Prevalence of T-2 Toxin in the Food and Beverages of Residents Living in a Kashin-Beck-Disease Area of Qamdo, Tibet.

It has been strongly suggested that selenium deficiency and T-2 toxin contamination have a strong relationship with the occurrence and development of Kashin-Beck disease (KBD). In order to provide information for understanding the high prevalence of KBD in Tibet, this study collected the responses to a cubital venous blood and dietary questionnaire of 125 subjects including 75 KBD patients and 50 healthy controls in a KBD-prevalent county (Luolong County) in Tibet, China. A total of 10 household local families were randomly selected in this area, and local diet samples of brick tea, Zanba powder, milk residue, and hulless Barley were collected from these residents. Selenium content in blood was detected by inductively coupled plasma mass spectrometry (ICP-MS). The T-2 toxin contamination level in food sample was assayed using an ELISA kit. The selenium levels of patients and controls were 42.0 ± 19.8 and 56.06 ± 22.4 µg/L, respectively. The serum selenium level in controls was higher than that in patients, but there was no significant difference, and the serum selenium level both in patients and controls in Tibet was lower than the normal range. The results of the dietary survey showed that the number of respondents who consumed butter tea was large; 46.67% of patients indicated that they drank buttered tea every day, which was significantly higher than in controls. The contents of T-2 toxin in Zanba powder, milk residue, hulless barley and drinking water samples were below the detection limit (0.05 µg/kg); this result was labeled Tr. Unexpectedly, the contents of T-2 toxin in brick tea were higher, with average levels of 424 ± 56 µg/kg in Detong village and 396 ± 24 µg/kg in Langcuo village. For the first time, we report the presence of an extremely high concentration of T-2 toxin in brick tea of Tibet.

LncRNA MIAT regulated by selenium and T-2 toxin increases NF-κB-p65 activation, promoting the progress of Kashin-Beck Disease.

LncRNA myocardial infarction associated transcript (MIAT) has been shown to be involved in osteoarthritis (OA), but its role in Kashin-Beck Disease (KBD) has rarely been reported. In this study, rats were administered with low selenium and/or T-2 toxin for 4 weeks to establish a KBD animal model. The serum selenium level, TNF-α and IL-1ß contents, phosphorylated p65 (p-p65) and MIAT expression were increased in each intervention group. Next, we isolated the primary epiphyseal chondrocytes, and found that selenium treatment reversed the effects of T-2 toxin on chondrocyte injury, p-p65 and MIAT expression. In addition, MIAT overexpression or T-2 toxin treatment led to increased cell death, apoptosis, inflammation, NF-κB-p65 pathway activation and MIAT expression, which was rescued by selenium treatment or MIAT siRNA transfection. Our results suggested that lncRNA MIAT regulated by selenium and T-2 toxin increased the activation of NF-κB-p65, thus being involved in the progress of KBD.

Activated FXR promotes xenobiotic metabolism of T-2 toxin and attenuates oxidative stress in broiler chicken liver.

The transmission of T-2 toxin and its metabolites into the edible tissues of poultry has potential effects on human health. The bile acid and xenobiotic system composes an intricate physiological network of chemoprotective and transporter-related functions, which ensures the detoxification and removal of harmful xenobiotic and endobiotic compounds from the body. This study revealed that cholic acid (CA), as one of the bile acids, promoted the metabolism of T-2 toxin in vivo by inducing the xenobiotic metabolism enzymes expression, thereby increasing the stress resistance and attenuating the oxidative stress. This study also indicated that dietary supplementation of 1% CA alleviated the mortality caused by T-2 toxin. Liver histology results demonstrated that CA supplementation significantly reduced inflammatory cell infiltration, sinusoidal expansion and congestion. Biochemistry results showed that the elevations of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and the increase in concentration of hydrogen peroxide (H2O2) in liver induced by the T-2 toxin were decreased by dietary supplementation of 1% CA. Additionally, CA supplementation led to the increase in superoxide dismutase (SOD) activity, but the decrease in catalase (CAT) activity in broiler chicken livers. Based on these findings, we propose that activation of FXR promotes T-2 toxin xenobiotic metabolism, and FXR plays a hepatoprotection role in liver injury induced by T-2 toxin.

T-2 Toxin-3α-glucoside in Broiler Chickens: Toxicokinetics, Absolute Oral Bioavailability, and in Vivo Hydrolysis.

Due to the lack of information on bioavailability and toxicity of modified mycotoxins, current risk assessment on these modified forms assumes an identical toxicity of the modified form to their respective unmodified counterparts. Crossover animal trials were performed with intravenous and oral administration of T-2 toxin (T-2) and T-2 toxin-3α-glucoside (T2-G) to broiler chickens. Plasma concentrations of T2-G, T-2, and main phase I metabolites were quantified using a validated liquid chromatography-tandem mass spectrometry method with a limit of quantitation for all compounds of 0.1 ng/mL. Resulting plasma concentration-time profiles were processed via two-compartmental toxicokinetic models. No T-2 triol and only traces of HT-2 were detected in the plasma samples after both intravenous and oral administration. The results indicate that T-2 has a low absolute oral bioavailability of 2.17 ± 1.80%. For T2-G, an absorbed fraction of the dose and absolute oral bioavailability of 10.4 ± 8.7% and 10.1 ± 8.5% were observed, respectively. This slight difference is caused by a minimal (and neglectable) presystemic hydrolysis of T2-G to T-2, that is, 3.49 ± 1.19%. Although low, the absorbed fraction of T2-G is 5 times higher than that of T-2. These differences in toxicokinetics parameters between T-2 and T2-G clearly indicate the flaw in assuming equal bioavailability and/or toxicity of modified and free mycotoxins in current risk assessments.

Prevalence of Selenium, T-2 Toxin, and Deoxynivalenol in Kashin-Beck Disease Areas in Qinghai Province, Northwest China.

The aim of this study was to determine the levels of selenium, T-2 toxin, and deoxynivalenol (DON) contamination in Kashin-Beck disease (KBD) areas and provide information for understanding the high prevalence of KBD in Qinghai Province. A total of 183 subjects were chosen in a KBD-prevalent county (Guide County) and a non-KBD county (Huangzhong County) in Qinghai Province, northwestern China, and the samples of wheat flour, soil, drinking water and blood, urine, and hair of children were collected from these residents. The selenium concentrations from all these sources were determined using atomic fluorescence spectrophotometry. The levels of T-2 toxin and DON contamination in the wheat flour samples were assayed using HPLC-MS/MS. The average selenium content in the soil, drinking water, and wheat flour samples from KBD areas were 26.93 ± 10.06 µg/kg, 0.097 ± 0.038 µg/L, and 9.50 ± 7.17 µg/kg, respectively. Among these, the selenium levels in the drinking water and wheat flour samples from the KBD endemic county were significantly lower than those from the non-KBD county. For the selenium nutrient status, only the hair selenium concentration of children from the KBD endemic county was significantly lower than that from the non-KBD county. The contents of T-2 toxin in all wheat samples were below the detection limit (0.4 µg/kg). The levels of DON contamination in wheat flour samples from KBD and non-KBD children's households within the KBD endemic county were relatively higher, with average levels of 302 ± 49 and 280 ± 48 µg/kg, respectively. The DON level of wheat flour samples from the children's households in the non-KBD county was significantly lower than that from the KBD endemic county. These results suggest that the lower selenium status in Guide County still remains. While the selenium nutritional status of the local children has improved to some extent, partly due to the introduction of food produce from nonlocal areas. DON contamination in the wheat flour may be involved in the fluctuating high prevalence rates of KBD in children in the KBD endemic Guide County in Qinghai Province.

Toxicokinetics of T-2 toxin and its major metabolites in broiler chickens after intravenous and oral administration.

T-2 toxin, one of the most toxic trichothecene mycotoxins, causes economic losses in animal production. Little information is available on the toxicokinetic parameters of T-2 toxin and its major metabolites (i.e., HT-2 toxin and T-2 triol) in broiler chickens. In this study, toxicokinetics of T-2 toxin and its major metabolites were evaluated in broiler chickens after a single intravenous (0.5 mg/kg b.w.) and multiple oral administrations (2.0 mg/kg b.w., every 12 h for 2 days). Plasma concentration profiles of T-2 toxin and its metabolites were analyzed by a noncompartmental model method. Following intravenous administration, the terminal elimination half-lives (t(1/2λz)) of T-2 toxin, HT-2 toxin, and T-2 triol were 17.33 ± 1.07 min, 33.62 ± 3.08 min, and 9.60 ± 0.50 min, respectively. Following multiple oral administrations, no plasma levels above the limit of quantification were observed for HT-2 toxin. The t(1/2λz) of T-2 toxin and T-2 triol was 23.40 ± 2.94 min and 87.60 ± 29.40 min, respectively. Peak plasma concentrations (Cmax ) of 53.10 ± 10.42 ng/mL (T-2 toxin) and 47.64 ± 9.19 ng/mL (T-2 triol) were observed at Tmax of 13.20 ± 4.80 min and 38.40 ± 15.00 min, respectively. T-2 toxin had a low absolute oral bioavailability (17.07%). Results showed that the T-2 toxin was rapidly absorbed and most of the T-2 toxin was extensively transformed to metabolites in broiler chickens.

Toxicokinetic study and absolute oral bioavailability of deoxynivalenol, T-2 toxin and zearalenone in broiler chickens.

Mycotoxins lead to economic losses in animal production. A way to counteract mycotoxicosis is the use of detoxifiers. The European Food Safety Authority stated that the efficacy of detoxifiers should be investigated based on toxicokinetic studies. Little information is available on the absolute oral bioavailability and the toxicokinetic parameters of deoxynivalenol, T-2 and zearalenone in broilers. Toxins were administered intravenously and orally in a two-way cross-over design. For deoxynivalenol a bolus of 0.75mg/kg BW was administered, for T-2 toxin 0.02mg/kg BW and for zearalenone 0.3mg/kg BW. Blood was collected at several time points. Plasma levels of the mycotoxins and their metabolite(s) were quantified using LC-MS/MS methods and toxicokinetic parameters were analyzed. Deoxynivalenol has a low absolute oral bioavailability (19.3%). For zearalenone and T-2 no plasma levels above the limit of quantification were observed after an oral bolus. Volumes of distribution were recorded, i.e. 4.99, 0.14 and 22.26L/kg for deoxynivalenol, T-2 toxin and zearalenone, respectively. Total body clearance was 0.12, 0.03 and 0.48L/minkg for deoxynivalenol, T-2 toxin and zearalenone, respectively. After IV administration, T-2 toxin had the shortest elimination half-life (3.9min), followed by deoxynivalenol (27.9min) and zearalenone (31.8min).

Quantitative determination of T-2 toxin, HT-2 toxin, deoxynivalenol and deepoxy-deoxynivalenol in animal body fluids using LC-MS/MS detection.

A sensitive and specific method for the quantitative determination of deoxynivalenol (DON), deepoxy-deoxynivalenol (DOM-1), T-2 toxin (T-2) and HT-2 toxin (HT-2) in animal body fluids (plasma and bile) using liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is presented. The extraction of plasma consisted of a deproteinization step using methanol, followed by a clean-up using an Oasis HLB solid-phase extraction column. For bile analysis, an extraction using a methanol/water mixture (70/30, v/v), followed by a liquid-liquid extraction using ethyl acetate, was performed. Chromatographic separation was achieved on a reversed-phase Nucleosil (100-5 C18 G100 × 3.0 mm) column. For the analysis of DON and DOM-1, a mixture of 0.1% acetic acid in water and methanol was used as the mobile phase. T-2 and its metabolite HT-2 were separated using 5mM ammonium acetate in a mixture of water/methanol/acetic acid. The mass spectrometer was operated in the negative or positive ESI selected reaction monitoring mode for DON and T-2 analysis, respectively. Calibration graphs (1-250 ng mL(-1)) were prepared for all matrices and correlation and goodness-of-fit coefficients were between 0.9978-1.000 and 2.96-11.77%, respectively. Limits of quantification were between 1 and 2.5 ng mL(-1) for all compounds. Limits of detection ranged from 0.01 to 0.63 ng mL(-1). The results for the within-day precision and accuracy fell within the ranges specified. The method has been successfully used for the quantitative determination of DON, DOM-1, T-2 and HT-2 in plasma and the semi-quantitative determination of the same compounds in bile from broiler chickens and pigs, respectively.

Effect of F-2 and T-2 fusariotoxins on experimental Cryptosporidium baileyi infection in chickens.

The course of Cryptosporidium baileyi infection in chickens fed with different doses of fusariotoxins was compared with that of control groups. F-2 toxin levels of 0.187-1.5 mg kg-1 and T-2 toxin levels of 0.187-6.0 mg kg-1 were investigated. The experimental animals were orally infected with 6 x 10(5) C. baileyi oocysts at 1 week of age. Total daily oocyst output was monitored by a quantitative method. Acquired immunity was tested at the age of 4 weeks, by ELISA and by a challenge infection with an equal number of oocysts, upon recovery from the primary infection. The results show that in chickens kept on the lower doses of F-2 and T-2 toxins, the parasite infection ran a similar course to that in the control groups, and the animals became resistant to re-infection. However, when higher doses (2.0-6.0 mg kg-1) of T-2 toxin were used, a depression of weight gain was observed with some other physiological parameters (PCV, weight of bursa, weight of thymus, skin thickness in PHA-P skin test) also indicating toxic effect and, simultaneously, the oocyst output decreased significantly and the patent period was slightly prolonged. Although certain modifications of the immune response could be revealed, the chickens became resistant to re-infection. Only early (1 week of age) parasite infection and 6 mg kg-1 T-2 toxin in the feed significantly depressed body weight gain and immunity.

A high-performance liquid chromatographic method for determining [3H]T-2 and its metabolites in biological fluids of the cynomolgus monkey.

High-performance liquid chromatography (HPLC) was used to separate, identify, and quantitate the trichothecin mycotoxin, T-2 (4 beta,15-diacetoxy-3 aopha-hydroxy-8 alpha [3-methyl-butyryloxy]-12,13-epoxy delta 9-trichothecin), and its metabolites in plasma and urine samples from cynomolgus monkeys treated with the toxin. A 15-min gradient elution system was developed to separate and measure radiolabeled T-2 mycotoxin and its metabolites. The HPLC technique for separating T-2 and its metabolites was compared with thin-layer chromatography. Samples from the in vitro metabolism of T-2 by plasma and urine were included as controls and as a measure of the toxin's stability in biological samples. Within 5 min, 22% of the plasma radiolabeled T-2 toxin was detected as metabolites after an intravenous administration of [3H] T-2 toxin to cynomolgus monkeys. By 24 h post-exposure, there was no parent T-2 toxin detected in plasma or urine. T-2 tetraol was the major metabolite detected in the plasma and urine of monkeys. Other metabolites observed in urine up to 5 days after exposure were 3'OH-T-2 and 3'OH-HT-2. We conclude that T-2 toxin was rapidly metabolized to more polar metabolites, which were eliminated in urine.

Acute effects of T-2 toxin on radioactive iron incorporation into circulating erythrocytes in mice.

The 24-h and 72-h incorporation of 59Fe into circulating erythrocytes in mice were strongly inhibited by a single subcutaneous dose of T-2 toxin given 1 h before the radioisotope. The system is extremely sensitive, since a significant effect was detected with T-2 toxin doses as low as 0.30 mg/kg, which is about one-tenth of the LD50 in the BALB/c strain used for the present study. In the treated animals no initial changes were observed in the blood 59Fe levels or in the rate of radioisotope clearance from plasma, indicating that the toxin does not interfere with iron absorption or transport. It is concluded that the inhibition observed reflects the damage produced by this toxin on reticulocytes and/or erythroblasts, and therefore this method could be of value as a very sensitive means of studying the risk of erythropoietic injury produced by dietary exposure to trichothecene mycotoxins.

Reversal of intracellular toxicity of the trichothecene mycotoxin T-2 with monoclonal antibody.

The trichothecene mycotoxin T-2 is a potent inhibitor of intracellular protein synthesis. We have previously shown that a mouse immunoglobulin G1 monoclonal antibody (15H6) specific for T-2 toxin can neutralize the in vitro protein synthesis inhibitory effect of the toxin in human B-lymphoblastoid cultures, and protect rats from lethal toxemia. We now report that these monoclonal antibodies can induce the net efflux of [3H]-T-2 toxin from poisoned human B-lymphoblastoid cells in vitro, and restore protein synthesis. Administration of the monoclonal antibodies (250 mg/kg) 30 min before infusion of a lethal dose (1 mg/kg) of T-2 toxin causes the sequestration of the toxin in the plasma compartment. When administered 35 min after T-2 toxin, a time when the bulk of toxin is in the tissues, the monoclonal antibodies facilitate the migration of toxin back into the plasma compartment. These data demonstrate that monoclonal antibodies can be of therapeutic value against an intracellular toxin.

Use of deuterated internal standards for quantitation of T-2 and HT-2 toxins in human blood by tandem mass spectrometry.

Deuterated acetyl derivatives (3-trideutero-acetyl-T-2 and 15-trideutero-HT-2) were prepared for use as internal standards for the quantitation of T-2 and HT-2 in blood by tandem mass spectrometry. The method used was multiple reaction monitoring (MRM), which essentially involves the selection of a parent ion for analysis followed by monitoring of the daughter ions generated by collision activated decomposition. The parent ions chosen for the trifluoroacetate derivative of T-2 and HT-2 were m/z+ 478 and 532, respectively. Both parents yield the same daughter ions, i.e., 180, 138, and 121. HT-2 and T-2 were added to blood extracts in amounts ranging from 1 to 20 ppb. The limit of detection is about 0.5 ppb with an effective detection limit of 1.0 ppb in a range of 1-20 ppb. The recovery is about 90%. This method can be used by veterinarians for purposes of diagnostics. It can be used for urine as well as blood.

Analysis of T-2 toxin in a biological matrix using multiple reaction monitoring.

The traditional analysis of a biological mixture by mass spectrometry involves the union of a gas liquid chromatograph with a mass spectrometer and analysis of the resolved effluent by either full scan or the recording of selected ions. The latter methodology is sensitive and selective but suffers from the interference presented by the biological matrix. With the advent of tandem mass spectrometry, greater flexibility in the elimination of the effects of a biological matrix is possible. The example used here is that of the trifluoroacetate derivative of T-2 toxin. Thus, a single or multiple selection of parent ions (m/z+ 478) is made and allowed to pass through the first analyzer (sectoring portion) of the tandem mass spectrometer into the second mass spectrometer, in this case a quadrupole. Here the parent fragment undergoes collision-activated decomposition, under the influence of argon gas and voltage (collision energy) into daughter ions which are detected by the third mass spectrometer (quadrupole). The daughters generated from the parent m/z+ 478 of T-2-TFA are 180, 138 and 121. They are unique and give definitive proof for the presence of T-2 toxin. There is the possibility that other 478 fragments may be present in the mixture that have the same retention time as T-2-TFA toxin. In this case, the daughters generated will be different from that of T-2. The method is called multiple reaction monitoring. It is highly accurate and can detect T-2-TFA in blood or urine at a concentration of 1 ppb.

Metabolism of T-2 toxin by blood cell carboxylesterases.

Human and rat blood hydrolysed T-2 toxin along two different pathways giving HT-2 toxin and neosolaniol as primary metabolites, respectively. Neosolaniol represents a metabolic pathway different from that obtained by liver. Rat erythrocytes formed neosolaniol as a primary metabolite whereas white blood cells hydrolysed T-2 toxin to HT-2 toxin. Human erythrocytes formed both HT-2 toxin and neosolaniol whereas all human white cells produced only HT-2 as the primary metabolite. The enzymes responsible for hydrolysis of T-2 toxin to HT-2 toxin in white blood cells and T-2 toxin to neosolaniol in red blood cells were all identified as carboxylesterases by use of specific inhibitors. The ratio between trichothecene hydrolysis and 4-nitrophenyl butyrate hydrolysis varied among the different cell fractions indicating that specific isoenzymes are involved.

Stability of T-2, HT-2, and T-2 tetraol in biological fluids.

The stabilities of tritium-labeled T-2, HT-2, and T-2 tetraol were studied in blood and urine at -70 degrees, 4 degrees, and 23 degrees C for 6 months in the presence of EDTA or NaF. Samples were counted with a radiochromatographic scanner and results indicated the stability of T-2 tetraol greater than T-2 greater than HT-2. Toxins were most stable when stored at -70 degrees C, in the presence of NaF, and in urine (pH 6). They were less stable in saline (control, pH 7) and least stable in blood (pH 8). These results suggest that urine and T-2 tetraol are the biological fluid and metabolite of choice for diagnostic purposes.

Rapid screening procedure for the detection of trichothecenes in plasma and urine.

A rapid and easy procedure to screen for trichothecenes in plasma and urine is presented. The toxins are extracted using a Clin-Elut column, hydrolyzed to their corresponding parent alcohols and cleaned up with a silica cartridge followed by derivatization for gas chromatographic analysis. The detection of any of the parent alcohols in plasma or urine would indicate an exposure to trichothecenes. Recoveries in urine are between 78 and 119% at levels of 50-1000 ng/ml and recoveries in plasma are between 80 and 116% at levels of 50-500 ng/ml. The limit of detection is better than 25 ppb.

Pharmacokinetics of T-2 toxin and its metabolite HT-2 toxin, after intravenous administration in dogs.

The pharmacokinetics of T-2 toxin and HT-2 toxin were investigated comparatively in five dogs, after iv administration of the toxins (0.4 mg/kg). T-2 toxin was very rapidly and almost completely biotransformed to HT-2 toxin (fm = 83.6 +/- 3.9%). The following mean pharmacokinetic parameters were determined in this study for T-2 toxin and HT-2 toxin, respectively: half-life 5.3 +/- 2.1 and 19.6 +/- 4.7 min, clearance 0.107 +/- 0.056 and 0.167 +/- 0.074 liters/min/kg, and volume of distribution 0.86 +/- 0.63 and 4.47 +/- 1.38 liters/kg. The high clearance values suggest that the metabolism of T-2 toxin and HT-2 toxin is carried out in blood and/or through diffusion by nonspecific carboxyesterases. The results of this study suggest the possibility of a sustenance of T-2 toxin metabolism by its binding to blood cells.

Pharmacokinetics of the trichothecene mycotoxin, T-2 toxin, in swine and cattle.

The pharmacokinetics of the trichothecene mycotoxin, T-2 toxin, were determined in growing gilts and heifers. Following intra-aortal administration in swine and intravenous administration in calves, the disappearance of the parent T-2 toxin followed a 2-compartment open model. Mean elimination phase half-lives were 13.8 and 17.4 min and mean apparent specific volumes of distribution were 0.366 and 0.376 l/kg in swine and calves, respectively. The fraction of T-2 toxin eliminated as parent compound in the urine was negligible. In spite of administration of a lethal oral dose in swine (2.4 mg/kg) and toxic oral doses (up to 3.6 mg/kg) in calves, no parent T-2 toxin was detected in plasma or urine. After intra-aortal administration in swine, tissue concentrations of T-2 toxin were consistently highest in lymphoid organs. Tissue residues of T-2 toxin were rapidly depleted such that, in spite of administration of a potentially lethal intra-aortal dose, no quantifiable T-2 toxin was present in any of the tissues collected at 4 hr after dosing. No T-2 toxin could be detected in liver, even at 1 hr after dosing.

Gas chromatographic assay with pharmacokinetic applications for monitoring T-2 and HT-2 toxins in plasma.

A gas-liquid chromatographic (GLC) method for monitoring T-2 and HT-2 toxins in plasma was developed. The procedure involved extraction of the toxins with ethyl acetate, chromatography on a C18 reversed-phase column and derivatization with heptafluorobutyric anhydride (HFBA). The T-2 and HT-2 HFBA derivatives were chromatographed on OV-17 at various temperatures and measured with an electron-capture detector. Iso-T-2 toxin and iso-HT-2 toxin were used as internal standards. Recoveries averaged 95.1 +/- 8.6% for T-2 toxin and 102.1 +/- 5.2% for HT-2 toxin at levels ranging from 40 to 120 ng/ml. The limits of detection were 30 and 5 ng/ml of T-2 and HT-2 toxin, respectively. The range of the assay covers plasma concentrations at which toxicity becomes manifest. The pharmacokinetic application of this GLC method is illustrated by simultaneous monitoring of T-2 and HT-2 toxins levels in plasma obtained after intravenous administration of T-2 toxin to a dog.