The Co-Occurrence of T-2 Toxin, Deoxynivalenol, and Fumonisin B1 Activated the Glutathione Redox System in the EU-Limiting Doses in Laying Hens.

Different mycotoxins in feed lead to combined exposure, increasing adverse effects on animal health. Trichothecene mycotoxins have been associated with inducing oxidative stress, which is neutralized by the glutathione system within the antioxidant defense, depending on the dose and duration of exposure. T-2 toxin, deoxynivalenol (DON), and fumonisin B1 (FB1) are commonly found in feed commodities simultaneously. In the present study, the intracellular biochemical and gene expression changes were investigated in the case of multi-mycotoxin exposure, focusing on certain elements of the glutathione redox system. In a short-term feeding trial, an in vivo study was performed with low (EU-proposed) doses: T-2/HT-2 toxin: 0.25 mg; DON/2-AcDON/15-AcDON.: 5 mg; FB1: 20 mg/kg feed, and high doses (twice the low dose) in laying hens. The multi-mycotoxin exposure affected the glutathione system; GSH concentration and GPx activity was higher in the liver in the low-dose group on day 1 compared to the control. Furthermore, the gene expression of antioxidant enzymes increased significantly on day 1 in both exposure levels compared to the control. The results suggest that when EU-limiting doses are applied, individual mycotoxins may have a synergistic effect in the induction of oxidative stress.

NRF-2α and mitophagy underlie enhanced mitochondrial functions and biogenesis induced by T-2 toxin in GH3 cells.

T-2 toxin is a natural contaminant in grain cereals produced by species of Fusarium. Studies indicate that T-2 toxin can positively affect mitochondrial function, but the underlying mechanism is unclear. In this study, we examined the role of nuclear respiratory factor 2α (NRF-2α) in T-2 toxin-activated mitochondrial biogenesis and the direct target genes of NRF-2α. Furthermore, we investigated T-2 toxin-induced autophagy and mitophagy, and the role of mitophagy in changes in mitochondrial function and apoptosis. It was found that T-2 toxin significantly increased NRF-2α levels and nuclear localization of NRF-2α was induced. NRF-2α deletion significantly increased the production of reactive oxygen species (ROS), abrogated T-2 toxin-induced increases in ATP and mitochondrial complex I activity, and inhibited the mitochondrial DNA copy number. Meanwhile, With chromatin immunoprecipitation sequencing (ChIP-Seq), various novel NRF-2α target genes were identified, such as mitochondrial iron-sulphur subunits (Ndufs 3,7) and mitochondrial transcription factors (Tfam, Tfb1m, and Tfb2m). Some target genes were also involved in mitochondrial fusion and fission (Drp1), mitochondrial translation (Yars2) and splicing (Ddx55), and mitophagy. Further studies showed that T-2 toxin induced Atg5 dependent autophagy and Atg5/PINK1-dependent mitophagy. In addition, mitophagy defects increase ROS production, inhibit ATP levels and the expression of genes related to mitochondrial dynamics, and promote apoptosis in the presence of T-2 toxins. Altogether, these results suggest that NRF-2α plays a critical role in promoting mitochondrial function and biogenesis through regulation of mitochondrial genes, and, interestingly, mitophagy caused by T-2 toxin positively affected mitochondrial function and protected cell survival against T-2 toxin.

Mitochondrial Damage Induced by T-2 Mycotoxin on Human Skin-Fibroblast Hs68 Cell Line.

T-2 toxin is produced by different Fusarium species and belongs to the group of type A trichothecene mycotoxins. T-2 toxin contaminates various grains, such as wheat, barley, maize, or rice, thus posing a risk to human and animal health. The toxin has toxicological effects on human and animal digestive, immune, nervous and reproductive systems. In addition, the most significant toxic effect can be observed on the skin. This in vitro study focused on T-2 toxicity on human skin fibroblast Hs68 cell line mitochondria. In the first step of this study, T-2 toxin's effect on the cell mitochondrial membrane potential (MMP) was determined. The cells were exposed to T-2 toxin, which resulted in dose- and time-dependent changes and a decrease in MMP. The obtained results revealed that the changes of intracellular reactive oxygen species (ROS) in the Hs68 cells were not affected by T-2 toxin. A further mitochondrial genome analysis showed that T-2 toxin in a dose- and time-dependent manner decreased the number of mitochondrial DNA (mtDNA) copies in cells. In addition, T-2 toxin genotoxicity causing mtDNA damage was evaluated. It was found that incubation of Hs68 cells in the presence of T-2 toxin, in a dose- and time-dependent manner, increased the level of mtDNA damage in both tested mtDNA regions: NADH dehydrogenase subunit 1 (ND1) and NADH dehydrogenase subunit 5 (ND5). In conclusion, the results of the in vitro study revealed that T-2 toxin shows adverse effects on Hs68 cell mitochondria. T-2 toxin induces mitochondrial dysfunction and mtDNA damage, which may cause the disruption of adenosine triphosphate (ATP) synthesis and, in consequence, cell death.

The role of MAPK/NF-κB-associated microglial activation in T-2 toxin-induced mouse learning and memory impairment.

T-2 toxin is a mycotoxin with multiple toxic effects and has emerged as an important food pollutant. Microglia play a significant role in the toxicity of various neurotoxins. However, whether they participate in the neurotoxicity of T-2 toxin has not been reported. To clarify this point, an in vivo mouse model of T-2 toxin (4 mg/kg) poisoning was established. The results of Morris water maze and open-field showed that T-2 toxin induced learning and memory impairment and locomotor inhibition. Meanwhile, T-2 toxin induced microglial activation, while inhibiting microglia activation by minocycline (50 mg/kg) suppressed the toxic effect of the T-2 toxin. To further unveil the potential mechanisms involved in T-2 toxin-induced microglial activation, an in vitro model of T-2 toxin (0, 2.5, 5, 10 ng/mL) poisoning was established using BV-2 cells. Transcriptomic sequencing revealed lots of differentially expressed genes related to MAPK/NF-κB pathway. Western blotting results further confirmed that T-2 toxin (5 ng/mL) induced the activation of MAPKs and their downstream NF-κB. Moreover, the addition of inhibitors of NF-κB and MAPKs reversed the microglial activation induced by T-2 toxin. Overall, microglial activation may contribute a considerable role in T-2 toxin-induced behavioral abnormalities, which could be MAPK/NF-κB pathway dependent.

MiR-214-3p may alleviate T-2 toxin-induced chondrocyte apoptosis and matrix degradation by regulating NF-κB signaling pathway in vitro.

T-2 toxin is part of the most toxic fungal secondary metabolites contaminating different kinds of grains. Previous studies have demonstrated that T-2 toxin can influence the survival of chondrocytes and extracellular matrix (ECM) composition. MiR-214-3p is essential for the homeostasis of chondrocytes and ECM. However, the molecular machinery underlying T-2 toxin-induced chondrocyte apoptosis and ECM degradation remain to be elucidated. The present study aimed to investigate the mechanism of miR-214-3p's involvement in T-2 toxin-induced chondrocyte apoptosis and ECM degradation. Meanwhile, the role of the NF-κB signaling pathway was scrutinized. C28/I2 chondrocytes were treated with 8 ng/ml of T-2 toxin for 24 h, after the pretreatment of miR-214-3p interfering RNAs for 6 h. Gene and protein levels involved in chondrocyte apoptosis and ECM degradation were assessed through RT-PCR and Western blotting. The apoptosis rate of chondrocyte was measured by flow cytometry. Results and data indicated that miR-214-3p was decreased in a dose-dependent manner at different concentrations of T-2 toxin. The enhancement of miR-214-3p could alleviate chondrocyte apoptosis and ECM degradation due to T-2 toxin exposure. The upregulation of miR-214-3p was associated with the decreased expression of apoptosis-promoting genes such as Bax and Cleaved-caspase3/caspase3 as well as the increased expression of anti-apoptotic genes such as Bcl2 and Survivin. Furthermore, miR-214-3p stimulated the relative protein expression of collagen Ⅱ but inhibited the expression of MMP13. Overexpressing miR-214-3p could suppress the relative protein expression of IKKß and phospho-p65/p65, thus blocking the activation of the NF-κB signaling pathway. The study suggested that the miR-214-3p attenuates T-2 toxin-induced chondrocyte apoptosis and ECM degradation through a potential NF-κB signaling pathway.

Toxicity and detoxification of T-2 toxin in poultry.

This review summarizes the updated knowledge on the toxicity of T-2 on poultry, followed by potential strategies for detoxification of T-2 in poultry diet. The toxic effects of T-2 on poultry include cytotoxicity, genotoxicity, metabolism modulation, immunotoxicity, hepatotoxicity, gastrointestinal toxicity, skeletal toxicity, nephrotoxicity, reproductive toxicity, neurotoxicity, etc. Cytotoxicity is the primary toxicity of T-2, characterized by inhibiting protein and nucleic acid synthesis, altering the cell cycle, inducing oxidative stress, apoptosis and necrosis, which lead to damages of immune organs, liver, digestive tract, bone, kidney, etc., resulting in pathological changes and impaired physiological functions of these organs. Glutathione redox system, superoxide dismutase, catalase and autophagy are protective mechanisms against oxidative stress and apoptosis, and can compensate the pathological changes and physiological functions impaired by T-2 to some degree. T-2 detoxifying agents for poultry feeds include adsorbing agents (e.g., aluminosilicate-based clays and microbial cell wall), biotransforming agents (e.g., Eubacterium sp. BBSH 797 strain), and indirect detoxifying agents (e.g., plant-derived antioxidants). These T-2 detoxifying agents could alleviate different pathological changes to different degrees, and multi-component T-2 detoxifying agents can likely provide more comprehensive protection against the toxicity of T-2.

YAP plays a protective role in T-2 toxin-induced inhibition of chondrocyte proliferation and matrix degradation.

Previous research has shown that T-2 toxin can damage cartilage, resulting in a disease phenotype similar to osteoarthritis. The precise molecular mechanism by which T-2 toxin causes chondrocyte injury, however, is unknown. The purpose of this study was to look into the role of YAP in T-2 toxin-induced rat chondrocyte injury. Based on research results, T-2 toxin decreased the levels of collagen II and PCNA while increasing the expression of matrix metalloproteinase MMP13. These findings supported the T-2 toxin's detrimental effect on chondrocytes. YAP's role in T-2 toxin-induced chondrocyte injury was also investigated. Total YAP and related nuclear proteins were found to decrease as the concentration of T-2 toxin increased. While PYAP expression was not significantly altered in response to T-2 toxin, the PYAP/YAP ratio decreased as the T-2 toxin concentration increased, implying that the HIPPO signaling pathway was activated. Furthermore, the YAP-specific inhibitor Verteporfin was used to investigate the role of YAP in T-2 toxin-induced chondrocyte injury. YAP inhibition increased MMP13 expression while decreasing COL2 and PCNA levels. In summary, the current study found that T-2 toxin decreased the levels of COL2 and PCNA while increasing the expression of MMP13 in chondrocytes after inhibiting YAP, providing a new insight into the mechanism of T-2 toxin-induced cartilage damage.

Direct T-2 Toxicity on Human Skin-Fibroblast Hs68 Cell Line-In Vitro Study.

T-2 toxin is produced by different Fusarium species, and it can infect crops such as wheat, barley, and corn. It is known that the T-2 toxin induces various forms of toxicity such as hepatotoxicity, nephrotoxicity, immunotoxicity, and neurotoxicity. In addition, T-2 toxin possesses a strong dermal irritation effect and can be absorbed even through intact skin. As a dermal irritant agent, it is estimated to be 400 times more toxic than sulfur mustard. Toxic effects can include redness, blistering, and necrosis, but the molecular mechanism of these effects still remains unknown. This in vitro study focused on the direct toxicity of T-2 toxin on human skin-fibroblast Hs68 cell line. As a result, the level of toxicity of T-2 toxin and its cytotoxic mechanism of action was determined. In cytotoxicity assays, the dose and time-dependent cytotoxic effect of T-2 on a cell line was observed. Bioluminometry results showed that relative levels of ATP in treated cells were decreased. Further analysis of the toxin's impact on the induction of apoptosis and necrosis processes showed the significant predominance of PI-stained cells, lack of caspase 3/7 activity, and increased concentration of released Human Cytokeratin 18 in treated cells, which indicates the necrosis process. In conclusion, the results of an in vitro human skin fibroblast model revealed for the first time that the T-2 toxin induces necrosis as a toxicity effect. These results provide new insight into the toxic T-2 mechanism on the skin.

Betulinic acid attenuates cognitive dysfunction, oxidative stress, and inflammation in a model of T-2 toxin-induced brain damage.

T-2 toxin is a mycotoxin that has harmful effects on the immune system and cognitive function. Betulinic acid (BA) is a plant-derived pentacyclic lupane-type triterpenoid which possesses a wide spectrum of bioactivities. The study was aimed to explore whether BA has a protective effect on cognitive impairment and oxidative stress caused by T-2 toxin. BA was suspended in 1% soluble starch by continuous intragastric administration for 14 days, then the brain damage in mice was induced by a single intraperitoneal injection of T-2 toxin (4 mg/kg). It was found that BA alleviated the reduction of discrimination index in T-2 toxin-treated mice, and enhanced dopamine (DA), 5-hydroxytryptamine (5-HT), and acetylcholine (ACH) levels of brain neurotransmitter. Meanwhile, BA pretreatment ameliorated oxidative stress through increase of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione (GSH) levels, and inhibition of the generation of reactive oxygen species (ROS) and malondialdehyde (MDA) in the brain of mice exposed to T-2 toxin. Moreover, BA reduced brain hemorrhage and ecchymosis, improved the mitochondrial morphology, enriched the number of organelles, and inhibited cell apoptosis in brain challenged with T-2 toxin. Furthermore, BA inhibited mRNA expression of pro-inflammatory cytokines such as interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α) as well as enhanced mRNA expression of anti-inflammatory cytokine such as IL-10 in the brain of T-2 toxin-triggered mice. Therefore, BA could improve the cognitive function, enhance the antioxidant capacity, and inhibit the secretion of proinflammatory cytokines in brain, thereby playing a preventive and protective role against brain damage caused by T-2 toxin.

Protective mechanisms of three antioxidants against T-2 toxin-induced muscle protein deterioration in shrimp.

BACKGROUND: Quercetin (Q), tea polyphenols (TP), and rutin (R) are widely used plant-derived active ingredients. They possess antioxidant, anti-inflammatory, and anti-tumor properties, and can reduce the muscle damage caused by mycotoxins. However, few studies have examined the protective mechanisms of quercetin, tea polyphenols, and rutin on muscle quality. To elucidate their protective mechanisms, shrimp were exposed to both T-2 toxin and these three antioxidants for 20 days in a dose-escalating trial. The changes in the protein composition of shrimp muscle were measured. The target proteins associated with T-2 and antioxidants were screened and identified by non-labeled quantitative proteomics. RESULTS: The T-2 toxin induced abnormal expression of 21 target proteins, leading to the deterioration of muscle proteins in shrimp. The three antioxidants ameliorated the T-2 toxin-induced damage to muscle proteins by increasing the sarcoplasmic and myofibrillar protein content and decreasing the alkali-soluble protein content. Quercetin had the strongest protective effect. The protective processes of these antioxidants involved the upregulation of target proteins involved in carbohydrate metabolism (enolase, malate dehydrogenase), protein translation (elongation factor 1-alpha and eukaryotic translation initiation factor 2 subunit alpha), and cytoskeleton component (actin 2, fast-type skeletal muscle actin 1). Quercetin regulated the largest number of target proteins, making it the best protective agent against T-2 toxin. CONCLUSION: The T-2 toxin (4.80-24.30 mg/kg feed) induced changes in target proteins and muscle composition of shrimp, leading to a deterioration in muscle proteins. Quercetin (2.00-32.00 g/kg feed) had significant protective effects against this deterioration in muscle protein in shrimp. © 2022 Society of Chemical Industry.

Mechanism of DNA methylation-mediated downregulation of O6-methylguanine-DNA methyltransferase in cartilage injury of Kashin-Beck disease.

OBJECTIVE: Kashin-Beck disease (KBD) is an endemic osteoarthropathy, in which excessive apoptosis of chondrocytes occurs. O6-methylguanine-DNA methyltransferase (MGMT), a DNA damage repair gene, plays an important role in apoptosis, but the mechanism is unclear in KBD cartilage injury. This study was to investigate the expression and promoter methylation of MGMT in KBD patients and its role in DNA damage and apoptosis of chondrocytes. METHODS: MGMT mRNA and protein level were detected by quantitative real-time PCR and immunohistochemistry. Demethylation of MGMT was carried out using 5-Aza-2'-deoxycytidine, and the methylation level of MGMT promoter was measured by quantitative methylation specific PCR. Next, small hairpin RNA was used to knockdown the expression of MGMT. Cell viability, apoptosis and DNA damage were determined by MTT assay, flow cytometry, Hoechst 33342 staining and alkaline comet assay following T-2 toxin and selenium treatment. RESULTS: MGMT protein expression and mRNA levels were decreased (P = 0.02, P = 0.007) and promoter methylation was increased (P = 0.008) in KBD patients. Meanwhile, MGMT level was upregulated by 5-Aza-2'-deoxycytidine in chondrocytes (P = 0.0002). DNA damage and apoptosis rates were increased in MGMT-silenced chondrocytes (all P < 0.0001). Furthermore, DNA damage and apoptosis were increased in chondrocytes treated with T-2 toxin (all P < 0.0001), but were decreased after selenium treatment (P < 0.0001, P = 0.01). Decreased mRNA level and increased methylation of MGMT were found in the T-2 toxin group (P = 0.005, P = 0.002), while selenium reversed it (P = 0.02, P = 0.004). CONCLUSIONS: MGMT might play a crucial part in the pathogenesis of KBD cartilage injury, which could provide a therapeutic target for KBD.

Human Biomonitoring of T-2 Toxin, T-2 Toxin-3-Glucoside and Their Metabolites in Urine through High-Resolution Mass Spectrometry.

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.

T-2 Toxin-The Most Toxic Trichothecene Mycotoxin: Metabolism, Toxicity, and Decontamination Strategies.

Among trichothecenes, T-2 toxin is the most toxic fungal secondary metabolite produced by different Fusarium species. Moreover, T-2 is the most common cause of poisoning that results from the consumption of contaminated cereal-based food and feed reported among humans and animals. The food and feed most contaminated with T-2 toxin is made from wheat, barley, rye, oats, and maize. After exposition or ingestion, T-2 is immediately absorbed from the alimentary tract or through the respiratory mucosal membranes and transported to the liver as a primary organ responsible for toxin's metabolism. Depending on the age, way of exposure, and dosage, intoxication manifests by vomiting, feed refusal, stomach necrosis, and skin irritation, which is rarely observed in case of mycotoxins intoxication. In order to eliminate T-2 toxin, various decontamination techniques have been found to mitigate the concentration of T-2 toxin in agricultural commodities. However, it is believed that 100% degradation of this toxin could be not possible. In this review, T-2 toxin toxicity, metabolism, and decontamination strategies are presented and discussed.

Glycolysis and Reactive Oxygen Species Production Participate in T-2 Toxin-Stimulated Chicken Heterophil Extracellular Traps.

T-2 toxin (T-2) is a kind of trichothecene toxin produced from Fusarium fungi, which is an environmental pollutant that endangers poultry and human health. Heterophil extracellular traps (HETs) are not only a form of chicken immune defense against pathogen infection but also involved in pathophysiological mechanisms of several diseases. However, the immunotoxicity of T-2 on HET formation in vitro has not yet been reported. In this study, heterophils were exposed to T-2 at doses of 20, 40, and 80 ng/mL for 90 min. Observation of the structure of HETs by immunofluorescence staining and the mechanism of HET formation was analyzed by inhibitors and PicoGreen. These results showed that T-2-triggered HET formation consisted of DNA, elastase, and citH3. Furthermore, T-2 increased reactive oxygen species (ROS) generation, and the formation of T-2-triggered HETs was also decreased by the inhibitors of glycolysis, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, p38 and extracellular signal-regulated kinase (ERK)1/2 signaling pathways, suggesting that T-2-induced HETs are associated with glycolysis, ROS production, ERK1/2 and p38 signaling pathways, and NADPH oxidase. Taken together, this study elucidates the mechanism of T-2-triggered HET formation, and it may provide new insight into understanding the immunotoxicity of T-2 to early innate immunity in chickens.

Comparative Analysis of Machine Learning Methods to Predict Growth of F. sporotrichioides and Production of T-2 and HT-2 Toxins in Treatments with Ethylene-Vinyl Alcohol Films Containing Pure Components of Essential Oils.

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 (aw) (0.99 and 0.96) regimes was assayed. GR in controls/treatments usually increased with increasing temperature, regardless of aw, but no significant differences concerning aw 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% (ED50, ED90, and ED100) ranged from 515 to 3330 µg/culture in Petri dish (25 g oat grains) depending on film type, aw, and temperature. ED90 and ED100 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.

A Two-Year Occurrence of Fusarium T-2 and HT-2 Toxin in Croatian Cereals Relative of the Regional Weather.

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.

A Comparative Transcriptome Analysis, Conserved Regulatory Elements and Associated Transcription Factors Related to Accumulation of Fusariotoxins in Grain of Rye (Secale cereale L.) Hybrids.

Detoxification of fusariotoxin is a type V Fusarium head blight (FHB) resistance and is considered a component of type II resistance, which is related to the spread of infection within spikes. Understanding this type of resistance is vital for FHB resistance, but to date, nothing is known about candidate genes that confer this resistance in rye due to scarce genomic resources. In this study, we generated a transcriptomic resource. The molecular response was mined through a comprehensive transcriptomic analysis of two rye hybrids differing in the build-up of fusariotoxin contents in grain upon pathogen infection. Gene mining identified candidate genes and pathways contributing to the detoxification of fusariotoxins in rye. Moreover, we found cis regulatory elements in the promoters of identified genes and linked them to transcription factors. In the fusariotoxin analysis, we found that grain from the Nordic seed rye hybrid "Helltop" accumulated 4 times higher concentrations of deoxynivalenol (DON), 9 times higher nivalenol (NIV), and 28 times higher of zearalenone (ZEN) than that of the hybrid "DH372" after artificial inoculation under field conditions. In the transcriptome analysis, we identified 6675 and 5151 differentially expressed genes (DEGs) in DH372 and Helltop, respectively, compared to non-inoculated control plants. A Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that DEGs were associated with glycolysis and the mechanistic target of rapamycin (mTOR) signaling pathway in Helltop, whereas carbon fixation in photosynthesis organisms were represented in DH372. The gene ontology (GO) enrichment and gene set enrichment analysis (GSEA) of DEGs lead to identification of the metabolic and biosynthetic processes of peptides and amides in DH372, whereas photosynthesis, negative regulation of catalytic activity, and protein-chromophore linkage were the significant pathways in Helltop. In the process of gene mining, we found four genes that were known to be involved in FHB resistance in wheat and that were differentially expressed after infection only in DH372 but not in Helltop. Based on our results, we assume that DH372 employed a specific response to pathogen infection that led to detoxification of fusariotoxin and prevented their accumulation in grain. Our results indicate that DH372 might resist the accumulation of fusariotoxin through activation of the glycolysis and drug metabolism via cytochrome P450. The identified genes in DH372 might be regulated by the WRKY family transcription factors as associated cis regulatory elements found in the in silico analysis. The results of this study will help rye breeders to develop strategies against type V FHB.

An update on T-2 toxin and its modified forms: metabolism, immunotoxicity mechanism, and human exposure assessment.

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.

Carry-over of some Fusarium mycotoxins in tissues and eggs of chickens fed experimentally mycotoxin-contaminated diets.

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, A1, B, B1) 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 B1 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 B1 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.

Effects of dietary T-2 toxin on gut health and gut microbiota composition of the juvenile Chinese mitten crab (Eriocheir sinensis).

The current study aims to investigate the effects of dietary T-2 toxin on the intestinal health and microflora in the juvenile Chinese mitten crab (Eriocheir sinensis) with an initial weight 2.00 ± 0.05 g. Juvenile crabs were fed with experimental diets supplemented with T-2 toxin at 0 (control), 0.6 (T1 group), 2.5 (T2 group) and 5.0 (T3 group) mg/kg diet for 8 weeks. Dietary T-2 toxin increased the malondialdehyde (MDA) content and the expression of Kelch-like ECH-associated protein 1 (keap1) gene while the expression of cap 'n' collar isoform C (CncC) decreased in the intestine. The activities of glutathione peroxidase (GSH-Px) and total anti-oxidation capacity (T-AOC) in the intestine increased only in the lower dose of dietary T-2. Dietary T-2 toxin significantly increased the mRNA expression of caspase-3, caspase-8, Bax and mitogen-activated protein kinase (MAPK) genes and the ratio of Bax to Bcl-2 accompanied with a reduction of Bcl-2 expression. Furthermore, T-2 toxin decreased the mRNA levels of antimicrobial peptides (AMPs), peritrophic membrane (PM1 and PM2) and immune regulated nuclear transcription factors (Toll-like receptor: TLR, myeloid differentiation primary response gene 88: Myd88, relish and lipopolysaccharide-induced TNF-α factor: LITAF). The richness and diversity of the gut microbiota were also affected by dietary T-2 toxin in T3 group. The similar dominant phyla in the intestine of the Chinese mitten crab in the control and T3 groups were found including Bacteroidetes, Firmicutes, Tenericutes and Proteobacteria. Moreover, the inclusion of dietary T-2 toxin of 4.6 mg/kg significantly decreased the richness of Bacteroidetes and increased the richness of Firmicutes, Tenericutes and Proteobacteria in the intestine. At the genus level, Dysgonomonas and Romboutsia were more abundant in T3 group than those in the control. However, the abundances of Candidatus Bacilloplasma, Chryseobacterium and Streptococcus in T3 group were lower than those in the control. This study indicates that T-2 toxin could cause oxidative damage and immunosuppression, increase apoptosis and disturb composition of microbiota in the intestine of Chinese mitten crab.