Structural Impact of Steroidal Glycoalkaloids: Barrier Integrity, Permeability, Metabolism, and Uptake in Intestinal Cells.

SCOPE: Potato tubers represent an essential food component all over the world and an important supplier of carbohydrates, fiber, and valuable proteins. However, besides their health promoting effects, potatoes contain α-solanine and α-chaconine, which are toxic steroidal glycoalkaloids (SGAs). Other solanaceous plants like eggplants and tomatoes produce SGAs as well, different in their chemical structure. This study aims to investigate toxic effects (cholinesterase inhibition, membrane, and barrier disruption), permeability, metabolism, and structure-activity relationships of SGAs. METHODS AND RESULTS: α-solanine, α-chaconine, α-solasonine, α-solamargine, α-tomatine, and their respective aglycones solanidine, solasodine, and tomatidine are analyzed using Ellman assay, cellular impedance spectroscopy, cell extraction, and Caco-2 intestinal model. Additionally, metabolism is analyzed by HPLC-MS techniques. The study observes dependencies of barrier disrupting potential and cellular uptake on the carbohydrate moiety of SGAs, while permeability and acetylcholinesterase (AChE) inhibition are dominated by the steroid backbone. SGAs show low permeabilities across Caco-2 monolayers in subtoxic concentrations. In contrast, their respective aglycones reveal higher permeabilities, but are extensively metabolized. CONCLUSION: Besides structure-activity relationships, this study provides new information on the overall effects of steroidal alkaloids on intestinal cells and closes a gap of knowledge for the metabolic pathway from oral uptake to final excretion.

Comparative metabolism of aflatoxin B1 in mouse, rat and human primary hepatocytes using HPLC-MS/MS.

Aflatoxin B1 (AFB1) is a highly hepatotoxic and carcinogenic mycotoxin produced by Aspergillus species. The compound is mainly metabolized in the liver and its metabolism varies between species. The present study quantified relevant AFB1- metabolites formed by mouse, rat, and human primary hepatocytes after treatment with 1 µM and 10 µM AFB1. The use of liquid chromatographic separation coupled with tandem mass spectrometric detection enabled the selective and sensitive determination of phase I and phase II metabolites of AFB1 over incubation times of up to 24 h. The binding of AFB1 to macromolecules was also considered. The fastest metabolism of AFB1 was observed in mouse hepatocytes which formed aflatoxin P1 as a major metabolite and also its glucuronidated form, while AFP1 occurred only in traces in the other species. Aflatoxin M1 was formed in all species and was, together with aflatoxin Q1 and aflatoxicol, the main metabolite in human cells. Effective epoxidation led to high amounts of DNA adducts already 30 min post-treatment, especially in rat hepatocytes. Lower levels of DNA adducts and fast DNA repair were found in mouse hepatocytes. Also, protein adducts arising from reactive intermediates were formed rapidly in all three species. Detoxification via glutathione conjugation and subsequent formation of the N-acetylcysteine derivative appeared to be similar in mice and in rats and strongly differed from human hepatocytes which did not form these metabolites at all. The use of qualitative reference material of a multitude of metabolites and the comparison of hepatocyte metabolism in three species using advanced methods enabled considerations on toxification and detoxification mechanisms of AFB1. In addition to glutathione conjugation, phase I metabolism is strongly involved in the detoxification of AFB1.

Polyphenols Cause Structure Dependent Effects on the Metabolic Profile of Human Hepatocarcinogenic Cells.

SCOPE: Although many beneficial health effects are attributed to polyphenols their influence on the human metabolome has not been elucidated yet. The ubiquitous occurrence of polyphenols in the human diet demands comprehensive knowledge about physiological and toxicological effects of these compounds on human cells. METHODS AND RESULTS: The human hepatocarcinogenic cell line HepG2 is used to elucidate the effects of 13 polyphenols and three respective phenolic degradation products on the human metabolome using HPLC-MS/MS. To investigate structure-activity-relationships, structurally related examples of polyphenols from different compound classes are selected. The analysis of catechins points toward a relation between the degree of hydroxylation and the extent of metabolic effects particularly on the urea cycle and the pentose phosphate pathway (PPP). A correlation between the modulation of the PPP and the stability of the compounds is demonstrated, which may be caused by reactive oxygen species (ROS). The incubation of flavones and alkenylbenzenes demonstrates reduced activity of methoxylated compounds and no impact of the B-ring position. CONCLUSION: In general, polyphenols induce a multitude of metabolic effects, for example, on energy metabolism, PPP, and urea cycle. These metabolic alterations may be related to the widely reported bioactivity of these compounds such as the anticarcinogenic effects.

Dose-dependent expression of GFI1 alters metabolism in the haematopoietic progenitors and MLL::AF9-induced leukaemic cells.

Growth factor independence 1 (GFI1) is a transcriptional repressor protein that plays an essential role in the differentiation of myeloid and lymphoid progenitors. We and other groups have shown that GFI1 has a dose-dependent role in the initiation, progression, and prognosis of acute myeloid leukaemia (AML) patients by inducing epigenetic changes. We now demonstrate a novel role for dose-dependent GFI1 expression in regulating metabolism in haematopoietic progenitor and leukaemic cells. Using in-vitro and ex-vivo murine models of MLL::AF9-induced human AML and extra-cellular flux assays, we now demonstrate that a lower GFI1 expression enhances oxidative phosphorylation rate via upregulation of the FOXO1- MYC axis. Our findings underscore the significance of therapeutic exploitation in GFI1-low-expressing leukaemia cells by targeting oxidative phosphorylation and glutamine metabolism.

Glycolytic interference blocks influenza A virus propagation by impairing viral polymerase-driven synthesis of genomic vRNA.

Influenza A virus (IAV), like any other virus, provokes considerable modifications of its host cell's metabolism. This includes a substantial increase in the uptake as well as the metabolization of glucose. Although it is known for quite some time that suppression of glucose metabolism restricts virus replication, the exact molecular impact on the viral life cycle remained enigmatic so far. Using 2-deoxy-d-glucose (2-DG) we examined how well inhibition of glycolysis is tolerated by host cells and which step of the IAV life cycle is affected. We observed that effects induced by 2-DG are reversible and that cells can cope with relatively high concentrations of the inhibitor by compensating the loss of glycolytic activity by upregulating other metabolic pathways. Moreover, mass spectrometry data provided information on various metabolic modifications induced by either the virus or agents interfering with glycolysis. In the presence of 2-DG viral titers were significantly reduced in a dose-dependent manner. The supplementation of direct or indirect glycolysis metabolites led to a partial or almost complete reversion of the inhibitory effect of 2-DG on viral growth and demonstrated that indeed the inhibition of glycolysis and not of N-linked glycosylation was responsible for the observed phenotype. Importantly, we could show via conventional and strand-specific qPCR that the treatment with 2-DG led to a prolonged phase of viral mRNA synthesis while the accumulation of genomic vRNA was strongly reduced. At the same time, minigenome assays showed no signs of a general reduction of replicative capacity of the viral polymerase. Therefore, our data suggest that the significant reduction in IAV replication by glycolytic interference occurs mainly due to an impairment of the dynamic regulation of the viral polymerase which conveys the transition of the enzyme's function from transcription to replication.

Intestinal Metabolism of Selected Steroidal Glycoalkaloids in the Pig Cecum Model.

Due to the presence of the steroidal glycoalkaloid solanine, the potato was chosen as Germany's poisonous plant of the year 2022. Steroidal glycoalkaloids are secondary plant metabolites which have been reported to induce toxic as well as beneficial health effects. Nevertheless, data regarding occurrence, toxicokinetics, and metabolism of steroidal glycoalkaloids is scarce, and substantially more research is required for a proper risk assessment. Therefore, the intestinal metabolism of solanine, chaconine, solasonine, solamargine, and tomatine was investigated using the ex vivo pig cecum model. All steroidal glycoalkaloids were degraded by the porcine intestinal microbiota, releasing the respective aglycon. Furthermore, the hydrolysis rate was strongly dependent on the linked carbohydrate side chain. Solanine and solasonine, which are linked to a solatriose, were metabolized significantly faster than the chaconine and solamargin, which are linked to a chacotriose. In addition, stepwise cleavage of the carbohydrate side chain and the formation of ß- and γ-intermediates were detected by HPLC-HRMS. The results provide valuable insights into the intestinal metabolism of selected steroidal glycoalkaloids and help to reduce uncertainties and improve risk assessment.

Enhancing the Performance of a Large Aperture Ultrasound System (LAUS): A Combined Approach of Simulation and Measurement for Transmitter-Receiver Optimization.

The Large Aperture Ultrasound System (LAUS) developed at BAM is known for its ability to penetrate thick objects, especially concrete structures commonly used in nuclear waste storage and other applications in civil engineering. Although the current system effectively penetrates up to ~9 m, further optimization is imperative to enhance the safety and integrity of disposal structures for radioactive or toxic waste. This study focuses on enhancing the system's efficiency by optimizing the transducer spacing, ensuring that resolution is not compromised. An array of twelve horizontal shear wave transducers was used to find a balance between penetration depth and resolution. Systematic adjustments of the spacing between transmitter and receiver units were undertaken based on target depth ranges of known reflectors at depth ranges from 5 m to 10 m. The trade-offs between resolution and artifact generation were meticulously assessed. This comprehensive study employs a dual approach using both simulations and measurements to investigate the performance of transducer units spaced at 10 cm, 20 cm, 30 cm, and 40 cm. We found that for depths up to 5 m, a spacing of 10 cm for LAUS transducer units provided the best resolution as confirmed by both simulations and measurements. This optimal distance is particularly effective in achieving clear reflections and a satisfactory signal-to-noise ratio (SNR) in imaging scenarios with materials such as thick concrete structures. However, when targeting depths greater than 10 m, we recommend increasing the distance between the transducers to 20 cm. This increased spacing improves the SNR in comparison to other spacings, as seen in the simulation of a 10 m deep backwall. Our results emphasize the critical role of transducer spacing in achieving the desired SNR and resolution, especially in the context of depth imaging requirements for LAUS applications. In addition to the transducer spacing, different distances between individual sets of measurement positions were tested. Overall, keeping the minimal possible distance between measurement position offsets provides the best imaging results at greater depths. The proposed optimizations for the LAUS in this study are primarily relevant to applications on massive nuclear structures for nuclear waste management. This research highlights the need for better LAUS efficiency in applications such as sealing structures, laying the foundation for future technological advances in this field.

Validation of Novel Ultrasonic Phased Array Borehole Probe by Using Simulation and Measurement.

Low-frequency ultrasonic testing is a well-established non-destructive testing (NDT) method in civil engineering for material characterization and the localization of cracks, reinforcing bars and delamination. A novel ultrasonic borehole probe is developed for in situ quality assurance of sealing structures in radioactive waste repositories using existing research boreholes. The aim is to examine the sealing structures made of salt concrete for any possible cracks and delamination and to localize built-in components. A prototype has been developed using 12 individual horizontal dry point contact (DPC) shear wave transducers separated by equidistant transmitter/receiver arrays. The probe is equipped with a commercially available portable ultrasonic flaw detector used in the NDT civil engineering industry. To increase the sound pressure generated, the number of transducers in the novel probe is increased to 32 transducers. In addition, the timed excitation of each transducer directs a focused beam of sound to a specific angle and distance based on the previously calculated delay time. This narrows the sensitivity of test volume and improves the signal-to-noise ratio of the received signals. In this paper, the newly designed phased array borehole probe is validated by beam computation in the CIVA software and experimental investigations on a half-cylindrical test specimen to investigate the directional characteristics. In combination with geophysical reconstruction methods, it is expected that an optimised radiation pattern of the probe will improve the signal quality and thus increase the reliability of the imaging results. This is an important consideration for the construction of safe sealing structures for the safe disposal of radioactive or toxic waste.

Metabolic profiling as a powerful tool for the analysis of cellular alterations caused by 20 mycotoxins in HepG2 cells.

Mycotoxins are secondary fungal metabolites which exhibit toxic effects in low concentrations. Several mycotoxins are described as carcinogenic or immunosuppressive, but their underlying modes of action especially on molecular level have not yet been entirely elucidated. Metabolic profiling as part of the omics methods is a powerful tool to study the toxicity and the mode of action of xenobiotics. The use of hydrophilic interaction chromatography in combination with targeted mass spectrometric detection enables the selective and sensitive analysis of more than 100 polar and ionic metabolites and allows the evaluation of metabolic alterations caused by xenobiotics such as mycotoxins. For metabolic profiling, the hepato-cellular carcinoma cell line HepG2 was treated with sub-cytotoxic concentrations of 20 mycotoxins. Moniliformin and citrinin significantly affected target elements of the citric acid cycle, but also influenced glycolytic pathways and energy metabolism. Penitrem A, zearalenone, and T2 toxin mainly interfered with the urea cycle and the amino acid homeostasis. The formation of reactive oxygen species seemed to be influenced by T2 toxin and gliotoxin. Glycolysis was altered by ochratoxin A and DNA synthesis was affected by several mycotoxins. The observed effects were not limited to these metabolic reactions as the metabolic pathways are closely interrelated. In general, metabolic profiling proved to be a highly sensitive tool for hazard identification in comparison to single-target cytotoxicity assays as metabolic alterations were already observed at sub-toxic concentrations. Metabolic profiling could therefore be a powerful tool for the overall evaluation of the toxic properties of xenobiotics.

Permeability of dopamine D2 receptor agonist hordenine across the intestinal and blood-brain barrier in vitro.

Hordenine, a bioactive food compound, has several pharmacological properties and has recently been identified as a dopamine D2 receptor (D2R) agonist. Since the pharmacokinetic profile of hordenine has been described to a limited extent, the present study focused on the transfer and transport of hordenine across the intestinal epithelium and the blood-brain barrier (BBB) in vitro. Hordenine was quickly transferred through the Caco-2 monolayer in only a few hours, indicating a rapid oral uptake. However, the high bioavailability may be reduced by the observed efflux transport of hordenine from the bloodstream back into the intestinal lumen and by first pass metabolism in intestinal epithelial cells. To determine the biotransformation rate of hordenine, the metabolite hordenine sulfate was synthesized as reference standard for analytical purposes. In addition, transfer studies using primary porcine brain capillary endothelial cells (PBCEC) showed that hordenine is able to rapidly penetrate the BBB and potentially accumulate in the brain. Thus, a D2R interaction of hordenine and activation of dopaminergic signaling is conceivable, assuming that the intestinal barrier can be circumvented by a route of administration alternative to oral uptake.

In Vitro Metabolism of Phenylspirodrimanes Derived from the Indoor Fungus Stachybotrys.

Fungi belonging to the genus Stachybotrys are frequently detected in water-damaged indoor environments, and a potential correlation between emerging health problems of inhabitants of affected housing and the fungi is controversially discussed. Secondary metabolites (i.e., mycotoxins) produced by Stachybotrys, such as the highly toxic macrocyclic trichothecenes (MCTs), are of potential concern to human health. The present study, however, focused on the potential effects of the more broadly and abundantly formed group of phenylspirodrimanes (PSDs). The phase I and II metabolism of four structurally different PSDs were investigated in vitro using hepatic models in combination with high-performance liquid chromatography high-resolution mass spectrometry (HPLC-HRMS) analysis. In addition to metabolite detection by HRMS, isolation and structure elucidation by nuclear magnetic resonance spectroscopy (NMR) was part of the conducted study as well.

Release of Small Phenolic Metabolites from Isotopically Labeled 13C Lignin in the Pig Cecum Model.

A diet with a high dietary fiber content is often recommended in today's nutrition due to several beneficial health effects related to its intake. Lignin as a part of dietary fiber is the second most abundant natural polymer and considered to be stable during digestion. However, some studies indicate a partial degradation during the intestinal metabolism. To further elucidate this hypothesis, the aim of this study was to investigate whether lignin is metabolized by the gut microbiota using the ex vivo pig cecum model. As potential lignin-derived metabolites might already naturally occur in the pig cecal matrix, an approach using isotopically labeled 13C lignin was chosen for this study. Ten small phenolic lignin degradation products and their time-dependent metabolism were identified via an untargeted HPLC-HRMS approach, and the quantity of the metabolites was estimated. From the results, we conclude that lignin is partially degraded releasing small phenolic metabolites.

Metabolic conjugation reduces in vitro toxicity of the flavonoid nevadensin.

The present study focuses on the association between metabolic capacity and toxicity of the natural occurring flavonoid nevadensin in vitro. Human colon (HT29), liver (HepG2) and bone marrow (KG1) carcinoma cells were used and strong cell line dependent differences in toxic effect strength were found. HepG2 and KG1 cells were more sensitive against nevadensin treatment in comparison to HT29 cells. High resolution mass spectrometry experiments showed that nevadensin is rapidly glucuronidated in HT29 cells, whereas KG1 cells do not metabolize nevadensin, thus glucuronidation was supposed to be a crucial metabolic pathway in vitro. To proof this suggestion, nevadensin glucuronides were isolated from pig liver microsomes und structurally elucidated via NMR spectroscopy. In HepG2 cells a cellular enrichment of nevadensin itself as well as nevadensin-7-O-glucuronide was determined by tandem mass spectrometry. A proteomic screening of uridine 5'-diphospho (UDP)-glucuronosyltransferase (UGT) in HT29 and HepG2 cells provided first hints that the isoforms UGT1A6 and UGT1A1 are responsible for nevadensin glucuronidation. Additionally, nevadensin was found to be a potent SULT inhibitor in HepG2 cells. In sum, the present study clearly illustrates the importance of obtaining detailed information about metabolic competence of cell lines which should be considered in the evaluation of toxic endpoints.

In Vitro Metabolism of Helenalin Acetate and 11α,13-Dihydrohelenalin Acetate: Natural Sesquiterpene Lactones from Arnica.

Arnica tincture is a herbal medicinal preparation with anti-inflammatory activity which is used traditionally for the topical treatment of blunt injuries as well as rheumatic muscle and joint complaints. Its main bioactive constituents are sesquiterpene lactones (STLs) of the helenalin and 11α,13-dihydrohelenalin types. Besides the mentioned activity, the tincture and its isolated STLs have antileishmanial activity. In a recent in vivo study, a treatment with Arnica tincture cured cutaneous Leishmaniasis (CL) in a golden hamster model. CL is a neglected tropical disease affecting more than two million people every year, for which new treatments are urgently needed. In order to use Arnica tincture on open CL lesions of human patients, it is important to know how the constituents are metabolized. Therefore, in vitro metabolism experiments with liver microsomes of different species (rat, pig and human) were performed with the Arnica STLs helenalin acetate and 11α,13-dihydrohelenalin acetate. Phase I and phase II metabolism experiments were performed, as well as a combination of both. Glutathione conjugation plays a major role in the metabolism of these STLs, as could be expected based on previous reports on their reactivity. Besides glutathione conjugates, several other metabolites were formed, e.g., water conjugates and hydroxides. Our results show for the first time a detailed picture of the metabolism of Arnica STLs. The fast and extensive formation of glutathione conjugates makes it unlikely that low absorbed levels of these compounds, as expected after dermal absorption from Arnica tincture, could be of toxicological concern.

Intestinal Absorption and Metabolism of the Tomato Imidazole Alkaloids N-Caprylhistamine-ß-glucoside and N-Caprylhistamine.

Histamine-based imidazole alkaloids N-caprylhistamine (HmC8) and N-caprylhistamine-ß-glucoside (HmC8-Glc) were recently identified as precursors for a tomato biomarker. As studies regarding metabolism and bioavailability are scarce, the present study aimed at the elucidation of intestinal absorption and metabolism using the Caco-2 model and the pig cecum model to mimic human intestinal conditions. The most abundant imidazole alkaloid HmC8-Glc was neither absorbed nor transferred across cellular barriers but extensively metabolized to HmC8 in the pig cecum model, whereas the aglycon HmC8 is subjected to transport and metabolic processes through the Caco-2 monolayer and metabolized to the bioactive neurotransmitter histamine by the intestinal microbiota. Deduced from the combined results of both methods, HmC8-Glc is not absorbed directly via the intestinal epithelium but requires a metabolic cleavage of the glycosidic bond by the gut microbiota. Because of the high bioavailability of the released HmC8 and histamine, HmC8 and its glucoside might also be involved in the intolerance to tomato products by histamine-intolerant consumers.

Spacer length and serum protein adsorption affect active targeting of trastuzumab-modified nanoparticles.

Receptor-mediated active targeting of nanocarriers is a widely investigated approach to specifically address cancerous cells and tissues in the human body. The idea is to use these formulations as drug carriers with enhanced specificity and therefore reduced systemic side effects. Until today a big obstacle to reach this goal remains the adsorption of serum proteins to the nanocarrier's surface after contact with biological fluids. In this context different nanoparticle characteristics could be beneficial for effective active targeting after formation of a protein corona which need to be identified. In this study trastuzumab was used as an active targeting ligand which was covalently attached to human serum albumin nanoparticles. For coupling reaction different molecular weight spacers were used and resulting physicochemical nanoparticle characteristics were evaluated. The in vitro cell association of the different nanoparticle formulations was tested in cell culture experiments with or without fetal bovine serum. For specific receptor-mediated cell interaction SK-BR-3 breast cancer cells with human epidermal growth factor receptor 2 (HER2) overexpression were used. MCF-7 breast cancer cells with normal HER2 expression served as control. Furthermore, serum protein adsorption on respective nanoparticles was characterized. The qualitative and quantitative composition of the protein corona was analyzed by SDS-PAGE and LC-MS/MS and the influence of protein adsorption on active targeting capability was determined.

Erratum to 'Spacer length and serum protein adsorption affect active targeting of trastuzumab-modified nanoparticles' [Biomaterials and Biosystems 5 (2022) 100032].

[This corrects the article DOI: 10.1016/j.bbiosy.2021.100032.].

Efflux at the Blood-Brain Barrier Reduces the Cerebral Exposure to Ochratoxin A, Ochratoxin α, Citrinin and Dihydrocitrinone.

Recent studies have implied that environmental toxins, such as mycotoxins, are risk factors for neurodegenerative diseases. To act directly as neurotoxins, mycotoxins need to penetrate or affect the integrity of the blood-brain barrier, which protects the mammalian brain from potentially harmful substances. As common food and feed contaminants of fungal origin, the interest in the potential neurotoxicity of ochratoxin A, citrinin and their metabolites has recently increased. Primary porcine brain capillary endothelial cells were used to investigate cytotoxic or barrier-weakening effects of ochratoxin A, ochratoxin α, citrinin and dihydrocitrinone. The transfer and transport properties of the mycotoxins across the barrier formed by porcine brain capillary endothelial cell monolayers were analysed using HPLC-MS/MS. High levels of Ochratoxin A caused cytotoxic and barrier-weakening effects, whereas ochratoxin α, citrinin and dihydrocitrinone showed no adverse effects up to 10 µM. Likely due to efflux transporter proteins, the transfer to the brain compartment was much slower than expected from their high lipophilicity. Due to their slow transfer across the blood-brain barrier, cerebral exposure of ochratoxin A, ochratoxin α, citrinin and dihydrocitrinone is low and neurotoxicity is likely to play a subordinate role in their toxicity at common physiological concentrations.

Identification of main influencing factors on the protein corona composition of PLGA and PLA nanoparticles.

Poly(DL-lactic-co-glycolic acid) and poly(DL-lactic acid) are widely used for the preparation of nanoparticles due to favorable characteristics for medical use like biodegradability and controllable degradation behavior. The contact with different media like human plasma or serum leads to the formation of a protein corona that determines the NP's in vivo processing. In this study, the impact of surface end group identity, matrix polymer hydrophobicity, molecular weight, and incubation medium on the protein corona composition was evaluated. Corona proteins were quantified using Bradford assay, separated by SDS-PAGE, and identified via LC-MS/MS. The acquired data revealed that surface end group identity had the most profound effect on corona composition in both quantitative and qualitative terms. Regarding matrix polymer hydrophobicity, adsorption profiles on NP systems with similar physicochemical characteristics resembled each other. The molecular weight of the matrix polymers proved to impact quantity, but not quality of corona bound proteins. The corona of plasma incubated NP showed adsorption of incubation medium-specific proteins but resembled those of serum incubated NP in terms of protein function, average mass and isoelectric point. Overall, the NP physicochemical properties proved to be easily adjustable determining factors of protein corona formation in physiological environments.

Intestinal Metabolism of α- and ß-Glucosylated Modified Mycotoxins T-2 and HT-2 Toxin in the Pig Cecum Model.

The type A trichothecene mycotoxins T-2 and HT-2 toxin are fungal secondary metabolites produced by Fusarium fungi, which contaminate food and feed worldwide. Especially as a result of the high toxicity of T-2 toxin and their occurrence together with glucosylated forms in cereal crops, these mycotoxins are of human health concern. Particularly, it is unknown whether and how these modified mycotoxins are metabolized in the gastrointestinal tract and, thus, contribute to the overall toxicity. Therefore, the comparative intestinal metabolism of T-2 and HT-2 toxin glucosides in α and ß configuration was investigated using the ex vivo pig cecum model, which mimics the human intestinal metabolism. Regardless of its configuration, the C-3 glycosidic bond was hydrolyzed within 10-20 min, releasing T-2 and HT-2 toxin, which were further metabolized to HT-2 toxin and T-2 triol, respectively. We conclude that T-2 and HT-2 toxin should be evaluated together with their modified forms for risk assessment.