Di-Glucosides of Zearalenone and α-/ß-Zearalenol by Recombinant Barley Glucosyltransferase HvUGT14077.

Zearalenone (ZEN) is an estrogenic mycotoxin occurring in Fusarium-infected cereals. Glucosylation is an important plant defense mechanism and generally reduces the acute toxicity of mycotoxins to humans and animals. Toxicological information about ZEN-glucosides is limited due to the unavailability of larger amounts required for animal studies. HvUGT14077, a recently-validated ZEN-conjugating barley UDP-glucosyltransferase was expressed in Escherichia coli, affinity purified, and characterized. HvUGT14077 possesses high affinity (Km = 3 µM) and catalytic efficiency (kcat/Km = 190 s-1·mM-1) with ZEN. It also efficiently glucosylates the phase-I ZEN-metabolites α-zearalenol and ß-zearalenol, with kcat/Km of 40 and 74 s-1·mM-1, respectively. HvUGT14077 catalyzes O-glucosylation at C-14 and C-16 with preference of 14-glucoside synthesis. Furthermore, relatively slow consecutive formation of 14,16-di-glucosides was observed; their structures were tentatively identified by mass spectrometry and for ZEN-14,16-di-glucoside confirmed by nuclear magnetic resonance spectroscopy. Recombinant HvUGT14077 allowed efficient preparative synthesis of ZEN-glucosides, yielding about 90% ZEN-14-glucoside and 10% ZEN-16-glucoside. The yield of ZEN-16-glucoside could be increased to 85% by co-incubation with a ß-glucosidase highly selective for ZEN-14-glucoside. Depletion of the co-substrate UDP-glucose was counteracted by a sucrose synthase based regeneration system. This strategy could also be of interest to increase the yield of minor glucosides synthesized by other glucosyltransferases.

Molecular insights on xenoestrogenic potential of zearalenone-14-glucoside through a mixed in vitro/in silico approach.

The mycotoxin zearalenone may contaminate food and feed worldwide upon infections by Fusarium spp. of plants and raw materials intended for human and animal consumption. Currently, contamination by zearalenone and congeners pose concern for health due to xenoestrogenic effects. However, while zearalenone and the main reduced metabolites are well-known xenoestrogens, some plant metabolites that may enter the food chain have been observed aside. Among them, zearalenone-14-glucoside may be abundant in the edible parts of infected plants, thereby entering significantly the human diet and animal feeding. On the basis of previous works, the lack of xenoestrogenicity for this compound per se was taken for granted, while neglecting the direct proof of estrogenic activity and considering the hydrolysis as a possible source of estrogenically active metabolites. The present work investigated the xenoestrogenicity of zearalenone-14-glucoside, in comparison to zearalenone, deepening the underlying molecular mechanisms through an integrated in vitro/in silico approach. On the basis of our results, zearalenone-14-glucoside effectively stimulated a xenoestrogenic response in cells, but such stimulus can be entirely attributable to the hydrolysis phenomenon, as the glycosylated form turned out to be unable to effectively bind and activate the estrogens receptors.