Cysteine Inhibits the Growth of Fusarium oxysporum and Promotes T-2 Toxin Synthesis through the Gtr/Tap42 Pathway.

Fusarium oxysporum is ubiquitous and can easily contaminate food during processing and storage, potentially producing T-2 toxin, which can pose a severe health risk to public health. Previous research on the presence of T-2 has focused on starch-rich foods, while protein- and amino acid-rich foods have received relatively little attention. In this study, the effects of amino acids on the growth of F. oxysporum and its T-2 production were investigated by gene deletion and complementation experiments. The results showed that amino acids, including aspartic acid, methionine, isoleucine, serine, phenylalanine, and cysteine, significantly inhibited the growth of F. oxysporum, while promoting T-2 synthesis, with cysteine having the most pronounced effect. The target of rapamycin complex 1 (TORC1) is a key pathway in response to a variety of amino acids, including cysteine. gtr2 and tap42 were found to be negative regulators of T-2 synthesis. The study highlights the elevated risk of T-2 production by F. oxysporum in cysteine-rich foods and the need to take appropriate measures to prevent and control the potential harm that such foods may present to public health. IMPORTANCE F. oxysporum and its T-2 contamination of food not only leads to food wastage but also poses a major food safety challenge to humans. The growth and T-2 production characteristics of F. oxysporum in high-protein substrates are considerably different from those in grains. Here, we show that the abundant free amino acids in a protein-rich food matrix are a key regulatory factor for the growth of, and toxin production by, F. oxysporum. Cysteine has the most pronounced effect on inhibiting mycelial growth and promoting T-2 synthesis through the TORC1 pathway. This implies that consumers tend to overlook T-2 contamination due to the poor growth of F. oxysporum in food rich in protein and amino acids, especially cysteine. Therefore, particular attention should be paid to the protection of those products.

Low-Concentration T-2 Toxin Attenuates Pseudorabies Virus Replication in Porcine Kidney 15 Cells.

Pseudorabies, caused by pseudorabies virus (PRV), is the main highly infectious disease that severely affects the pig industry globally. T-2 toxin (T2), a significant mycotoxin, is widely spread in food and feeds and shows high toxicity to mammals. The potential mechanism of the interaction between viruses and toxins is of great research value because revealing this mechanism may provide new ideas for their joint prevention and control. In this study, we investigated the effect of T2 on PRV replication and the mechanism of action. The results showed that at a low dose (10 nM), T2 had no significant effect on porcine kidney 15 (PK15) cell viability. However, this T2 concentration alleviated PRV-induced cell injury and increased cell survival time. Additionally, the number of PK15 cells infected with PRV significantly reduced by T2 treatment. Similarly, T2 significantly decreased the copy number of PRV. Investigation of the mechanism revealed that 10 nM T2 significantly inhibits PRV replication and leads to downregulation of oxidative stress- and apoptosis-related genes. These results suggest that oxidative stress and apoptosis are involved in the inhibition of PRV replication in PK15 cells by low-concentration T2. Taken together, we demonstrated the protective effects of T2 against PRV infection. A low T2 concentration inhibited the replication of PRV in PK15 cells, and this process was accompanied by downregulation of the oxidative stress and apoptosis signaling pathways. Our findings partly explain the interaction mechanism between T2 and PRV, relating to oxidative stress and apoptosis, though further research is required.

T-2 Toxin Induces Oxidative Stress at Low Doses via Atf3ΔZip2a/2b-Mediated Ubiquitination and Degradation of Nrf2.

T-2 toxin is mainly produced by Fusarium species, which is an extremely toxic mycotoxin to humans and animals. It is well known that T-2 toxin induces oxidative stress, but the molecular mechanism is still unknown. In this study, we found that T-2 toxin significantly promoted reactive oxygen species (ROS) accumulation in MCF-7 cells at low doses which maintains cell viability at least 80%. Further analysis showed that T-2 toxin downregulated the expression of the master regulator of antioxidant defense gene, nuclear factor erythroid 2-related factor (Nrf2), and its targeted antioxidant genes. Overexpression of Nrf2 or its target gene heme oxygenase 1 (HO1) significantly blocked the ROS accumulation in MCF-7 cells under T-2 toxin treatment. Moreover, we found that T-2 toxin downregulated the antioxidant genes via inducing the expression of ATF3ΔZip2a/2b. Importantly, overexpression of ATF3ΔZip2a/2b promoted the ubiquitination and degradation of Nrf2. Altogether, our results demonstrated that T-2 toxin-induced ROS accumulation via ATF3ΔZip2a/2b mediated ubiquitination and degradation of Nrf2, which provided a new insight into the mechanism of T-2 toxin-induced oxidative stress.

Role of DNA methylation-related chromatin remodeling in aryl hydrocarbon receptor-dependent regulation of T-2 toxin highly inducible Cytochrome P450 1A4 gene.

Mycotoxins are toxic secondary metabolites produced by food-contaminating fungi, which lead to global epigenetic changes and cause toxicity to both farm animals and humans. However, whether mycotoxins induce gene-specific epigenetic alterations associated with inducible downstream gene expression is unclear as are the underlying regulatory mechanisms. Here, we found that T-2 toxin and its deacetylated metabolites but not deoxynivalenol (DON) or other representative mycotoxins highly induced the expression of cytochrome P450 1A4 (CYP1A4) in both Leghorn male hepatoma (LMH) cells and chicken primary hepatocytes, and this effect was related to the regulation of both aryl hydrocarbon receptor (AhR) and DNA methylation. We used methylation-sensitive restriction enzyme digestion-qPCR (MSRE-qPCR) and chromatin immunoprecipitation (ChIP) assays and found that the binding of DNA methyltransferase 1 (DNMT1) and histone deacetylase 2 (HDAC2) to highly methylated CpG island 3-2 at the enhancer of CYP1A4 was accompanied by the recruitment of the repressive histone modification marker H3K27me3, inducing a silent state. In turn, T-2 toxin stimulation enriched the binding of AhR to demethylated CpG island 3-2, which facilitated p300 and H3K9ac recruitment and ultimately generated an activated chromatin structure at the enhancer by increasing the active histone modification markers, including H3K4me3, H3K27ac, and H3K14ac. Interestingly, T-2 toxin-induced AhR activation also facilitated RNA polymerase II binding to CpG island 2, which may form a transcriptionally active chromatin structure at the promoter and ultimately transactivate CYP1A4. Our findings provide novel insights into the epigenetic regulation of T-2 toxin-induced gene expression.

L-Arginine protects mouse Leydig cells against T-2 toxin-induced apoptosis in vitro.

To explore the protective mechanism of L-arginine against T-2 toxin-induced apoptosis in mouse Leydig cells, we investigated whether L-arginine can prevent T-2 toxin-induced apoptosis in mouse Leydig cells and explored the underlying mechanisms. Leydig cells were isolated and cultured with control, T-2 toxin (10 nM), L-arginine (0.25, 0.5, and 1.0 mM), and T-2 toxin (10 nM T-2 toxin) + L-arginine (0.25, 0.5, or 1.0 mM) for 24 h. Cells and supernatants were harvested to examine proliferation of the cells, the apoptosis rate, activity of caspase-3 and mitochondria, and the gene expression levels of Bcl-2, Bax, PARP, and caspase-3. Results showed that proliferation and mitochondrial activity of Leydig cells were inhibited by administration of T-2 toxin. Bcl-2 gene expression levels was decreased, while the gene expression levels of Bax and PARP were increased, which could trigger mitochondria-mediated apoptosis, activate downstream caspase-3, and then increased caspase-3 at both activity and gene expression levels. The expression of the Bcl-2 gene was upregulated and the expression of Bax, caspase-3, and PARP gene were downregulated when L-arginine was added to the cultured cells. The results of this study showed that L-arginine could block T-2 toxin-induced apoptosis in mouse Leydig cells by regulating specific intracellular death-related pathways.

Preparation of T­2 toxin­containing pH­sensitive liposome and its antitumor activity.

T­2 toxin is a type A trichothecene mycotoxin. In order to reduce the side effects of T­2 toxin and increase the tumor targeting ability, a pH­sensitive liposome of T­2 toxin (LP­pHS­T2) was prepared and characterized in the present study. The cytotoxicity of LP­pHS­T2 on A549, Hep­G2, MKN­45, K562 and L929 cell lines was tested by 3­(4,5­dimethylthiazolyl­2)­2,5­diphenyltetrazolium bromide assay, with T­2 toxin as the control. The apoptotic and migratory effects of LP­pHS­T2 on Hep­G2 cells were investigated. The preparation process of LP­pHS­T2 involved the following parameters: Dipalmitoyl phosphatidylcholine: dioleoylphosphatidylethanolamine, 1:2; total phospholipid concentration, 20 mg/ml; phospholipid:cholesterol, 3:1; 4­(2­hydroxyethyl)­1­piperazineethanesulfonic acid buffer (pH 7.4), 10 ml; drug:lipid ratio, 2:1; followed by ultrasound for 10 min and extrusion. The encapsulation efficiency reached 95±2.43%. The average particle size of LP­pHS­T2 after extrusion was 100 nm; transmission electron microscopy showed that the shape of LP­pHS­T2 was round or oval and of uniform size. The release profile demonstrated a two­phase downward trend, with fast leakage of T­2 toxin in the first 6 h (~20% released), followed by sustained release up to 48 h (~46% released). From 48­72 h, the leakage rate increased (~76% released), until reaching a minimum at 72 h. When LP­pHS­T2 was immersed in 0.2 mol/l disodium phosphate­sodium dihydrogen phosphate buffers (pH 6.5), the release speed was significantly increased and the release rate reached 91.2%, demonstrating strong pH sensitivity. Overall, antitumor tests showed that LP­pHS­T2 could promote the apoptosis and inhibit the migration of Hep­G2 cells. The present study provided a new approach for the development of T­2 toxin­based anti­cancer drugs.

Protective effects of dietary arginine against oxidative damage and hepatopancreas immune responses induced by T-2 toxin in Chinese mitten crab (Eriocheir sinensis).

T-2 toxin is a secondary metabolite produced by Fusarium spp. that is a major cereal and animal feed contaminant. T-2 toxin has numerous adverse effects on animals, including hepatotoxicity. Arginine (Arg) is closely associated with the regulation of immune responses and antioxidant activity in tissues. The objective of the present study was to evaluate the protective effects of dietary Arg against oxidative damage and immune responses of the hepatopancreas induced by T-2 toxin in Chinese mitten crab. According to the results, 3.17% Arg in the diet decreased alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase activity in the haemolymph significantly, when compared with the levels of activity in the T-2 toxin group. Arg supplementation also increased superoxide dismutase and glutathione peroxidase activity, while decreasing malondialdehyde concentrations in the hepatopancreas, when compared with the levels in the T-2 toxin group. In addition, 3.17% Arg in the diet increased acid phosphatase and alkaline phosphatase activity in the hepatopancreas, as well as albumin concentrations in the haemolymph, when compared with the T-2 toxin group. Dietary Arg also regulated the expression of antioxidant enzyme-related genes (mitochondrial manganese superoxide dismutase, cytosolic manganese superoxide dismutase, and catalase) and immune related genes (prophenoloxidase, NF-κB-like transcription factor Relish, and lipopolysaccharide-induced TNF-α factor) to alleviate the damage associated with the T-2 toxin. Furthermore, Arg ameliorated damage to the hepatopancreas microstructure in the crabs. The results of the present study indicate that dietary Arg could enhance the antioxidant and immune capacity of Chinese mitten crab against oxidative damage and immune injury to the hepatopancreas induced by T-2 toxin.

Aromatic hydrocarbon receptor regulates chicken cytochrome P450 1A5 transcription: A novel insight into T-2 toxin-induced gene expression and cytotoxicity in LMH cells.

T-2 toxin is a secondary metabolite produced by the Fusarium genus and is highly toxic to both farmed animals and humans. In our previous study, we found that chicken cytochrome P450 1A5 (CYP1A5) can be significantly induced by T-2 toxin in chicken primary hepatocytes and catalyze T-2 toxin into a more toxic product, 3'-OH-T-2. Here, we showed that T-2 toxin also induced the expression of CYP1A5 in LMH cells at both the mRNA and protein levels, and this can be strongly inhibited by both resveratrol and siRNA targeting the aryl hydrocarbon receptor (AhR), indicating the involvement of AhR in T-2 toxin-induced transcriptional activation of CYP1A5. We further showed that T-2 toxin induced the expression of AhR and promoted the translocation of AhR into the nucleus as well as its binding to the proximal xenobiotic-responsive element (XRE) in the 5'-flanking region of CYP1A5, which mediates both the basal expression and the transcriptional activation of CYP1A5. Interestingly, CYP1A5 induction mediated by AhR enhances the cytotoxicity of T-2 toxin by reducing cell viability, activating oxidative stress and inducing DNA damage as well as apoptosis. Our findings provide novel insight into T-2 toxin-induced gene expression and cytotoxicity and may provide a novel target to reduce latent harm to chickens.

Combined Inhibitory Effects of Citrinin, Ochratoxin-A, and T-2 Toxin on Aquaporin-2.

Aquaporins form a large family of transmembrane protein channel that facilitates selective and fast water transport across the cell membrane. The inhibition of aquaporin channels leads to many water-related diseases such as nephrogenic diabetes insipidus, edema, cardiac arrest, and stroke. Herein, we report the molecular mechanism of mycotoxins (citrinin, ochratoxin-A, and T-2 mycotoxin) inhibition of aquaporin-2 (AQP2) and arginine vasopressin receptor 2. Molecular docking, molecular dynamics simulations, quantum chemical calculations, residue conservation-coupling analysis, sequence alignment, and in vivo studies were utilized to explore the binding interactions between the mycotoxins and aquaporin-2. Theoretical studies revealed that the electrostatic interactions induced by the toxins pulled the key residues (187Arg, 48Phe, 172His, and 181Cys) inward, hence reduced the pore diameter and water permeation. The permeability coefficient of the channel was reduced from native ((3.32 ± 0.75) × 10-14 cm3/s) to toxin-treated AQP2 ((1.08 ± 0.03) × 10-14 cm3/s). The hydrogen bonds interruption and formation of more hydrogen bonds with toxins also led to the reduced number of water permeation. Further, in vivo studies showed renal damages and altered level of aquaporin expression in mycotoxin-treated Mus musculus. Furthermore, the multiple sequence alignments among the model organism along with evolutionary coupling analysis provided the information about the interdependences of the residues in the channel.

T-2 toxin downregulates LHCGR expression, steroidogenesis, and cAMP level in human cumulus granulosa cells.

Our goals were to investigate whether environmentally relevant doses of T-2 toxin can affect human ovarian granulosa cells' function and to reveal the potential mechanism of T-2 toxin's action. Results showed that T-2 toxin strongly attenuated luteinizing hormone/choriogonadotropin receptor (LHCGR) mRNA expression in follicle-stimulating hormone (FSH)-stimulated human cumulus granulosa cells. Addition of human chorionic gonadotropin was not able to elicit maximal response of ovulatory genes amphiregulin, epiregulin, and progesterone receptor. T-2 toxin reduced mRNA levels of CYP19A1 and steroidogenic acute regulatory protein (STAR) and lowered FSH-stimulated estradiol and progesterone production. Mechanistic experiments demonstrated that T-2 toxin decreased FSH-stimulated cyclic adenosine monophosphate (cAMP) production. Addition of total PDE inhibitor 3-isobutyl-1-methylxanthine prevented T-2 toxin's action on LHCGR, STAR, and CYP19A1 mRNA expression in FSH-stimulated human cumulus granulosa cells. Furthermore, T-2 toxin partially decreased 8-bromoadenosine 3'5'-cyclic monophosphate (8-Br-cAMP)-stimulated LHCGR and STAR, but did not affect 8-Br-cAMP-stimulated CYP19A1 mRNA expression in human cumulus granulosa cells. Overall, our data indicate that environmentally relevant dose of T-2 toxin decreases steroidogenesis and ovulatory potency in human cumulus granulosa cells probably through activation of PDE, thus posing a significant risk for female fertility.

The aggravating effect of selenium deficiency on T-2 toxin-induced damage on primary cardiomyocyte results from a reduction of protective autophagy.

Selenium deficiency and T-2 toxin exposure may contribute to the development of Keshan disease characterized by congestive cardiomyopathy. The aim of this study was to explore the role of autophagy in the aggravation of selenium deficiency on T-2 toxin-induced damages on primary cardiomyocyte. Our present study demonstrated that 0.25-1 µM T-2 toxin damaged primary cardiomyocytes and selenium deficiency exacerbated T-2 toxin-induced damages by measuring the levels of MTT, lactate dehydrogenase and cleaved-caspase 3. T-2 toxin triggered autophagy in primary cardiomyocytes, as indicated by markedly increased expressions of LC3-Ⅱ and Beclin-1 mRNA levels. Rapamycin (autophagy agonist) treatment increased autophagy levels and decreased the cytotoxicity caused by T-2 toxin while 3-methyladenine (autophagy inhibitor) treatment reduced autophagy levels and enhanced the cytotoxicity of T-2 toxin, suggesting that autophagy protect primary cardiomyocytes from the cytotoxicity of T-2 toxin. Selenium deficiency lowered cytoprotective autophagy in the primary cardiomyocytes treated by T-2 toxin. It can be concluded that autophagy induced by T-2 toxin plays a role in protecting primary cardiomyocyte, but selenium deficiency decreases the protective autophagy and then exacerbate T-2 toxin-induced damages. Our finding may partly interpret the combination effects of selenium deficiency and T-2 toxin on the development of Keshan disease.

Expression Profiles of Selenium-Related Genes in Human Chondrocytes Exposed to T-2 Toxin and Deoxynivalenol.

The combination of excess mycotoxin exposure and selenium deficiency has been widely considered as a cause of Kashin-Beck disease (KBD). The present study aimed to investigate the expression profiles of selenium-related genes in human chondrocytes after exposure to T-2 toxin and deoxynivalenol (DON) and to preliminarily identify the potential biological functions of the identified genes. Gene expression profiling was performed on human chondrocytes treated with 0.01 µg/ml T-2 toxin and 1.0 µg/ml DON by using Affymetrix Human Gene Arrays. The 1660 selenium-related genes were derived from the Comparative Toxicogenomics Database. Gene-term enrichment analysis was conducted by the DAVID gene annotation tool. Our results showed that 69 and 191 selenium-related genes were differentially expressed after T-2 toxin and DON treatment, respectively. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these identified genes were involved in various biological functions, such as the GO terms in response to oxidative stress, cell cycle arrest, and apoptotic process and the KEGG metabolic, FoxO signaling, and p53 signaling pathways. Our results may help explain the mechanisms of interaction between mycotoxins and selenium following human chondrocyte damage and reveal the potential roles of environmental risk factors in cartilage lesions during KBD development.

The Impact of T-2 Toxin on Vasoactive Intestinal Polypeptide-Like Immunoreactive (VIP-LI) Nerve Structures in the Wall of the Porcine Stomach and Duodenum.

T-2 toxin is a secondary metabolite of some Fusarium species. It is well-known that this substance can harmfully impact living organisms. Among others, thanks to the ability of crossing the blood-brain barrier, T-2 toxin can affect the central nervous system. Mycotoxins mostly get into the organism through the digestive tract; therefore, first of all they have to break the intestinal barrier, wherein the important component is the enteric nervous system (ENS). However, knowledge about the impact of T-2 toxin on the ENS is rather scant. As a result of the influence of various physiological and pathological agents, ENS can undergo adaptive and reparative processes which manifest as changes in the immunoreactivity of perikaryons for neuronal active substances. So, the aim of the present investigation was to study how low doses of T-2 toxin affect vasoactive intestinal polypeptide-like immunoreactive (VIP-LI) nervous structures in the ENS of the porcine stomach and duodenum. Obtained results have shown that T-2 toxin causes an percentage increase of VIP-LI nerve cells and nerve fibers in every enteric plexus in both fragments of gastrointestinal tract studied. This shows that even low doses of T-2 toxin can have an influence on living organisms.

T-2 mycotoxin treatment of newborn rat pups does not significantly affect nervous system functions in adulthood.

T-2 toxin is primarily produced by Fusarium sp. abundant under temperate climatic conditions. Its main harmful effect is the inhibition of protein synthesis. Causing oxidative stress, it also promotes lipid peroxidation and changes plasma membrane phospholipid composition; this may lead to nervous system alterations. The aim of the present study was to examine whether a single dose of T-2 toxin administered at newborn age has any long-lasting effects on nervous system functions. Rat pups were treated on the first postnatal day with a single intraperitoneal dose of T-2 toxin (0.2 mg/bwkg). Body weight of treated pups was lower during the second and third week of life, compared to littermates; later, weight gain was recovered. At young adulthood, behavior was tested in the open field, and no difference was observed between treated and control rats. Field potential recordings from somatosensory cortex and hippocampus slices did not reveal any significant difference in neuronal network functions. In case of neocortical field EPSP, the shape was slightly different in treated pups. Long-term synaptic plasticity was also comparable in both groups. Seizure susceptibility of the slices was not different, either. In conclusion, T-2 toxin did not significantly affect basic nervous system functions at this dose.

T-2 toxin induces cytotoxicity and disrupts tight junction barrier in SerW3 cells.

T-2 toxin, which is produced in grain and grain products as a secondary metabolite by Fusarium species, is also potentially dangerous for human health. Up to date, no study was reported the cytotoxicity of T-2 toxin on SerW3 cells in the perspective of junctional barriers. This study focused on revealing the cytotoxic effects of T-2 on Sertoli cells associated with cell junctional barriers. In the present study, SerW3 cells were exposed to T-2 toxin at 12, 120 and 1200ng/ml doses for 24 and 48h. Cytotoxicity tests including cell viability (MTT), lactate dehydrogenase (LDH) cytotoxicity test and trypan blue exclusion assay were performed. Occludin, ZO-1, N-cadherin and ß-catenin were immunolabelled, expressions of occludin and N-cadherin were determined by western blotting. SerW3 cell barrier integrity was measured by transepithelial electrical resistance (TEER). Cytotoxicity caused by T-2 toxin increased in a dose dependent manner, expressions of proteins and TEER measurement decreased. This study may underlie the early targets of T-2 toxin on SerW3 cells mimicking blood-testis barrier in vitro.

HT-2 toxin affects development of porcine parthenotes by altering DNA and histone methylation in oocytes matured in vitro.

T-2 toxin is a type A mycotoxin produced by various Fusarium species, while HT-2 toxin is a major metabolite of T-2 toxin. Both T-2 toxin and HT-2 toxin are known to have deleterious effects on animals. Our previous work showed that HT-2 treatment caused the failure of porcine oocyte maturation. In this study, we reported that HT-2 also affected porcine embryo development. In HT-2 toxin treated group, all the percentages of embryos in 2-cell, 4-cell and blastocyst stage were significantly lower compared with those in control groups. We then explored the causes from the epigenetic modification aspect of the oocytes. The analysis of fluorescence intensity showed that 5-methyl cytosine (5 mC) level was increased after exposure to HT-2 toxin in porcine oocytes, indicating that the general DNA methylation level increased in the treated porcine oocytes. In addition, histone modifications were also affected, since our results showed that H3K4me2 and H3K9me2 levels were increased in the oocytes from HT-2-treated group. Therefore, our results indicated that HT-2 toxin decreased porcine embryo developmental competence through altering the epigenetic modifications of oocytes.

Preparation of T-2-glucoronide with Rat Hepatic Microsomes and Its Use along with T-2 for Activation of the JAK/STAT Signaling Pathway in RAW264.7 Cells.

T-2 toxin (T-2), one of the most toxic trichothecene A-type mycotoxins, is biotransformed in animal tissues to modified T-2s (mT-2s) including T-2-glucuronide (T-2-GlcA). In this study, the optimal conditions for T-2-GlcA synthesis were established, and the JAK/STAT pathway in RAW264.7 cells was used to study the toxicity of T-2-GlcA. Because many mT-2 standards are not readily available, optimal conditions for T-2-GlcA synthesis in vitro were established by incubating T-2 with rat liver microsomes, UDPGA, and 0.2% Triton X-100 for 90 min. qRT-PCR and Western blot results showed 21- and 760-fold increases in IL-6 mRNA expression induced by T-2-GlcA and T-2, respectively. Similar differences were observed in JAK3, SOCS2/3, and CIS mRNA expression. T-2-GlcA induced a dose-responsive decrease in STAT1 mRNA expression, whereas the result with T-2 was the opposite. Moreover, the phosphorylation of STAT3 induced by T-2-GlcA was higher than that by T-2, whereas the phosphorylation of STAT1 was to the contrary. Overall, the results show that T-2-GlcA was somewhat toxic, but activation of the JAK/STAT pathway in RAW264.7 was higher by T-2.

Increased Chondrocyte Apoptosis in Kashin-Beck Disease and Rats Induced by T-2 Toxin and Selenium Deficiency.

OBJECTIVE: To investigate chondrocyte apoptosis and the expression of biochemical markers associated with apoptosis in Kashin-Beck disease (KBD) and in an established T-2 toxin- and selenium (Se) deficiency-induced rat model. METHODS: Cartilages were collected from the hand phalanges of five patients with KBD and five healthy children. Sprague-Dawley rats were administered a selenium-deficient diet for 4 weeks prior to T-2 toxin exposure. The apoptotic chondrocytes were observed by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Caspase-3, p53, Bcl-2, and Bax proteins in the cartilages were visualized by immunohistochemistry, their protein levels were determined by Western blotting, and mRNA levels were determined by real-time reverse transcription polymerase chain reaction. RESULTS: Increased chondrocyte apoptosis was observed in the cartilages of children with KBD. Increased apoptotic and caspase-3-stained cells were observed in the cartilages of rats fed with normal and Se-deficient diets plus T-2 toxin exposure compared to those in rats fed with normal and Se-deficient diets. Caspase-3, p53, and Bax proteins and mRNA levels were higher, whereas Bcl-2 levels were lower in rats fed with normal or Se-deficiency diets supplemented with T-2 toxin than the corresponding levels in rats fed with normal diet. CONCLUSION: T-2 toxin under a selenium-deficient nutritional status induces chondrocyte death, which emphasizes the role of chondrocyte apoptosis in cartilage damage and progression of KBD.

Effects of T-2 Toxin on Pacific White Shrimp Litopenaeus vannamei: Growth, and Antioxidant Defenses and Capacity and Histopathology in the Hepatopancreas.

Modified-masked T-2 toxin (mT-2) formed during metabolism in edible aquatic animals may go undetected by traditional analytical methods, thereby underestimating T-2 toxicity. The effects of T-2 on growth and antioxidant capacity and histopathological changes in the hepatopancreas were studied in Pacific white shrimp Litopenaeus vannamei exposed for 20 d to 0, 0.5, 1.2, 2.4, 4.8, and 12.2 mg/kg of T-2 in their feed. The concentration of mT-2 in the hepatopancreas was detected by liquid chromatography-tandem mass spectrophotometry before and after trifluoroacetic acid (TFA) treatment that converted mT-2 to free T-2. A dose-dependent increase in mT-2 concentration was observed in the hepatopancreas. Dietary exposure to T-2 significantly decreased (P < 0.05) shrimp growth and survival rate compared with the controls. The malondialdehyde (MDA) concentration was significantly increased in shrimp exposed to feed with ≥2.4 mg/kg T-2 (P < 0.05). The antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GPx), total antioxidant capacity (T-AOC), and also glutathione (GSH) content increased in shrimp dosed with 2.4-4.8 mg/kg T-2 but declined at the highest dose (12.2 mg/kg), probably indicating an inability to cope with high concentrations of reactive oxygen species (ROS) as evident from a marked increase in MDA (P < 0.05) culminating in cellular toxicity. Histopathological changes in the hepatopancreas were dose dependent, with cell autophagy evident at the highest exposure dose. This is the first report in shrimp of a dose-dependent increase in ROS, SOD enzyme activity, and T-AOC at low T-2 exposures, and associated histopathological changes in the hepatopancreas, in response to dietary T-2. Received January 26, 2016; accepted October 9, 2016.

COMPARISON OF THE CYTOTOXIC EFFECTS OF ZEARALENON AND T-2 TOXIN ON THE HORSES AND OXEN GERM CELL IN VITRO BEFORE AND AFTER CRYOPRESERVATION.

The article presents the results of the studies of cytotoxic effect of zearalenone and T-2 toxin on sperm of horses and bulls during incubation and after thawing according to the technology of sperm obtaining and cryopreservation in Kharkov. We first have shown in vitro toxic effects of different concentrations of zearalenone and T-2 toxin (from 0.5 to 0.01 mM) on the membrane stability, as well as quantitative and qualitative indicators of semen in stallions and bulls before and after freezing and thawing. It has been found that the biological activity of the native sperm in 1 h after addition of 0.5 mM zearalenone is not changed, and after thawing is reduced by 19.4 %, or 0.69 ball (P < 0.01) compared to control without toxin. T-2 toxin at a concentration of 0.5 mM reduced the activity of the native sperm after an hour of incubation by 0.59 ball (P < 0.01) and decreased it after thawing decreased by 60.28 %, or 2.14 ball (P < 0.001). 1 h of incubation with zearalenone at concentration of 0.25 mM did not affect the activity of the native sperm, but the activity after freeze­thaw deteriorated by 12.2 %, or 0.41 ball (P < 0.05). T-2 toxin at a concentration of 0.25 mM reduced activity of native sperm after 1 h exposure by 0.46 ball (P < 0.05), and after thawing spermatozoa degrade it by 1.77 ball (P < 0.001) compared to control without toxins. In our study, the lowest concentration giving a significant decrease in sperm parameters was 0.01 mM. The negative impact of zearalenone in such a dose on the native sperm was absent, and sperm after thawing only slightly reduced activity and perezhivayemost by 0.09 points and 0.27 hours, respectively. The simultaneous addition of zearalenone and T-2 toxin at the same dose of 0.01 mM resulted in a decrease in biological perezhivayemost and absolute indicator of native sperm perezhivayemost by 0.36 points and 0.64 hours (P < 0.05), respectively.