Development of a three-compartment toxicokinetic model for T-2 toxin in shrimp by blindfold particle swarm optimization algorithm.

Tricothecenes-2 toxin (T-2) is a major mycotoxin that is widely distributed in aquatic feeds and poses a huge challenge to the aquatic industry, but there is scant information on the toxicokinetics of T-2 in aquatic animals. Here, we describe the development of a three-compartment toxicokinetic model for the absorption, distribution, metabolism and elimination (ADME) of T-2 in shrimp. The three compartments were central (the hemolymph), slow metabolizing and fast metabolizing compartments to account for the varying ADME rates of T-2 in different shrimp organs. The toxicokinetic model was solved by the blindfold particle swarm optimization algorithm, and the values for the model equation parameters were obtained by applying the experimental data of T-2 concentrations in shrimp. The model had a good fit with the experimental data. It was revealed through the model that after i.m. administration, T-2 was rapidly absorbed into the hemolymph and distributed into shrimp organs. The hepatopancreas and intestine belonged to the fast and muscle to the slow metabolizing compartments, respectively, while the hemolymph had no capacity to metabolize T-2. The T-2 elimination rates in the hepatopancreas and intestine were similar and quite high while that in the muscle was very low. The methods used in developing and solving the model could be used for similar toxicokinetic and pharmacokinetic studies of other animals.

Interactions between T-2 toxin and its metabolites in HepG2 cells and in silico approach.

The T-2 toxin (T-2) is commonly metabolized to HT-2 toxin (HT-2), Neosolaniol (NEO), T2-triol and T2-tetraol and they can modify the toxicity of T-2. In this study, T-2 and its modified forms were evaluated by in vitro and in silico methods. The in vitro cytotoxicity individually was evaluated by MTT and Total Protein Content (PC) assays in human hepatocarcinoma (HepG2) cells. The order of IC50 was T-2 tetraol > T-2 triol > NEO > T-2 = HT-2. The T-2 and HT-2 evidenced the highest cytotoxic effect in HepG2 cells individually. No differences were observed in binary combinations tested and the two mycotoxins in the mixture tested individually. The T-2+HT-2 combination showed the highest toxic potential with the lowest IC50 value of 34.42 ± 0.58 nM at 24 h. All binary combinations exhibited antagonistic interactions. The ADME and toxicity profile of mycotoxins were obtained by the in silico admetSAR predictive model which determines the metabolic and toxicological approaches in order to know if these mycotoxins might be taken into consideration to support a more realistic and adequate risk assessment.

Assessing Global Human Exposure to T-2 Toxin via Poultry Meat Consumption Using a Lifetime Physiologically Based Pharmacokinetic Model.

Residue depletion of T-2 toxin in chickens after oral gavage at 2.0 mg/kg twice daily for 2 days was determined in this study. A flow-limited physiologically based pharmacokinetic (PBPK) model was developed for lifetime exposure assessment in chickens. The model was calibrated with data from the residue depletion study and then validated with independent data. A local sensitivity analysis was performed, and 16 sensitive parameters were subjected to Monte Carlo analysis. The population PBPK model was applied to estimate daily intake values of T-2 toxin in different countries based on reported consumption factors and the guidance value of 0.25 mg/kg in feed for chickens by the European Food Safety Authority (EFSA). The predicted daily intakes in different countries were all lower than the EFSA's total daily intake, suggesting that the EFSA's guidance value has minimal risk. This model provides a foundation for scaling to other mycotoxins and other food animal species.

Biotransformation enzyme activities and phase I metabolites analysis in Litopenaeus vannamei following intramuscular administration of T-2 toxin.

T-2 toxin (T-2) is a type-A trichothecene produced by Fusarium that causes toxicity to animals. T-2 contamination of grain-based aquatic feed is a concern for the industries related to edible aquatic crustacean species such as the shrimp industry because it can lead to serious food safety issues. T-2, its metabolites, and selected phase I (EROD, CarE) and phase II (GST, UGT, SULT) detoxification enzymes in hemolymph and tissues were monitored at 0, 5, 10 15, 30, 45, and 60 min following T-2 intramuscular administration (3 mg/kg bw) in shrimp (Litopenaeus vannamei). Marked increases of EROD activity in hepatopancreas and CarE activity in hemolymph, gill, hepatopancreas and intestine were observed followed by increases in phase II enzymes (GST, UGT, SULT) in hepatopancreas, hemolymph, intestine and gill, which remained elevated for an extended period. Time-dependent decrease in shrimp tissue T-2 concentration was observed. HT-2 increased up to 15 min. Most other T-2 metabolites were detected but not T-2 tetraol. Enzyme responses on exposure to T-2 were tissue-specific and time-dependent. Detection results indicated that HT-2 may not be the only important metabolite in aquatic crustacean species. Further investigation into T-2 metabolite toxicity is needed to fully understand the food safety issues related to T-2.

Effect of T-2 toxin-injected shrimp muscle extracts on mouse macrophage cells (RAW264.7).

Following intramuscular injections of 0.1 mL, 3 mg kg-1 BW-1(1/10 LD50) T-2 toxin (T-2), the tissue concentration of T-2 in shrimp was quantitatively detected using LC-MS/MS. The biological half-time (t1/2) of T-2 in blood was 40.47 ± 0.24 min. The highest number of intramuscular T-2 shrimp could tolerate when given at blood t1/2 intervals was 4. The shrimps which were injected 5 T-2 died. The T-2 toxin highest accumulation was 0.471 ± 0.012 ng g-1 BW-1. The effect of toxic shrimp muscle subjected to different processing conditions (high pressure, trifluoroacetic acid, acid and alkali digestions, artificial digestive juice [to simulate exposure to gastric and intestinal juices]) on mouse macrophage cells (RAW267.4) were evaluated by the MTT assay. The inhibition ratio of 2% muscle extract on RAW267.4 was 85.70 ± 2.63%. The immunocytotoxicity of muscle extracts to RAW264.7 was highest in muscle extracts subjected to physical and chemical digestion (high pressure > NaOH > trifluoroacetic acid > 0.02 M HCl > 0.2 M HCl > controls), and also artificial digestion (artificial intestinal juice > artificial gastric juice > N type intestinal juice > N type gastric liquid > controls). Results showed that high-pressure and artificial intestinal juice were most effective in the release of modified T-2 to free T-2 thus enhancing toxicity. These results can be interpreted as measurement of T-2 in food being of little value because of enhanced toxicity of T-2-contaminated food as they pass through the gastrointestinal tract.

Comparison of T-2 Toxin and HT-2 Toxin Distributed in the Skeletal System with That in Other Tissues of Rats by Acute Toxicity Test.

Twelve healthy rats were divided into the T-2 toxin group receiving gavage of 1 mg/kg T-2 toxin and the control group receiving gavage of normal saline. Total relative concentrations of T-2 toxin and HT-2 toxin in the skeletal system (thighbone, knee joints, and costal cartilage) were significantly higher than those in the heart, liver, and kidneys (P < 0.05). The relative concentrations of T-2 toxin and HT-2 toxin in the skeletal system (thighbone and costal cartilage) were also significantly higher than those in the heart, liver, and kidneys. The rats administered T-2 toxin showed rapid metabolism compared with that in rats administered HT-2 toxin, and the metabolic conversion rates in the different tissues were 68.20%-90.70%.

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.

Metabolism of HT-2 Toxin and T-2 Toxin in Oats.

The Fusarium mycotoxins HT-2 toxin (HT2) and T-2 toxin (T2) are frequent contaminants in oats. These toxins, but also their plant metabolites, may contribute to toxicological effects. This work describes the use of 13C-assisted liquid chromatography-high-resolution mass spectrometry for the first comprehensive study on the biotransformation of HT2 and T2 in oats. Using this approach, 16 HT2 and 17 T2 metabolites were annotated including novel glycosylated and hydroxylated forms of the toxins, hydrolysis products, and conjugates with acetic acid, putative malic acid, malonic acid, and ferulic acid. Further targeted quantitative analysis was performed to study toxin metabolism over time, as well as toxin and conjugate mobility within non-treated plant tissues. As a result, HT2-3-O-ß-d-glucoside was identified as the major detoxification product of both parent toxins, which was rapidly formed (to an extent of 74% in HT2-treated and 48% in T2-treated oats within one day after treatment) and further metabolised. Mobility of the parent toxins appeared to be negligible, while HT2-3-O-ß-d-glucoside was partly transported (up to approximately 4%) through panicle side branches and stem. Our findings demonstrate that the presented combination of untargeted and targeted analysis is well suited for the comprehensive elucidation of mycotoxin metabolism in plants.

Mouse tissue distribution and persistence of the food-born fusariotoxins Enniatin B and Beauvericin.

The fusariotoxins Enniatin B (Enn B) and Beauvericin (Bea) have recently aroused interest as food contaminants and as potential anticancer drugs. However, limited data are available about their toxic profile. Aim of this study was to investigate their pharmacological behavior in vivo and their persistence in mice. Therefore, liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to analyze the distribution of Enn B and Bea in selected tissue samples and biological fluids originating from mice treated intraperitoneally with these cyclohexadepsipeptides. Overall, no toxicological signs during life time or pathological changes were observed. Moreover, both fusariotoxins were found in all tissues and serum but not in urine. Highest amounts were measured in liver and fat demonstrating the molecules tendency to bioaccumulate in lipophilic tissues. While for Bea no metabolites could be detected, for Enn B three phase I metabolites (dioxygenated-Enn B, mono- and di-demethylated-Enn B) were found in liver and colon, with dioxygenated-Enn B being most prominent. Consequently, contribution of hepatic as well as intestinal metabolism seems to be involved in the overall metabolism of Enn B. Thus, despite their structural similarity, the metabolism of Enn B and Bea shows distinct discrepancies which might affect long-term effects and tolerability in humans.

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.

Liquid chromatography-tandem mass spectrometry method for toxicokinetics, tissue distribution, and excretion studies of T-2 toxin and its major metabolites in pigs.

A rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantitatively analyzing T-2 toxin and its major metabolites (HT-2 toxin and T-2 triol) in swine biological samples. For all matrices, liquid-liquid extraction (ethyl acetate or acetonitrile) and Varian Bond-Elut MycoSep cartridges for solid phase extraction were used for sample preparation. The analytes were separated via a Zorbax XDB-C18 column and were detected using LC-MS/MS with an electrospray ionization interface in positive ion mode. The resulting calibration curves offered satisfactory linearity (r(2)>0.992) within the test range. The limits of quantification for T-2 toxin, HT-2 toxin, and T-2 triol were 1ng/mL (µg/kg), 2ng/mL (µg/kg), and 5ng/mL (µg/kg), respectively. The recovery rates in different matrices ranged from 74.3% to 102.4%, and the interday and intraday precisions were all less than 10.2% for the target analytes. The developed method was successfully applied to toxicokinetics, tissue distribution, and excretion studies of T-2 toxin and its major metabolites after intravenous (i.v.) administration in pigs. The results provide important information for evaluating and controlling human exposure to residual T-2 toxin and its major metabolites in animal-derived food.

T-2 toxin is hydroxylated by chicken CYP3A37.

T-2 toxin (T-2) is an acute toxic trichothecene mycotoxin produced mainly by Fusarium species, detected in many crops including oats, wheat and barley, in animal feed and food. It is important to know the metabolic pathway and kinetics of T-2 in food animals given that T-2 can cause serious adverse effects on human health. In this study, we investigated the metabolic capacity of chicken CYP3A37 in the metabolism of T-2 using reconstituted bacteria produced enzymes. Our results showed that chicken CYP3A37 is able to convert T-2 to 3'-OH T-2 with an apparent Km of 15.29 µM, and T-2 hydroxylation activity of CYP3A37 is strongly inhibited by ketoconazole (IC50=0.11 µM). We also observed that chicken CYP3A37 can catalyze erythromycin N-demethylation, another CYP3A-specific activity. These findings imply that chicken CYP3A37 may have a broad substrate spectrum, similar to its human homologue CYP3A4.

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).

Fusariotoxin transfer in animal.

Mycotoxin fusariotoxins, essentially represented by trichothecenes, zearalenone and fumonisins, are widely scattered in cereals and their products. Human and animals are particularly concerned by toxicity consecutive to oral chronic exposure. Human exposure can be direct via cereals or indirect via products of animals having eaten contaminated feed. As this alimentary risk is considered as a major problem in public health, it is thus of great importance to determine bioavailability, metabolic pathways and distribution of these mycotoxins in animal and human organism. Most studies indicate that fusariotoxins can be rapidly absorbed in the small intestine but the mechanisms involved remain unclear. Except NIV, fusariotoxins can be partly metabolised into more hydrophilic molecules in digestive tract or liver. Fumonisins present different behaviour as they seem very few and slowly absorbed and metabolised. The main part of absorbed fusariotoxins shows a rapid elimination within 24h after ingestion, followed by a slower excretion of small amounts. However, traces of fusariotoxins or their derivates can be found in animal products. This manuscript, reviewing literature published on fusariotoxin transfer, highlights that too little data are available to correctly appreciate fusariotoxin transfer in organism. Further studies focusing on mechanisms involved in the transfer are needed before clarifying risk assessment for human health.

Structural characterization of metabolites after the microbial degradation of type A trichothecenes by the bacterial strain BBSH 797.

Contamination of feed with trichothecenes, a group of Fusarium mycotoxins, leads to losses in performance due to their immunosupressive effects and the negative effect on the gastrointestinal system in animal production. A possible way of detoxification is microbial degradation, which was the focus of this study. A bacterial strain--BBSH 797--which can degrade some mycotoxins of the trichothecene group, has already been isolated. It transforms deoxynivalenol (DON) into its metabolite DOM-1, the non-toxic deepoxide of DON. Analogous to the microbial degradation of DON, the transformation of six different type A trichothecenes was observed. The metabolites appearing were characterized by GC-MS after derivatization with TRI-SIL TBT. Two metabolites were additionally, identified by liquid chromatography-mass spectrometry with particle beam interface (LC-PB-MS) with electron impact (EI)-ionization mode. The major finding was that scirpentriol was completely transformed into its non-toxic metabolite deepoxy scirpentriol, while the mycotoxin T-2 triol underwent a more complicated metabolism. According to the study, T-2-triol was degraded into its non-toxic deepoxy form and into T-2 tetraol, which was then further metabolized to deepoxy T-2 tetraol. GC-MS after derivatization with TRI-SIL TBT was suitable for the structural characterization of trichothecenes and their degradation products. Besides the mass spectra of already known degradation products, spectra of new metabolites could be recorded by LC-PB-MS.

Effect of T-2 toxin on blood-brain barrier permeability monoamine oxidase activity and protein synthesis in rats.

Systemic exposure to T-2 toxin disrupts brain biogenic monoamine metabolism. Although the mechanisms underlying these neurochemical perturbations are unclear, we have suggested that they are a reflection of increased blood-brain barrier (BBB) permeability, or altered protein synthesis that affects brain enzyme activities. Accordingly, BBB permeability, in vitro protein synthesis and in vitro monoamine oxidase (MAO) activity were examined in rats after either acute, or 7-day exposure to T-2. Membrane permeability was assessed from the recovery of systemically administered [14C]mannitol and [14C]dextran with [3H]water as the diffusible reference, either 2 hr post-intraperitoneal (i.p.) injections of 0, 0.2 and 1 mg T-2/kg body weight or following a 7-day exposure to diets containing 0 and 10 ppm T-2. Protein synthesis, determined by [14C]leucine incorporation, and MAO activity, determined by H2O2 production, were observed either 2 hr post-ip injection of 0 and 1 mg T-2/kg body weight or following a 7-day exposure to diets containing 0, 2.5 and 10 ppm T-2. Permeability increases were observed in all brain regions examined for mannitol, but not for dextran following T-2 i.p. The effect of dietary T-2 was more modest, affecting mannitol uptake in two brain regions, the cerebellum and pons plus medulla regions. Protein synthesis was significantly decreased by i.p. administration of T-2, while dietary treatment significantly reduced MAO enzyme activity. Collectively, the effect of T-2 toxin on BBB permeability, protein synthesis and MAO enzyme activity may account for the neurochemical imbalance observed in T-2 intoxication.

Effects of sterigmatocystin and T-2 toxin on the induction of unscheduled DNA synthesis in primary cultures of human gastric epithelial cells.

Primary cultures of human gastric epithelial cells were tested for induction of unscheduled DNA synthesis (UDS) by sterigmatocystin (ST) and T-2 toxin. Autoradiographic results indicated that ST (10(-6)-10(-4)M) induced UDS in the presence of S9 activation system. The repair rates were 24-91% (net grains > or = 3) and 2-71% (net grains > or = 5). T-2 toxin did not induce UDS in this study.

Comparative fate of the tritiated trichothecene mycotoxin, T-2 toxin, in chickens and ducks.

A tritiated preparation of the trichothecene mycotoxin, T-2 toxin, was administered as a single oral dose to 21-day-old male broiler (Hubbard x Hubbard) chickens and White Pekin ducks. There were few significant differences between the two species in metabolism, tissue retention, and excretion of T-2 toxin and its metabolites. On the basis of the data obtained, the differences in toxicological sensitivity to T-2 toxin known to exist between these two species cannot likely be attributed to differences in the metabolism or elimination of T-2 toxin from the body.