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Background: Kashin-Beck disease (KBD) is an endemic osteoarthropathy characterized by excessive chondrocytes apoptosis. T-2 toxin exposure has been proved to be its etiology. Connective tissue growth factor (CTGF) exerts a profound influence on cartilage growth and metabolism. We investigated the potential role of CTGF in KBD development and examined CTGF alterations under T-2 toxin stimulation., Methods: The levels of CTGF and chondrocyte apoptosis-related markers in cartilage and primary chondrocytes from KBD and control groups were measured using qRT-PCR, Western blotting, immunohistochemistry, and immunofluorescence. We analyzed expression changes of these genes in response to T-2 toxin. Apoptosis rates of chondrocytes induced by T-2 toxin were measured by flow cytometry and TUNEL assay. The active pharmaceutical ingredient targeting CTGF was screened through Comparative Toxicogenomics Database, and molecular docking was performed using AutoDock Tools., Results: The CTGF levels in KBD cartilage and chondrocytes were significantly elevated and positively associated with the levels of apoptosis-related genes. T-2 toxin exposure increased CTGF and apoptosis-related gene levels in chondrocytes, with apoptosis rates rising alongside T-2 toxin concentration. Curcumin was identified as targeting CTGF and exhibited effective binding. It could down-regulate CTGF, apoptosis-related genes, such as Cleaved caspase 3 and BAX, and also significantly reduce apoptosis rate in chondrocytes treated with T-2 toxin., Conclusion: CTGF plays a crucial role in the development of KBD. Curcumin has shown potential in inhibiting CTGF levels and reducing chondrocyte apoptosis, highlighting its promise as a therapeutic agent for preventing cartilage damage in KBD. Our findings provided valuable insights into the pathogenesis of KBD and could promote the development of novel therapeutic strategies for this debilitating disease., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

HT-2 toxin is a type of mycotoxin which is shown to affect gastric and intestinal lesions, hematopoietic and immunosuppressive effects, anorexia, lethargy, nausea. Recently, emerging evidences indicate that HT-2 also disturbs the reproductive system. In this study, we investigated the impact of HT-2 toxin exposure on the organelles of porcine oocytes. Our results found that the abnormal distribution of endoplasmic reticulum increased after HT-2 treatment, with the perturbation of ribosome protein RPS3 and GRP78 expression; Golgi apparatus showed diffused localization pattern and GM130 localization was also impaired, thereby affecting the Rab10-based vesicular transport; Due to the impairment of ribosomes, ER, and Golgi apparatus, the protein supply to lysosomes was hindered, resulting in lysosomal damage, which further disrupted the LC3-based autophagy. Moreover, the results indicated that the function and distribution of mitochondria were also affected by HT-2 toxin, showing with fragments of mitochondria, decreased TMRE and ATP level. Taken together, our study suggested that HT-2 toxin exposure induces damage to the organelles for endomembrane system, which further inhibited the meiotic maturation of porcine oocytes., Competing Interests: Declaration of competing interest The absence of any conflicts of interest in relation to this article has been explicitly acknowledged by the authors., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Kashin-Beck disease (KBD) is an endemic, environmentally associated cartilage disease. Previous studies have shown that the environmental suspected pathogenic factors of KBD, T-2 toxin and low selenium, are involved in the regulation of inflammation, oxidative stress and autophagy in some tissues and organs. In cartilage diseases, the level of cellular autophagy determines the fate of the chondrocytes. However, whether autophagy is involved in KBD cartilage lesions, and the role of low selenium and T-2 toxins in KBD cartilage injury and autophagy are still unclear. This work took the classical AMPK/mTOR/ULK1 autophagy regulatory pathway as the entry point to clarify the relationship between the environmental suspected pathogenic factors and chondrocyte autophagy. Transmission electron microscopy was used to observe the autophagy of chondrocytes in KBD patients. qRT-PCR and western blot were used to analyze the expression of AMPK/mTOR/ULK1 pathway and autophagy markers. The rat model of KBD was established by low selenium and T-2 toxin, the autophagy in rat cartilage was detected after 4- and 12-week interventions. Chondrocyte autophagy was found in KBD, and the AMPK/mTOR/ULK1 pathway was down-regulated. In the rat model, the pathway showed an up-regulated trend when low selenium and T-2 toxin, were treated for a short time or low concentration, and autophagy level increased. However, when low selenium and T-2 toxin were treated for a long time or at high concentrations, the pathway showed a down-regulated trend, and the autophagy level was reduced and even defective. In conclusion, in the process of KBD cartilage lesion, chondrocyte autophagy level may increase in the early stage, and decrease in the late stage with the progression of lesion. Low selenium and T-2 toxins may affect autophagy by AMPK/mTOR/ULK1 pathway., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest. All authors participated in drafting the article and critically revised important content, and endorsed the final version. Han and Deng had full access to all the data in the study and took responsibility for the integrity of the data and the accuracy of the data analysis., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Mycotoxins are toxic, fungal secondary metabolites that contaminate agricultural commodities, food, and feed. Among them, T-2, HT-2, and diacetoxyscirpenol (DAS; the major type A trichothecene) are primarily produced from Fusarium species. These mycotoxins exert numerous toxicological effects in animals and humans, such as dermatotoxicity, haematotoxicity, hepatotoxicity, nephrotoxicity, neurotoxicity, and immunotoxicity. In the present study, human Jurkat T cells were used as a model to investigate apoptotic cell death induced by T-2, HT-2, and DAS. The results showed that T-2, HT-2, and DAS decreased cell viability and increased production of Reactive Oxygen Species in a time- and dose-dependency. Based on their IC 50 values, they could be ranked in decreasing order of cytotoxicity as T-2 > HT-2 > DAS. All tested mycotoxins caused DNA fragmentation, up-regulated cytochrome C, caspase 3, and caspase 9 mRNA levels, and down-regulated the relative expression of Bcl-2 and caspase 8. The effects of these trichothecenes on apoptosis were determined based on flow cytometry. At the IC 50 concentrations, the percentages of apoptotic cells were significantly higher than for the controls. Taken together, these data suggested that T-2, HT-2, and DAS could induce apoptosis through the mitochondrial apoptotic pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Trichothecenes produced by Fusarium species are commonly detected in oats. However, the ratios of the concentrations of free trichothecenes and their conjugates and how they are impacted by different interacting environmental conditions are not well documented. This study aims to examine the effect of water activity (0.95 and 0.98 a w ) and temperature (20 and 25 °C) stress on the production of T-2 and HT-2 toxins, deoxynivalenol and their conjugates, as well as diacetoxyscirpenol (DAS). Multiple mycotoxins were detected using liquid chromatography-tandem mass spectrometry from 64 contaminated oat samples. The highest concentrations of HT-2-glucoside (HT-2-Glc) were observed at 0.98 a w and 20 °C, and were higher than other type A trichothecenes in the natural oats' treatments. However, no statistical differences were found between the mean concentrations of HT-2-Glc and HT-2 toxins in all storage conditions analysed. DAS concentrations were generally low and highest at 0.95 a w and 20 °C, while deoxynivalenol-3-glucoside levels were highest at 0.98 a w and 20 °C in the naturally contaminated oats. Emerging mycotoxins such as beauvericin, moniliformin, and enniatins mostly increased with a rise in water activity and temperature in the naturally contaminated oats treatment. This study reinforces the importance of storage a w and temperature conditions in the high risk of free and modified toxin contamination of small cereal grains.

The present study assessed the ability of Trichoderma to combat F. sporotrichioides, focusing on their antagonistic properties. Tests showed that Trichoderma effectively inhibited F. sporotrichioides mycelial growth, particularly with T. atroviride strains. In co-cultures on rice grains, Trichoderma almost completely reduced the biosynthesis of T-2 and HT-2 toxins by Fusarium. T-2 toxin-α-glucoside (T-2-3α-G), HT-2 toxin-α-glucoside (HT-2-3α-G), and HT-2 toxin-β-glucoside (HT-2-3β-G) were observed in the common culture medium, while these substances were not present in the control medium. The study also revealed unique metabolites and varying metabolomic profiles in joint cultures of Trichoderma and Fusarium, suggesting complex interactions. This research offers insights into the processes of biocontrol by Trichoderma, highlighting its potential as a sustainable solution for managing cereal plant pathogens and ensuring food safety., (© 2024. The Author(s).)

Fusarium poae is commonly detected in field surveys of Fusarium head blight (FHB) of cereal crops and can produce a range of trichothecene mycotoxins. Although experimentally validated reports of F. poae strains producing T-2/HT-2 trichothecenes are rare, F. poae is frequently generalized in the literature as a producer of T-2/HT-2 toxins due to a single study from 2004 in which T-2/HT-2 toxins were detected at low levels from six out of forty-nine F. poae strains examined. To validate/substantiate the observations reported from the 2004 study, the producing strains were acquired and phylogenetically confirmed to be correctly assigned as F. poae ; however, no evidence of T-2/HT-2 toxin production was observed from axenic cultures. Moreover, no evidence for a TRI16 ortholog, encoding a key acyltransferase shown to be necessary for T-2 toxin production in other Fusarium species, was observed in any of the de novo assembled genomes of the F. poae strains. Our findings corroborate multiple field-based and in vitro studies on FHB-associated Fusarium populations which also do not support the production of T-2/HT-2 toxins with F. poae and therefore conclude that F. poae should not be generalized as a T-2/HT-2 toxin producing species of Fusarium .

The aim of this study was to simultaneously determine T-2 and HT-2 toxins and the α and β anomers of their glucosides to assess their content in wheat and oat grains harvested in Poland (2020-2022). Of 298 wheat samples, only 14 (5%) contained the sum of the T-2 and HT-2 toxins (average 34.2 μg/kg; 10.6-67.7 μg/kg). In oat ( n = 129), these compounds were detected much more frequently (70% of samples) at an average level of 107.5 μg/kg (6.9-949.1 μg/kg). The sum of T-2 and HT-2 glucosides was detectable in 3% of the wheat (average 16.3 μg/kg; 7.1-39.4 μg/kg) and 65% of the oat samples (average 35.1 μg/kg; 4.0-624.1 μg/kg). Following the study, T-2-3-α-glucoside was identified as the only naturally occurring anomer, while both anomers of HT-2-3-glucosides were detected with higher contents and occurrence rates of HT-2-3-β-glucoside than the α anomer of this compound.

Fusarium is a genus that mostly consists of plant pathogenic fungi which are able to produce a broad range of toxic secondary metabolites. In this study, we focus on a type A trichothecene-producing isolate (15-39) of Fusarium sporotrichioides from Lower Austria. We assessed the secondary metabolite profile and optimized the toxin production conditions on autoclaved rice and found that in addition to large amounts of T-2 and HT-2 toxins, this strain was able to produce HT-2-glucoside. The optimal conditions for the production of T-2 toxin, HT-2 toxin, and HT-2-glucoside on autoclaved rice were incubation at 12 °C under constant light for four weeks, darkness at 30 °C for two weeks, and constant light for three weeks at 20 °C, respectively. The HT-2-glucoside was purified, and the structure elucidation by NMR revealed a mixture of two alpha-glucosides, presumably HT-2-3- O -alpha-glucoside and HT-2-4- O -alpha-glucoside. The efforts to separate the two compounds by HPLC were unsuccessful. No hydrolysis was observed with two the alpha-glucosidases or with human salivary amylase and Saccharomyces cerevisiae maltase. We propose that the two HT-2-alpha-glucosides are not formed by a glucosyltransferase as they are in plants, but by a trans-glycosylating alpha-glucosidase expressed by the fungus on the starch-containing rice medium.

The aim of this study is to determine the levels of deoxynivalenol (DON), HT-2 toxin (HT2), T-2 toxin (T2), and ochratoxin A (OTA) in bee products (bee pollen, propolis, honey and royal jelly) available in Turkey. In addition, exposure and health risk assessments were performed to identify the potential health risk of these mycotoxins. The mycotoxins were analyzed by high-performance liquid chromatography (HPLC) with a UV detector and positive samples were confirmed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The most common mycotoxins in all bee products were DON and T-2 toxin, with mean concentrations of 1.601 and 0.704 µg/per kg dry sample, respectively, followed by OTA and HT-2 toxin. It was determined that the mycotoxins taken as a result of consuming bee products in specified amounts do not pose a risk to health., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The contamination of oats with Fusarium toxins poses a high risk for food safety. Among them, trichothecenes are the most frequently reported in European oats, especially in northern countries. The environmental conditions related to the climate change scenario might favour a distribution shift in Fusarium species and the presence of these toxins in Southern European countries. In this paper, we present an ambitious work to determine the species responsible for trichothecene contamination in Spanish oats and to compare the results in the United Kingdom (UK) using a metataxonomic approach applied to both oat grains and soil samples collected from both countries. Regarding T-2 and HT-2 toxin producers, F. langsethiae was detected in 38% and 25% of the oat samples from the UK and Spain, respectively, and to the best of our knowledge, this is the first report of the detection of this fungus in oats from Spain. The relevant type B trichothecene producer, F. poae , was the most frequently detected Fusarium species in oats from both origins. Other important trichothecene producers, such as the Fusarium tricinctum species complex or Fusarium cerealis , were also frequently detected in oat fields. Many Fusarium toxins, including T-2 and HT-2 toxins, deoxynivalenol, or nivalenol, were detected in oat samples. The results obtained in this work revealed a clear change in the distribution of trichothecene producers and the necessity to establish the potential of these species to colonize oats and their ability to produce mycotoxins.

Mycotoxin contamination of food is a constant global concern. There has been a scientific debate in Europe on the validation of accredited detection methods for type A trichothecenes T-2 and HT-2 and the restriction on dangerous concentrations. The issue is of great importance as this type of mycotoxin is frequently found in spring cereals grown in Lithuania. The aim of this study was to optimise and validate a method for the determination of T-2/HT-2 toxin concentrations in oats harvested in 2015-2018 and to observe the changes in the concentrations of both toxins in oat flour during 3- and 6-week storage at different temperatures and increased relative air humidity. All of the oat grain samples (100%) collected in 2015-2018 tested positive for contamination with type A trichothecenes. The highest mean co-contamination by T-2 + HT-2 (260.4 ± 140.9 µg/kg) and the highest concentration (594.6 µg/kg) were determined in 2018 when warm and wet weather conditions prevailed during oat flowering. The effect of long-term storage (6 weeks) on T-2 and HT-2 toxin production manifested itself only when the samples had been stored under cooler conditions (8 °C). The most important factors which impacted the variation of the concentrations of type A trichothecenes in flour were ambient temperature and storage time.

Oats ( Avena sativa L.) are well known for their nutritional properties but are susceptible to the growth of different Fusarium fungi resulting in mycotoxin contamination of harvested oats. In this study, oat samples from harvest years 2011 to 2017 were preselected for their suitability as milling oats for food purposes with DON contents below 1750 µg/kg. The reduction of DON, T-2 and HT-2 toxins during the commercial de-hulling process was analysed. While the average reduction for the sum of T-2 and HT-2 toxins in large oat kernels was 85%, the reduction for thin kernels was 66%. The reduction for DON was about 60% and did not differ for the two kernel fractions. In laboratory de-hulling experiments, milling oat samples and de-hulled oat kernels with known DON, T-2 and HT-2 toxin content were correlated with the associated DNA amount of Fusarium graminearum , Fusarium culmorum and Fusarium langsethiae . The reduction of the Fusarium DNA amount after de-hulling was comparable to the reduction of the associated mycotoxins. Notably, the correlation between F. langsethiae DNA amounts and the sum of T-2 and HT-2 toxin contents was R 2 = 0.69 in milling oats and it rose to R 2 = 0.85 in de-hulled oat kernels. In laboratory tests, at least one third of the initial levels of DON and the sum of T-2 and HT-2 toxins could be removed by polishing off the first parts of the outer layers; two thirds remained in the polished oat kernels. These observations indicate that de-hulling alone may not be completely sufficient to remove mycotoxin contamination in oats. These findings are of high importance in the discussion of determining legal maximum levels for DON or the sum of T-2 and HT-2 toxins in intermediate and final products.

Over recent decades, the Norwegian cereal industry has had major practical and financial challenges associated with the occurrence of Fusarium head blight (FHB) pathogens and their associated mycotoxins in cereal grains. Deoxynivalenol (DON) is one of the most common Fusarium -mycotoxins in Norwegian oats, however T-2 toxin (T2) and HT-2 toxin (HT2) are also commonly detected. The aim of our study was to rank Nordic spring oat varieties and breeding lines by content of the most commonly occurring Fusarium mycotoxins (DON and HT2 + T2) as well as by the DNA content of their respective producers. We analyzed the content of mycotoxins and DNA of seven fungal species belonging to the FHB disease complex in grains of Nordic oat varieties and breeding lines harvested from oat field trials located in the main cereal cultivating district in South-East Norway in the years 2011-2020. Oat grains harvested from varieties with a high FHB resistance contained on average half the levels of mycotoxins compared with the most susceptible varieties, which implies that choice of variety may indeed impact on mycotoxin risk. The ranking of oat varieties according to HT2 + T2 levels corresponded with the ranking according to the DNA levels of Fusarium langsethiae, but differed from the ranking according to DON and Fusarium graminearum DNA. Separate tests are therefore necessary to determine the resistance towards HT2 + T2 and DON producers in oats. This creates practical challenges for the screening of FHB resistance in oats as today's screening focuses on resistance to F. graminearum and DON. We identified oat varieties with generally low levels of both mycotoxins and FHB pathogens which should be preferred to mitigate mycotoxin risk in Norwegian oats.

Milk thistle [ Silybum marianum (L.) Gaertn.] achieved a significant increase in interest over the past few years from local and foreign pharmaceutical corporations. The silymarin complex of constituents extracted from milk thistle achenes provides compelling health benefits primarily thanks to antioxidant activities and hepatoprotective effects. However, consuming mycotoxin-contaminated plant material can cause immunosuppression and hepatotoxic problems. The aim of this study was to develop and validate a method for the determination of mycotoxin content in milk thistle. Fusarium toxins as T-2 and HT-2 toxins in grown milk thistle harvested from a breeding station in the Czech Republic during 2020-2021 were studied. The analysis of T-2 and HT-2 toxins was performed by UPLC-MS/MS after immunoaffinity columns EASI-EXTRACT ® T-2 & HT-2 clean up. All analysed samples of milk thistle were contaminated with T-2 toxin and HT-2 toxin. The content of T-2 toxin in the samples from 2020 was in the range of 122.7-290.2 µg/kg and HT-2 toxin 157.0-319.0 µg/kg. In 2021, the content of T-2 toxin was in the range of 28.8-69.9 µg/kg and HT-2 toxin was 24.2-75.4 µg/kg. The results show that the climatic conditions of the year of harvesting have a highly statistically significant effect on the content of T-2 and HT-2 toxins in milk thistle.

Wheat straw is commonly used as a cellulose source in mushroom compost and could be a secondary source of mycotoxin contamination in the food chain. We cultivated edible Agaricus bisporus and Pleurotus ostreatus on T-2/HT-2 artificially-contaminated mushroom compost and developed and in-house validated an UHPLC-MS/MS method for determination of T-2, HT-2, T2-triol and T2-tetraol in mushroom compost and mushroom basidiocarp. A rapid phase I metabolization of T-2 and HT-2 in mushroom compost was observed. In Agaricus bisporus , basidiocarps 8-15 µg kg -1 accumulation of HT-2 calculated on wet weight was measured. No detectable mycotoxins were found in Pleurotus ostreatus basidiocarp.

T-2 toxin usually co-occurs with HT-2 toxin and neosolaniol (NEO) in the grains and feed. Our previous studies found that T-2 toxin and its metabolites' binary or ternary combination exposure to porcine Leydig cells (LCs) displayed synergism in certain range of dosage and cannot be predicted based on individual toxicity. However, the possible mechanism of these mycotoxins' combined exposure to cell lesions remains unknown. Based on 50% cell viability, the mechanism of apoptosis in porcine Leydig cells was investigated after exposure to T-2, HT-2, NEO individual and binary or ternary combinations. Compared with control, the adenosine triphosphate (ATP) content decreased, reactive oxygen species (ROS) level increased, and mitochondrial membrane potential (MMP) decreased in all treated groups. Additionally, the cell apoptosis rates were significantly increased in test groups ( p < 0.05), and the B-cell lymphoma 2 (Bcl-2) Associated X (Bax)/Bcl-2 ratio and the expression of caspase 3, caspase 8, cytochrome c (Cytc) in the treated group are all significantly higher than the control group. Moreover, the expression of Cytc and caspase 8 gene in NEO and T-2+NEO groups was significantly higher than that in other individual and combined groups. It can be concluded that the toxicities of T-2, HT-2, and NEO individually and in combination can induce apoptosis related to the oxidative stress and mitochondrial damage, and the synergistic effect between toxins may be greater than a single toxin effect, which is beneficial for assessing the possible risk of the co-occurrences in foodstuffs to human and animal health.

The metabolic profile of T-2 toxin (T-2) and its modified form T-2-3-glucoside (T-2-3-Glc) remain unexplored in human samples. Therefore, the present study aimed to investigate the presence of T-2, T-2-3-Glc and their respective major metabolites in human urine samples ( n = 300) collected in South Italy through an ultra-high performance liquid chromatography (UHPLC) coupled to Q-Orbitrap-HRMS methodology. T-2 was quantified in 21% of samples at a mean concentration of 1.34 ng/mg Crea (range: 0.22-6.54 ng/mg Crea). Almost all the major T-2 metabolites previously characterized in vitro were tentatively found, remarking the occurrence of 3'-OH-T-2 (99.7%), T-2 triol (56%) and HT-2 (30%). Regarding T-2-3-Glc, a low prevalence of the parent mycotoxin (1%) and its metabolites were observed, with HT-2-3-Glc (17%) being the most prevalent compound, although hydroxylated products were also detected. Attending to the large number of testing positive for T-2 or its metabolites, this study found a frequent exposure in Italian population.

The efficacy of ethylene-vinyl alcohol copolymer films (EVOH) incorporating the essential oil components cinnamaldehyde (CINHO), citral (CIT), isoeugenol (IEG), or linalool (LIN) to control growth rate (GR) and production of T-2 and HT-2 toxins by Fusarium sporotrichioides cultured on oat grains under different temperature (28, 20, and 15 °C) and water activity (a w ) (0.99 and 0.96) regimes was assayed. GR in controls/treatments usually increased with increasing temperature, regardless of a w , but no significant differences concerning a w were found. Toxin production decreased with increasing temperature. The effectiveness of films to control fungal GR and toxin production was as follows: EVOH-CIT > EVOH-CINHO > EVOH-IEG > EVOH-LIN. With few exceptions, effective doses of EVOH-CIT, EVOH-CINHO, and EVOH-IEG films to reduce/inhibit GR by 50%, 90%, and 100% (ED 50 , ED 90 , and ED 100 ) ranged from 515 to 3330 µg/culture in Petri dish (25 g oat grains) depending on film type, a w , and temperature. ED 90 and ED 100 of EVOH-LIN were >3330 µg/fungal culture. The potential of several machine learning (ML) methods to predict F. sporotrichioides GR and T-2 and HT-2 toxin production under the assayed conditions was comparatively analyzed. XGBoost and random forest attained the best performance, support vector machine and neural network ranked third or fourth depending on the output, while multiple linear regression proved to be the worst.

In the UK and Northern Europe, ripening oats can become contaminated with T-2 and HT-2 mycotoxins, produced mainly by Fusarium langsethiae. There are indicative levels related to the maximum limits for oat grain for these toxins. The objectives of this study were to examine the effect of interacting conditions of temperature (10-30 °C) and water activity (a w , 0.995-0.90) on (a) lag times prior to growth, (b) growth and (c) T-2 and HT-2 toxins by two strains of F. langsethiae isolated from oats in the UK and compare this with the type strain (Fl201059) which has been genomically sequenced, and (d) develop (and validated with published data) a probabilistic models for impacts of temperature × a w on growth and toxin production. All three strains had an optimum a w range and temperature of 0.995-0.98 and 25 °C for growth. For T-2 + HT-2 production these were 0.995 a w and 20 °C. Overall, the type strain produced higher amounts of T-2 + HT-2 with a HT-2/T-2 ratio of up to 76. Using this study data sets and those from the literature, probabilistic models were developed and validated for growth and T-2 + HT-2 toxin production in relation to temperature × a w conditions. These models, when applied in stored oats, will be beneficial in determining the conditions on the relative level of risk of contamination with these two toxins in the context of the EU indicative maximum levels., (Copyright © 2021 Elsevier B.V. All rights reserved.)

HT-2 toxin (HT-2), a mycotoxin produced by Fusarium species, is detected in a variety of cereal grain-based human food and animal feed. Apart from its well-established immunotoxicity and haematotoxicity, it also causes reproductive disorders. In the present study, we revealed the adverse effects of HT-2 on early oogenesis at the foetal stage. Pregnant mice were orally administered with HT-2 for 3 days at mid-gestation. Oocytes from female foetuses exposed to HT-2 displayed defects in meiotic prophase, including unrepaired DNA damage, elevated recombination levels, and reduced expression of meiotic-related genes. Subsequently, increased oxidative stress was observed in the foetal ovaries exposed to HT-2, along with the elevated levels of reactive oxygen species, malondialdehyde, catalase, and superoxide dismutase 1/2, thereby resulting in impaired mitochondrial membrane potential and cell apoptosis. Furthermore, pre-treatment with urolithin A, a natural compound with antioxidant activities, partially reversed the delayed meiotic process by alleviating oxidative stress. Since early oogenesis is essential to determine female fertility in adult life, this study indicated that brief maternal exposure to HT-2 toxin may compromise the fertility of a developing female foetus., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Wheat (Triticum aestivum) is susceptible to mycotoxin contamination, which can result in significant health risks and economic losses. This research examined the ability of air atmospheric cold plasma (air-ACP) treatment to reduce pure and spiked T-2 and HT-2 mycotoxins' concentration on wheat grains. This study also evaluated the effect of ACP treatment using different gases on wheat grain germination parameters. The T-2 and HT-2 mycotoxin solutions applied on round cover-glass were placed on microscopy slides and wheat grains (0.5 g) were individually spiked with T-2 and HT-2 on their surfaces. Samples were then dried at room temperature (∼24 °C) and treated by air-ACP for 1 to 10 min. Ten minutes of air-ACP treatment significantly reduced pure T-2 and HT-2 concentrations by 63.63% and 51.5%, respectively. For mycotoxin spiked on wheat grains, 10 min air-ACP treatment significantly decreased T-2 and HT-2 concentrations up to 79.8% and 70.4%, respectively. No significant change in the measured quality and color parameters was observed in the ACP-treated samples. Wheat grain germination parameters were not significantly different, when treated with ACP using different gases. Air-ACP treatment and ACP treatment using 80% nitrogen + 20% oxygen improved the germination of wheat grains by 10% and 6%, respectively. This study demonstrated that ACP is an innovative technology with the potential to improve the safety of wheat grains by reducing T-2/HT-2 mycotoxins with an additional advantage of improving their germination. PRACTICAL APPLICATION: Atmospheric cold plasma (ACP) technology has a huge potential to degrade mycotoxins in food grains. This study evaluated the efficacy of ACP to reduce two major mycotoxins (T-2 and HT-2 toxins) in wheat grains. The results of this study will help to develop and scale-up the ACP technology for mycotoxin degradation in grains., (© 2021 Institute of Food Technologists®.)

HT-2 toxin is widely found in moldy crops and is the major metabolite of T-2 toxin, which has been shown to exert various toxic effects in farm animals. However, little is known about the effects of HT-2 toxin on male reproduction, particularly spermatogenesis. This study aims to investigate the toxic effects of HT-2 toxin on goat spermatogonial stem cells (SSCs) and related autophagy-regulated mechanisms. Our results showed that HT-2 toxin exposure resulted in decreased cell viability and proliferation, disrupted SSCs self-renewal, and reduced germ cell-related gene expression. HT-2 toxin exposure also induced oxidative stress and cell apoptosis, as shown by ROS accumulation, increased antioxidant enzyme activity levels, decreased the mitochondrial membrane potential, and increased caspase-9 mRNA and Bcl/bax protein levels. Additionally, HT-2 toxin exposure increased the expression of the autophagy-inducing genes Atg5, Atg7 and Beclin1 and the number of autophagosomes, which indicated that HT-2 toxin induced autophagy in the goat SSCs. Moreover, we also examined a possible mechanism by which HT-2 toxin exposure induced higher expression of AMPK, mTOR and ULK at both the mRNA and protein levels. our results indicated that HT-2 toxin caused apoptosis and autophagy by activating AMPK-mTOR-ULK1 pathway, which further affected SSCs viability., Competing Interests: Declaration of competing interest The authors have declared that they have no potential conflicts of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

A fast high performance liquid chromatography tandem mass spectrometry multi-method based on an ACN-precipitation extraction was developed for the analysis of 41 (modified) mycotoxins in beer. Validation according to the performance criteria defined by the European Commission (EC) in Commission Decision no. 657/2002 revealed good linearity (R 2  > 0.99), repeatability (RSDr < 15%), reproducibility (RSDR < 15%), and recovery (79-100%). Limits of quantification ranging from 0.04 to 75 µg/L were obtained. Matrix effects varied from -67 to +319% and were compensated for using standard addition. In total, 87 beer samples, produced worldwide, were analyzed for the presence of mycotoxins with a focus on modified mycotoxins, whereof 76% of the samples were contaminated with at least one mycotoxin. The most prevalent mycotoxins were deoxynivalenol-3-glucoside (63%), HT-2 toxin (15%), and tenuazonic acid (13%). Exposure estimates of deoxynivalenol and its metabolites for German beer revealed no significant contribution to intake of deoxynivalenol., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Within the European Union (EU), edible insects need to be approved as "Novel Food" according to Regulation (EU) 2015/2283 and must comply with the requirements of European food law with regard to microbiological and chemical food safety. Substrates used for feeding insects are susceptible to the growth of Fusarium spp. and consequently to contamination with trichothecene mycotoxins. Therefore, the current study aimed to investigate the influence of T-2 and HT-2 toxins on the larval life cycle of yellow mealworm (Tenebrio molitor (L.)) and to study the transfer of T-2, HT-2, T-2 triol and T-2 tetraol in the larvae. In a 4-week feeding study, T. molitor larvae were kept either on naturally (oat flakes moulded with Fusarium sporotrichioides) or artificially contaminated oat flakes, each at two levels (approximately 100 and 250 μg/kg total T-2 and HT-2). Weight gain and survival rates were monitored, and mycotoxins in the feeding substrates, larvae and residues were determined using LC-MS/MS. Larval development varied between the diets and was 44% higher for larvae fed artificially contaminated diets. However, the artificially contaminated diets had a 16% lower survival rate. No trichothecenes were detected in the surviving larvae after harvest, but T-2 and HT-2 were found both in the dead larvae and in the residues of naturally and artificially contaminated diets.

The T-2 toxin (T-2) is commonly metabolized to HT-2 toxin (HT-2), Neosolaniol (NEO), T2-triol and T2-tetraol and they can modify the toxicity of T-2. In this study, T-2 and its modified forms were evaluated by in vitro and in silico methods. The in vitro cytotoxicity individually was evaluated by MTT and Total Protein Content (PC) assays in human hepatocarcinoma (HepG2) cells. The order of IC 50 was T-2 tetraol > T-2 triol > NEO > T-2 = HT-2. The T-2 and HT-2 evidenced the highest cytotoxic effect in HepG2 cells individually. No differences were observed in binary combinations tested and the two mycotoxins in the mixture tested individually. The T-2+HT-2 combination showed the highest toxic potential with the lowest IC 50 value of 34.42 ± 0.58 nM at 24 h. All binary combinations exhibited antagonistic interactions. The ADME and toxicity profile of mycotoxins were obtained by the in silico admetSAR predictive model which determines the metabolic and toxicological approaches in order to know if these mycotoxins might be taken into consideration to support a more realistic and adequate risk assessment., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

To investigate into the T-2 and HT-2 toxin occurrence, 240 samples of unprocessed cereals (maize, wheat, barley, and oats) were sampled from different fields located in three Croatian regions during 2017-2018. In all samples, sum concentrations of T-2/HT-2 toxin were determined using the ELISA method, while the LC-MS/MS was used as a confirmatory method for both mycotoxins in positive samples (>LOD) and the establishment of T-2 over HT-2 toxin ratios. The results showed oats to be the most contaminated cereal, with T-2/HT-2 toxins detected in 70.0% of samples, followed by barley (40.9%), maize (26.8%) and wheat (19.2%), with the mean T-2/HT-2 ratio ranging from 1:2.7 in maize to 1:4.4 in oats. Sum T-2/HT-2 concentrations in two maize samples were higher than the indicative level recommended by the European Commission, necessitating subsequent investigations into the conditions under which these poorly investigated mycotoxins are produced. Statistically significantly ( p < 0.05) higher concentrations of T-2/HT-2 toxin were determined in oats throughout study regions as compared to those found in wheat, but not maize and barley, while the concentrations of these mycotoxins were related to the regional weather in Croatia.

An analytical method for the simultaneous determination of trichothecenes-namely, nivalenol (NIV), deoxynivalenol (DON) and its acetylated derivatives (3- and 15-acetyl-DON), T-2 and HT-2 toxins-and zearalenone (ZEN) in wheat, wheat flour, and wheat crackers was validated through a collaborative study involving 15 participants from 10 countries. The validation study, performed within the M/520 standardization mandate of the European Commission, was carried out according to the IUPAC (International Union of Pure and Applied Chemistry) International Harmonized Protocol. The method was based on mycotoxin extraction from the homogenized sample material with a mixture of acetonitrile-water followed by purification and concentration on a solid phase extraction column. High-performance liquid chromatography coupled with tandem mass spectrometry was used for mycotoxin detection, using isotopically labelled mycotoxins as internal standards. The tested contamination ranges were from 27.7 to 378 μg/kg for NIV, from 234 to 2420 μg/kg for DON, from 18.5 to 137 μg/kg for 3-acetyl-DON, from 11.4 to 142 μg/kg for 15-acetyl-DON, from 2.1 to 37.6 μg/kg for T-2 toxin, from 6.6 to 134 μg/kg for HT-2 toxin, and from 31.6 to 230 μg/kg for ZEN. Recoveries were in the range 71-97% with the lowest values for NIV, the most polar mycotoxin. The relative standard deviation for repeatability (RSD r ) was in the range of 2.2-34%, while the relative standard deviation for reproducibility (RSD R ) was between 6.4% and 45%. The HorRat values ranged from 0.4 to 2.0. The results of the collaborative study showed that the candidate method is fit for the purpose of enforcing the legislative limits of the major Fusarium toxins in wheat and wheat-based products.

A method to determine T-2 and HT-2 toxins, both in feed and food, is described. The method is based on sample clean-up by immunoaffinity columns, followed by triple quadrupole LC-MS/MS determination. It was in-house validated in compliance with the European Union legislation, in the concentration range 5.0-750 µg kg -1 and accredited according to the ISO/IEC 17025 standard, for the scope of official control of feed and food. The method was applied to monitor the contamination levels of T-2 and HT-2 toxins in 105 feed samples, both raw materials and compound feed and 17 cereals and derived products for human consumption, collected in Southern Italy between 2015 and 2019. T-2 toxin was quantified in 32 feed (30.5%) and in 2 food samples (11.8%). HT-2 toxin was quantified in 65 feed (61.9%) and in 3 food (17.6%) samples.

Fusarium mycotoxins are fungal contaminants found in different crops intended for human and animal consumption. Due to the co-occurrence of several of mycotoxins, the present study aimed at examining the transfer of these toxins into tissues of broiler chickens and eggs of laying hens fed contaminated diets. After an adaptation period, the chickens were fed contaminated diets containing mg/kg levels of deoxynivalenol (DON), enniatins (ENN A, A 1 , B, B 1 ) and beauvericin (BEA) and high μg/kg levels of HT-2 toxin (HT-2), T-2 toxin (T-2) and zearalenone (ZEN) during a repletion period of two weeks, followed by a depletion period of two weeks. DON, ZEN, T-2 and HT-2 were not carried out into the skin and the liver of broiler chickens. ENN B (20.5 ± 6.6 μg/kg) and BEA (162 ± 55 μg/kg) were found in the liver, while in the skin their respective concentrations were 50 ± 17 μg/kg and 120 ± 16 μg/kg during the first week of the repletion period. Carry-over rates into liver and skin were higher for BEA (1.6% and 1.2%, respectively) than for ENNs (0.1 and 0.4%, respectively). During the depletion period, ENNs and BEA were eliminated from the skin and the liver. ENN B, ENN B 1 and BEA were carried over into eggs at 0.1%, 0.05% and 0.44% upon 2-3 days of feeding the contaminated diet, respectively. These transfers were fully eliminated 9-10 days after feeding the control diet again. These results indicate the transfer of ENN B, ENN B 1 and BEA from feed to chicken offal, meat products and eggs at a very low degree, thus marginally contribute to the total dietary intake of these fusariotoxins for consumers. Nevertheless, taking precautionary measures in the field, harvest, transport and storage of the raw materials is required to keep the mycotoxin concentration in feed below the safe levels., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

T-2 toxin is the most toxic trichothecene mycotoxin, and it exerts potent toxic effects, including immunotoxicity, neurotoxicity, and reproductive toxicity. Recently, several novel metabolites, including 3',4'-dihydroxy-T-2 toxin and 4',4'-dihydroxy-T-2 toxin, have been uncovered. The enzymes CYP3A4 and carboxylesterase contribute to T-2 toxin metabolism, with 3'-hydroxy-T-2 toxin and HT-2 toxin as the corresponding primary products. Modified forms of T-2 toxin, including T-2-3-glucoside, exert their immunotoxic effects by signaling through JAK/STAT but not MAPK. T-2-3-glucoside results from hydrolyzation of the corresponding parent mycotoxin and other metabolites by the intestinal microbiota, which leads to enhanced toxicity. Increasing evidence has shown that autophagy, hypoxia-inducible factors, and exosomes are involved in T-2 toxin-induced immunotoxicity. Autophagy promotes the immunosuppression induced by T-2 toxin, and a complex crosstalk between apoptosis and autophagy exists. Very recently, "immune evasion" activity was reported to be associated with this toxin; this activity is initiated inside cells and allows pathogens to escape the host immune response. Moreover, T-2 toxin has the potential to trigger hypoxia in cells, which is related to activation of hypoxia-inducible factor and the release of exosomes, leading to immunotoxicity. Based on the data from a series of human exposure studies, free T-2 toxin, HT-2 toxin, and HT-2-4-glucuronide should be considered human T-2 toxin biomarkers in the urine. The present review focuses on novel findings related to the metabolism, immunotoxicity, and human exposure assessment of T-2 toxin and its modified forms. In particular, the immunotoxicity mechanisms of T-2 toxin and the toxicity mechanism of its modified form, as well as human T-2 toxin biomarkers, are discussed. This work will contribute to an improved understanding of the immunotoxicity mechanism of T-2 toxin and its modified forms.

Fusarium mycotoxins are common contaminants in cereals and often co-occur with plant-derived mycotoxin sugar conjugates. Several of these modified mycotoxins are not degraded in the small intestine and hence carried through to the large intestine where microbial transformation may occur. This study aims to assess the gastrointestinal stability of the trichothecenes HT-2 toxin (HT-2), HT-2-β-glucoside (HT-2-Glc), diacetoxyscirpenol (DAS), DAS-α-glucoside (DAS-Glc) and fumonisin B1 (FB 1 ), N -(1-deoxy-d-fructos-1-yl) fumonisin-B 1 (NDF-FB 1 ). All tested modified mycotoxins were stable under upper gastrointestinal (GI) conditions. In faecal batch culture experiments, HT-2-Glc was hydrolysed efficiently and no further microbial biotransformation of HT-2 was observed. DAS-Glc hydrolysis was slow and DAS was de-acetylated to 15-monoacetoxyscripenol. NDF-FB 1 was hydrolysed at the slowest rate and FB 1 accumulation varied between donor samples. Our results demonstrate that all tested modified mycotoxins are stable in the upper GI tract and efficiently hydrolysed by human gut microbiota, thus potentially contributing to colonic toxicity. Hence the microbial biotransformation of any novel modified mycotoxins needs to be carefully evaluated.

T-2 and its major metabolite HT-2 toxin are naturally occurring contaminants in cereals, with the highest concentrations determined in oats. Because of their toxicity and resistance to conventional methods used in mycotoxin degradation, development of new effective procedures for reduction of T-2/HT-2 toxin levels is needed. The aim of this study was to investigate the effect of different gas types within various timeframes on the efficiency of T-2 and HT-2 toxin degradation by low pressure dielectric barrier discharge (DBD) plasma in oat flour. Although humidity of the sample influences the intensity of the plasma, oxygen atoms efficiently oxidize the sample and produce CO and N 2 . Before and after treatment, T-2 and HT-2 toxin concentrations were analysed by the confirmatory liquid chromatography tandem-mass spectrometry (LC-MS/MS) method. The highest reduction of T-2 and HT-2 toxin of 43.25% and 38.54%, respectively, was achieved using nitrogen plasma and this proved to be time-dependent., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The design and application of an inkjet-printed electrochemically reduced graphene oxide microelectrode for HT-2 mycotoxin immunoenzymatic biosensing is reported. A water-based graphene oxide ink was first formulated and single-drop line working microelectrodes were inkjet-printed onto poly(ethylene 2,6-naphthalate) substrates, with dimensions of 78 μm in width and 30 nm in height after solvent evaporation. The printed graphene oxide microelectrodes were electrochemically reduced and characterized by Raman and X-ray photoelectron spectroscopies in addition to microscopies. Through optimization of the electrochemical reduction parameters, differential pulse voltammetry were performed to examine the sensing of 1-naphthol (1-N), where it was revealed that reduction times had significant effects on electrode performance. The developed microelectrodes were then used as an immunoenzymatic biosensor for the detection of HT-2 mycotoxin based on carbodiimide linking of the microelectrode surface and HT-2 toxin antigen binding fragment of antibody (anti-HT2 (10) Fab). The HT-2 toxin and anti-HT2 (10) Fab reaction was reported by anti-HT2 immune complex single-chain variable fragment of antibody fused with alkaline phosphatase (anti-IC-HT2 scFv-ALP) which is able to produce an electroactive reporter - 1-N. The biosensor showed detection limit of 1.6 ng ∙ mL -1 and a linear dynamic range of 6.3 - 100.0 ng ∙ mL -1 within a 5 min incubation with 1-naphthyl phosphate (1-NP) substrate., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 β 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.

T-2 toxin, one of the most toxic mycotoxins, is commonly presented along with its metabolites, HT-2 toxin, neosolaniol (NEO), T-2 triol, and T-2 tetraol in foodstuff and feed. The aim of this study was to evaluate the cytotoxic effects of T-2 toxin alone and in combination with its metabolites on porcine Leydig cells. Based on the determination of cell viability with CCK-8, toxicological interactions were investigated using Combination Index method. The cytotoxic potency of five tested mycotoxins individual and their mixtures all showed with a dose-dependent manner. In view of IC 50 values, the decreasing cytotoxicity of mycotoxins was ranking: T-2 toxin > HT-2 toxin > T-2 triol > NEO > T-2 tetraol. Combinations of T-2+HT-2, T-2+NEO, and HT-2+NEO displayed synergism at low doses but antagonism at high doses, while the ternary combination of T-2+HT-2+NEO revealed adverse situation from antagonism to synergism. All binary and ternary combinations of T-2 toxin, T-2 triol, and T-2 tetraol exhibited antagonistic interactions. Our results suggest that the co-occurrence of T-2 toxin and its metabolites might pose a slight threat to reproductive health due to antagonistic interactions. However, the synergy observed should be not ignored especially at low doses of mycotoxins co-occurrence in the diet., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A dietary exposure assessment to sum of deoxynivalenol (DON) forms, sum of T-2/HT-2 toxins (T2/HT2) and zearalenone (ZEA) was conducted for Czech children 4-6 years and Czech men and women 18-59 years. Retail foods (25 different commodities, n = 336) were assessed by LC-MS/MS methods. The 95th percentile chronic exposure to sum of DON forms was determined in children from 648 to 1030 ng/kg bw/day (LB/lower bound/and UB/upper bound/), in men from 362 to 923 ng/kg bw/day and in women from 272 to 490 ng/kg bw/day. The 95th percentile chronic exposure to sum T2/HT2 was determined in children from 6.5 to 31 ng/kg bw/day, in men from 1.9 to 11.2 ng/kg bw/day and in women from 2.5 to 11.5 ng/kg bw/day. The 95th percentile chronic exposure to ZEA was determined in children from 11.9 to 24.9 ng/kg bw/day, in men from 5.9 to 27.5 ng/kg bw/day and in women from 4.8 to 12.6 ng/kg bw/day. The risk linked with the mean and the 95th percentile chronic exposure (LB scenario) to the sum of DON forms, sum of T2/HT2 and ZEA is considered to be out of health concern for the selected population groups., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

T-2 and HT-2 widely found in food products can seriously affect human and animal health. In this study, sterilized corn was inoculated with F. poae and incubated to allow fungal growth before being examined via liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) to determine the concentrations of T-2/HT-2. Broilers were then fed with a mix of moldy corn and normal feed at different ratios to obtain different toxin doses. After 35 days, the contaminated feed was replaced with mycotoxin-free feed and the distribution and concentration of residual toxins in the tissues and organs of the chickens were examined at different time points. The results showed that at the time of feed replacement (0 h), T-2 residue was present at significantly higher concentrations in the lungs and small intestines than in other tissues (P < 0.05). In addition, T-2 concentrations increased in a dose-dependent manner in the tissues of chickens in the low-, medium-, and high-dose groups; however, the differences in concentration between the groups were not statistically significant. The HT-2 content (0 h) in the livers and small intestines was significantly higher than that in other tissues (P < 0.05). At 48 h post-feed replacement, the concentration of T-2 dropped below detectable levels in all tissues while HT-2 could still be detected at 192 h post-feed replacement. Thus, this study reveals the distribution and persistence of residual T-2/HT-2 from moldy feed in broilers, providing a reference for the detection of these toxins in animal-derived food products and a theoretical basis for formulating food-safety and quality standards., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cereal foods are commonly contaminated with multiple mycotoxins resulting in frequent human mycotoxin exposure. Children are at risk of high-level exposure because of their high cereal intake relative to body weight. Hence, this study aims to assess multimycotoxin exposure in UK children using urinary biomarkers. Spot urines ( n = 21) were analyzed for multimycotoxins (deoxynivalenol, DON; nivalenol, NIV; ochratoxin A, OTA; zearalenone, ZEN; α-zearalenol, α-ZEL; β-zearalenol, β-ZEL; T-2 toxin, T-2; HT-2 toxin, HT-2; and aflatoxin B 1 and M 1, AFB 1 , AFM 1 ) using liquid chromatography-coupled tandem mass spectrometry. Urine samples frequently contained DON (13.10 ± 12.69 ng/mL), NIV (0.36 ± 0.16 ng/mL), OTA (0.05 ± 0.02 ng/mL), and ZEN (0.09 ± 0.07 ng/mL). Some samples (1-3) contained T-2, HT-2, α-ZEL, and β-ZEL but not aflatoxins. Dietary mycotoxin estimation showed that children were frequently exposed to levels exceeding the tolerable daily intake (52 and 95% of cases for DON and OTA). This demonstrates that UK children are exposed to multiple mycotoxins through their habitual diet.

Thyroid hormone triiodothyronine (T 3 ) plays an important role in coordinated endochondral ossification and hypertrophic differentiation of the growth plate, while aberrant thyroid hormone function appears to be related to skeletal malformations, osteoarthritis, and Kashin-Beck disease. The T-2 toxin, present extensively in cereal grains, and one of its main metabolites, HT-2 toxin, are hypothesized to be potential factors associated with hypertrophic chondrocyte-related osteochondropathy, known as the Kashin-Beck disease. In this study, we investigated the effects of T 3 and HT-2 toxin on human chondrocytes. The immortalized human chondrocyte cell line, C-28/I2, was cultured in four different groups: controls, and cultures with T 3 , T 3 plus HT-2 and HT-2 alone. Cytotoxicity was assessed using an MTT assay after 24-h-exposure. Quantitative RT-PCR was used to detect gene expression levels of collagen types II and X , aggrecan and runx2 , and the differences in runx2 were confirmed with immunoblot analysis. T 3 was only slightly cytotoxic, in contrast to the significant, dose-dependent cytotoxicity of HT-2 alone at concentrations ≥ 50 nM. T 3 , together with HT-2, significantly rescued the cytotoxic effect of HT-2. HT-2 induced significant increases in aggrecan and runx2 gene expression, while the hypertrophic differentiation marker, type X collagen , remained unchanged. Thus, T 3 protected against HT-2 induced cytotoxicity, and HT-2 was an inducer of the pre-hypertrophic state of the chondrocytes.

The present study aimed at elucidating the uptake and biotransformation of T2 and HT2 toxins in five cultivars of durum wheat, by means of cultured plant organs. An almost complete absorption of T2 toxin (up to 100 µg) was noticed after 7 days, along with the contemporaneous formation of HT2 in planta, whereas HT2 showed a slower uptake by uninfected plant organs. Untargeted MS-analysis allowed to identify a large spectrum of phase I and phase II metabolites, resulting in 26 T2 and 23 HT2 metabolites plus tentative isomers. A novel masked mycotoxin, 3-acetyl-HT2-glucoside, was reported for the first time in wheat. The in vitro approach confirmed its potential to both investigate the contribution of plant metabolism in the biosynthesis of masked mycotoxins and to foresee the development of biocatalytic tools to develop nature-like mixtures to be used as reference materials.

Cereal grains are essential ingredient in food, feed and industrial processing. One of the major causes of cereal spoilage and mycotoxin contamination is the presence of toxigenic Fusarium spp. Nanoparticles have immense applications in agriculture, nutrition, medicine or health but their possible impact on the management of toxigenic fungi and mycotoxins have been very little explored. In this report, the potential of silver nanoparticles (AgNPs) (size 14-100 nm) against the major toxigenic Fusarium spp. affecting crops and their effect on mycotoxin accumulation is evaluated for the first time. The studied Fusarium spp. (and associated mycotoxins) were F. graminearum and F. culmorum (deoxynivalenol, 3-acetyldeoxynivalenol and zearalenone), F. sporotrichioides and F. langsethiae (T-2 and HT-2 toxins), F. poae (nivalenol), F. verticillioides and F. proliferatum (fumonisins B 1 and B 2 ) and F. oxysporum (mycotoxins no detected). The factors fungal species, AgNP dose (range 2-45 μg/mL), exposure time (range 2-30 h) and their interactions significantly influence spore viability, lag period and growth rate (GR) in subsequent cultures in maize-based medium (MBM) of all the studied species. The effective lethal doses (ED 50 , ED 90 and ED 100 ) to control spore viability and GR were in the range 1->45 μg/mL depending on the remaining factors. At high exposure times (20-30 h), the three effective doses ranged 1-30 μg/mL for all the studied species. At the end of the incubation period (10 days) mycotoxin levels in MBM cultures inoculated with fungal spores from treatments were strongly related with the size reached by the colony at that time. None of the treatments produced stimulation in conidia germination, GR or mycotoxin biosynthesis with respect to controls. Thus, the antifungal effect of the assayed AgNPs against the tested Fusarium spp. suggests that AgNPs could be a new antifungal ingredient in bioactive polymers (paints, films or coating) likely to be implemented in the agro-food sector for controlling these important toxigenic Fusarium spp. and their main associated mycotoxins., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This paper describes a methodology for hazard assessment of groups of related substances for which toxicity data are insufficient, and which utilises, next to conventional toxicological assessments and mechanistic information, the derivation of relative toxicity potency factors (RPFs). Zearalenone (ZEN) and T-2 toxin (T2) and HT-2 toxin (HT2) and their modified forms have been used as examples. A tolerable daily intake (TDI) for ZEN of 0.25 μg/kg bw was established. In vitro and in vivo studies suggested that modified forms of ZEN act via the same mode of action as ZEN (oestrogenicity). Results from in vivo uterotrophic assays were used to establish RPFs, allowing inclusion the different modified forms in a group TDI with ZEN. A TDI for the sum of T2/HT2 of 0.02 μg/kg bw per day and an acute reference dose (ARfD) of 0.3 μg/kg bw for the sum of T2/HT2 was established. In vitro studies show that phase I metabolites of T2/HT2 act via a similar mode of action as their parent compounds, namely protein synthesis inhibition with immune- and haematotoxicity. The phase I metabolites as well as conjugates of T2/HT2 and their phase I metabolites can be included in a group TDI with T2/HT2 applying RPFs., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Two garlic-derived compounds, Propyl Propane Thiosulfonate (PTS) and Propyl Propane Thiosulfinate (PTSO), were examined for their efficacy against mycotoxigenic Fusarium species ( F. graminearum , F. langsethiae , F. verticillioides ). The objectives were to assess the inhibitory effect of these compounds on growth and mycotoxin production in vitro , and in situ in artificially inoculated wheat, oats and maize with one isolate of each respectively, at different water activity (a w ) conditions when stored for up to 20 days at 25 °C. In vitro , 200 ppm of either PTS or PTSO reduced fungal growth by 50-100% and mycotoxin production by >90% depending on species, mycotoxin and a w conditions on milled wheat, oats and maize respectively. PTS was generally more effective than PTSO. Deoxynivalenol (DON) and zearalenone (ZEN) were decreased by 50% with 80 ppm PTSO. One-hundred ppm of PTS reduced DON and ZEN production in wheat stored at 0.93 a w for 20 days, although contamination was still above the legislative limits. Contrasting effects on T-2/HT-2 toxin contamination of oats was found depending on a w , with PTS stimulating production under marginal conditions (0.93 a w ), but at 0.95 a w effective control was achieved with 100 ppm. Treatment of stored maize inoculated with F. verticilliodies resulted in a stimulation of total fumonsins in most treatments. The potential use of such compounds for mycotoxin control in stored commodities is discussed.

This study deals with the influence of food matrix components on the degradation of the mycotoxins T-2 toxin (T-2) and HT-2 toxin (HT-2) and with the binding of T-2 to starch during thermal food processing. Both mycotoxins were heated in a simulated food environment and subsequently analyzed via HPLC-HRMS to generate degradation curves and to draw conclusions regarding the thermal degradation under food processing conditions. Thermal degradation increased generally with increasing time and temperature with a maximum degradation rate of 93% (T-2) and 99% (HT-2). Furthermore, HRMS data were exploited to screen the samples for degradation products. In model heating experiments, T-2 was bound to 1-O-methyl-α-D-glucopyranoside, a model compound that was used to simulate starch. The formed reaction products were isolated and identified by NMR, giving detailed insights into a potential binding of T-2 to starch. In the next step, further model heating experiments were performed, which proved the covalent binding of T-2 to starch. Finally, the amount of matrix-bound T-2 was estimated roughly in a semi-quantitative approach in the model heating experiments as well as during cookie-making via GC-MS analysis of the isovaleric acid ester moiety of T-2, released after alkaline hydrolysis.

This study examined the effect of climate change (CC) abiotic factors of temperature (20, 25, 30 °C), water activity (a w ; 0.995, 0.98) and CO 2 exposure (400, 1000 ppm) may have on (a) growth, (b) gene expression of biosynthetic toxin genes (Tri5, Tri6, Tri16), and (c) T-2/HT-2 toxins and associated metabolites by Fusarium langsethiae on oat-based media and in stored oats. Lag phases and growth were optimum at 25 °C with freely available water. This was significantly reduced at 30 °C, at 0.98 a w and 1000 ppm CO 2 exposure. In oat-based media and stored oats, Tri5 gene expression was reduced in all conditions except 30 °C, 0.98 a w , elevated CO 2 where there was a significant (5.3-fold) increase. The Tri6 and Tri16 genes were upregulated, especially in elevated CO 2 conditions. Toxin production was higher at 25 °C than 30 °C. In stored oats, at 0.98 a w , elevated CO 2 led to a significant increase (73-fold) increase in T2/HT-2 toxin, especially at 30 °C. Nine T-2 and HT-2 related metabolites were detected, including a new dehydro T-2 toxin (which correlated with T-2 production) and the conjugate, HT-2 toxin, glucuronide. This shows that CC factors may have a significant impact on growth and mycotoxin production by F. langsethiae., (Copyright © 2019. Published by Elsevier Ltd.)

T-2 and HT-2 toxins and their main modified forms (T-2 glucoside and HT-2 glucoside) may co-occur in cereals and cereal-based products. A fluorescence polarization immunoassay (FPIA) was developed for the simultaneous determination of T-2 toxin, HT-2 toxin and relevant glucosides, expressed as sum. The developed FPIA, using a HT-2-specific antibody, showed high sensitivity (IC 50 = 2.0 ng/mL) and high cross-reactivity (100% for T-2 toxin and 80% for T-2 and HT-2 glucosides). The FPIA has been used to develop two rapid and easy-to-use methods using two different extraction protocols, based on the use of organic (methanol/water, 90:10, v / v ) and non-organic (water) solvents, for the determination of these toxins in wheat. The two proposed methods showed analytical performances in terms of sensitivity (LOD 10 µg/kg) recovery (92-97%) and precision (relative standard deviations ≤13%), fulfilling the criteria for acceptability of an analytical method for the quantitative determination of T-2 and HT-2 toxins established by the European Union. Furthermore, the methods were then validated in accordance with the harmonized guidelines for the validation of screening methods included in the Regulation (EU) No. 519/2014. The satisfactory analytical performances, in terms of intermediate precision (≤25%), cut-off level (80 and 96 µg/kg for the two methods) and rate of false positives (<0.1%) confirmed the applicability of the proposed methods as screening method for assessing the content of these toxins in wheat at the EU indicative levels reported for T-2 and HT-2 toxins.

Introduction and Objective: Mycotoxins are toxic metabolites produced by certain types of fungi, causing pathological changes in humans and animals. The aim of this study was to assess the degree of contamination of selected cereal grains, bran and cereal products intended for children, with mycotoxins using GCxGC-TOF-MS technique. The study involved mycotoxins belonging to the type A and B trichothecenes group, including T-2 toxin (T-2), HT-2 toxin (HT-2), scirpenol (SCI), 15-monoacetoxyscirpenol (15-MAS), diacetoxyscirpenol (DAS), triacetoxyscirpenol (TAS), fusarenon-X (FUS-X), nivalenol (NIV), deoxynivalenol (DON), 3-acetyl-DON (3-Ac-DON), 15-acetyl-DON (15-Ac-DON). The study also assessed the effect of conditions in which the samples were stored, including temperature (6°C and 28°C) and time (14 and 28 days), on fungal growth and mycotoxin production. Among all studied compounds, only DAS and HT-2 toxins were detected in tested samples, with the exception of products intended for children. Measured HT-2 mycotoxin content in tested samples was in the range 83.9 - 196.4 µg kg -1 ., Results and Conclusions: Experiments with storage conditions showed a statistically significant increase in the HT-2 toxin level after 14 days of storage in all samples, irrespective of temperature. Prolonged storage (additional 14 days) did not cause significant changes in the HT-2 content. Further analyses showed a statistically significant effect of storage temperature on HT-2 toxin levels only in cereal products intended for children after both 14 and 28 days. Interestingly, lower temperature (6°C) was more optimal then higher temperature (28°C) for the HT-2 toxin production. No significant effect of storage temperature on HT-2 level was observed for cereal grains and bran.

In this report, we have investigated the apoptosis and autophagy of chondrocytes induced by the T-2 and HT-2 toxins. The viability of chondrocytes was measured by the MTT assay. Malondialdehyde (MDA) and superoxide dismutase (SOD) kits were used to measure the oxidative stress of chondrocytes. The apoptosis of chondrocytes was measured using flow cytometry. Hoechst 33258 and MDC staining agents were introduced to analyze apoptosis and autophagy induction in chondrocytes, respectively. Protein expression of Bax, caspase-9, caspase-3, and Beclin1 was examined by western blotting analysis. The T-2 and HT-2 toxins significantly decreased the viability of chondrocytes in a time-dependent manner. The level of oxidative stress in chondrocytes induced by the T-2 toxin was significantly higher when compared with that of the HT-2 toxin. The apoptosis rate of chondrocytes induced by the T-2 toxin increased from 3.26 ± 1.03%, 18.38 ± 1.28%, 34.5 ± 1.40% to 49.67 ± 5.31%, whereas apoptosis rate of chondrocytes induced by the HT-2 toxin increased from 3.82 ± 1.03%, 11.61 ± 1.27%, 25.72 ± 2.95% to 36.28 ± 2.81% in 48 h incubation time. Hoechst 33258 staining confirmed that apoptosis of chondrocytes induced by the T-2 toxin was significantly higher than that observed when the chondrocytes were incubated with the HT-2 toxin. MDC staining revealed that the autophagy rate of chondrocytes induced by the T-2 toxin increased from 6.38% to 63.02%, whereas this rate induced by the HT-2 toxin changed from 6.08% to 53.33%. The expression levels of apoptosis and autophagy related proteins, Bax, caspase-9, caspase-3, and Beclin1 in chondrocytes induced by the T-2 toxin were significantly higher when compared with those levels induced by the HT-2 toxin.

β-zearalenol (β-zol) and HT-2 are mycotoxins which cause apoptosis and oxidative stress in mammalian reproductive cells. Melatonin is an endogenous antioxidant involved in apoptosis and oxidative stress-related activities. This study investigated the effects of β-zol and HT-2 on bovine ovarian granulosa cells (BGCs), and how melatonin may counteract these effects. β-zol and HT-2 inhibited cell proliferation in a dose-dependent manner, and induced apoptosis of BGCs. They also yielded upregulation of the apoptosis-related genes Bax/Bcl-2 and Caspase3 and phosphorylation of p38MAPK. Increases in intracellular ROS were observed along with higher levels of mRNA anti-oxidation markers SOD1, SOD2, and CAT. SOD1, SOD2, malonaldehyde (MDA), and glutathione peroxidase (GSH-px) activities increased, as did the levels of SOD1 and SOD2 proteins. All of these effects were reduced or entirely attenuated in BGCs pre-treated with melatonin. Our results demonstrate that melatonin has protective effects against mycotoxin-induced apoptosis and oxidative stress in BGCs., (Copyright © 2019 Elsevier B.V. All rights reserved.)