Mycotoxin found in Fusarium

This review focuses on the toxicity and metabolism of T-2 toxin and analytical methods used for the determination of T-2 toxin. Among the naturally occurring trichothecenes in food and feed, T-2 toxin is a cytotoxic fungal secondary metabolite produced by various species of Fusarium. Following ingestion, T-2 toxin causes acute and chronic toxicity and induces apoptosis in the immune system and fetal tissues. T-2 toxin is usually metabolized and eliminated after ingestion, yielding more than 20 metabolites. Consequently, there is a possibility of human consumption of animal products contaminated with T-2 toxin and its metabolites. Several methods for the determination of T-2 toxin based on traditional chromatographic, immunoassay, or mass spectroscopy techniques are described. This review will contribute to a better understanding of T-2 toxin exposure in animals and humans and T-2 toxin metabolism, toxicity, and analytical methods, which may be useful in risk assessment and control of T-2 toxin exposure [1].

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 [2].

Metabolism of T-2 [1]
T-2 is usually metabolized and eliminated after ingestion. In general, T-2 is soluble in water, and the major metabolic reactions are usually hydrolysis, hydroxylation, de-epoxidation, and conjugation. The most typical metabolites of T-2 are HT-2 toxin (hydrolysis), T-2 triol, T-2 tetraol, neosolaniol (NEO), 3′-hydroxy HT-2, 3′-hydroxy T-2, 3′-hydroxy T-2 triol, and dihydroxy HT-2 (Figure 2) and de-epoxy-3′-hydroxy T-2 and de-epoxy-3′-hydroxy HT-2 (Figure 3). Because human consumption of animal products contaminated with T-2 and its metabolites is a possibility, distribution and metabolism studies of T-2 toxin in animals could provide important information for both evaluating and controlling human exposure to residual T-2 metabolites in foods of animal origin.

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Liver Metabolic Systems [1]
Studies on the metabolism of T-2 have been carried out on liver S-9 fractions and microsomes. During initial studies, HT-2 was considered as the sole metabolite in bovine and human liver homogenates and in liver microsomes of various animals. However, new metabolites of T-2 were subsequently detected. Upon incubation of T-2 in a rat liver S-9 fraction, T-2 was rapidly converted into HT-2, T-2 tetraol, and two unknown metabolites, which were designated TMR-1 and TMR-2. TMR-1 was characterized as 4-deacetylneosolaniol (Figure 2) by spectroscopic analysis. TMR-1, TMR-2, and T-2 tetraol were also found in the incubation mixture of HT-2, which suggested that the three compounds were converted from T-2 via HT-2 by hydrolysis at the C-4 position. Along with these metabolites, four other metabolites of T-2 were detected in homogenates of mouse and monkey livers. These new metabolites included neosolaniol, 15-deacetylneosolaniol, 3′-hydroxy T-2, and 3′-hydroxy HT-2 (Figure 2). The hydroxylation reactions were enhanced by treating mice with phenobarbital (PB). Liver microsomes from rat, chicken, and mouse can biotransform T-2 into a variety of metabolites including HT-2, neosolaniol, 4-deacetylneosolaniol, T-2 triol, 3′-hydroxy T-2, and 3′-hydroxy HT-2 (Figure 2), and two unidentified compounds, RLM-2 and RLM-3. These two additional compounds were tentatively identified as isomers of 3′-hydroxy T-2 by gas chromatography−mass spectrometry (GC-MS). The major metabolite in microsomal preparations from both control and PB-induced microsomes was HT-2. Following incubation of PB-induced chickens with T-2, 3′-hydroxy T-2 was the major metabolite. However, 30 and 79% of the added T-2 remained unchanged 60 min after incubation in PB-induced and control chickens, respectively. The significant increase in hydroxylated metabolites formed by liver microsomes from PB-induced animals may be caused by cytochrome P-450 catalysis. This effect has been described in liver homogenates prepared from mice and monkeys and has been used to enhance the in vitro production of 3′-hydroxy metabolites by liver S-9 fractions isolated from pigs and rats. Treatment of a rat hepatic S-9 preparation with T-2 yielded primarily 3′-hydroxy T-2 (Figure 2) as the major metabolic product (>85%), and a new minor metabolite, RLM-3, was identified by GC-MS and 1H and 13C NMR experiments as 4′-hydroxy T-2. In work by Pace, [3H]T-2 was administered to perfused rat livers and used to study the metabolism and clearance of T-2. T-2 was metabolized and eliminated as 3′-hydroxy HT-2, 3′-hydroxy T-2 triol, 4-deacetylneosolaniol, and T-2 tetraol (Figure 2) and as glucuronide conjugates of HT-2, 3′-hydroxy HT-2, and T-2 tetraol. (90) The biochemical pathways of T-2 metabolism in perfused rat liver were the same as those observed in vivo. Again, bile was observed to be the major excretion route of T-2 and its metabolites, and the perfusion model was considered to be useful as a tool for the isolation of minor metabolites for structural analysis.

Animals [2]
Twenty 5-week-old clinically healthy chickens weighing 1.3–1.4 kg were used in this study. The broilers were kept under conditions of controlled temperature (25 °C) and humidity (45%). Chickens were allowed a 7-day acclimation period prior to the study initiation. T-2 toxin-free feeds and drinks were supplied ad libitum. The composition of the feeds was 56.8% corn, 25.0% soybean meal, 8% bran wheat, and other ingredients. They could meet the daily nutrition of the chickens.

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Experimental design [2]
Twenty chickens were randomly divided into two groups. Twelve chickens in group A were administered with T-2 toxin by a single intravenous bolus injection (0.5 mg/kg b.w.); eight chickens in group B received T-2 toxin orally at a dosage of 2.0 mg/kg b.w., every 12 h for 2 days. The solutions for intravenous (i.v.) and oral administrations were freshly prepared daily at a concentration of 2 mg/mL by dissolving T-2 toxin standard in ethyl alcohol—sterilized deionized water (1:1) mixture. T-2 toxin was administered intravenously into the right wing vein of the chickens in group A and was administered orally directly to the craw of the chickens in group B using a thin plastic tube attached to a syringe.
Blood samples (1.0 mL) were collected from the left wing vein of each chicken in groups A and B. Samples were collected into a heparinized tube through a needle at the following time points: 0, 2.5, 5, 7.5, 10, 15, 20, 30, 45 min, 1, 1.5, and 2 h postadministration for the i.v. administration and 0, 5, 10, 15, 20, 30, 45 min, 1, 1.5, 2, 2.5, 3, 4.5, and 6 h postadministration after the last dose for the oral administration. Blood samples were centrifuged at 1500 g for 10 min. The supernatants were stored frozen at −20 °C until analysis.

Absorption, Distribution and Excretion
T2-trihomycete is readily absorbed by pigs and rats through the skin and intestines. T-2 toxin can be transmitted through the milk of lactating cows and pigs. It is estimated that eggs from chickens orally administered 1 mg/kg body weight of T-2 toxin daily for 8 consecutive days (equivalent to 1.6 mg/kg T-2 toxin in feed) contain 0.9 μg of the substance. Following oral administration of (3)H-T2-trihomycete (1 mg/kg body weight) to mice and rats, the radioactive material was recovered in feces (55%) and urine (15%) within 72 hours. The substance was distributed to the liver, kidneys, and other organs without specific accumulation. Following oral administration of (3)HT-2 toxin to mice and rats, the toxin rapidly distributed to tissues and was excreted in feces and urine. In mice, the concentration of the radiolabeled substance in plasma peaked 30 minutes after oral administration… and also peaked after intramuscular injection in guinea pigs… After the addition of (3)HT-2 toxin to chick feed, peak levels were observed in blood, plasma, abdominal fat, heart, kidneys, gizzard, liver, and the remainder of the carcass within 4 hours, and in muscle, skin, bile, and gallbladder within 12 hours… The distribution of T-2 toxin in pig tissues was similar to that in chickens…

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Metabolism/Metabolites
Two major metabolites were isolated from the urine of lactating cows orally administered 180 mg of T-2 toxin. They were 3'-hydroxy-HT2 toxin and 3'-hydroxy-T2 toxin, respectively.
Human liver enzymes can deacetylate T2-trihomycete to HT2-trihomycete in vitro.
After oral administration of (3)H-T2-trihomycete (1 mg/kg body weight) to mice and rats, 55% of the radioactive material was recovered in feces and 15% in urine within 72 hours. Analysis of the radioactive material recovered from rat feces revealed that 2.7% of the dose was excreted as unmetabolized T2-trihomyceteene, and 7.5% as 4-O-deacetylated T2-trihomyceteene (HT2-trihomyceteene)... The remaining fecal excretion products were not identified. In urine, HT2-trihomyceteene (1.4% of the total dose) and 8-hydroxydiacetoxyscarbenol (1.8%) were identified; three unidentified metabolites were also isolated. The epoxide moiety appears to be crucial to its toxicological activity; the liver may detoxify T2-triterpenes via epoxide hydrolases. In vitro experiments showed that rat liver homogenate metabolized T2-triterpenes to HT2-triterpenes, T2-triterpenestetraol, 4-deacetylated neosoraniol, and neosoraniol. The same metabolite was also obtained from HT2-triterpenes, indicating that T2-triterpenes preferentially hydrolyze at the C-4 position to generate NT2-triterpenes. Triterpenes are sesquiterpene toxins produced by Fusarium species. Due to the high stability of these fungal toxins, there is great interest in microbial transformation methods capable of removing toxins from contaminated grains or cereal products. We tested 23 yeast species belonging to the clade Trichomonascus (Ascomycota, subphylum Yeast), including 4 Mucor species and 19 asexual species currently classified as Blastobotrys, to evaluate their ability to convert the trihomocytoxin T-2 into a less toxic product. These yeast species were capable of three types of biotransformation: acetylation to 3-acetyl T-2 toxin, glycosylation to T-2 toxin 3-glucoside, and removal of the isovaleryl group to generate neosoranilol. Some yeast species were capable of more than one type of biotransformation. Three species of Bacillus have been shown to convert T-2 toxin into T-2 toxin 3-glucoside, a compound identified as a cryptic fungal toxin found in Fusarium-infected grains. This is the first report of the production of trifidax toxin glycosides using a whole-cell microbial method. The potential large-scale application of T-2 toxin 3-glucoside will contribute to toxicity testing and the development of methods for detecting this compound in agricultural products and other products. More complete metabolite/metabolite data for T-2 toxin (a total of 6 metabolites) can be found on the HSDB record page.
Biological Half-Life
The plasma half-life of T-2 toxin is less than 20 minutes. Incubation of 9000 g of supernatant from a homogenate of human or bovine liver at 37°C for 75 minutes converts T-2 toxin into 3'-hydroxy-HT2. The metabolic rate of T-2 toxin in humans (half-life 20 minutes) is faster than in cattle (half-life 40 minutes). The toxicity of this metabolite is comparable to that of T-2 toxin, and it induces vomiting almost as quickly as T-2 toxin.

Toxicity Data
LC50 (rat) = 20 mg/m³/10 min

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[1]. T-2 toxin, a trichothecene mycotoxin: review of toxicity, metabolism, and analytical methods. J Agric Food Chem. 2011 Apr 27;59(8):3441-53.

[2]. Toxicokinetics of T-2 toxin and its major metabolites in broiler chickens after intravenous and oral administration. J Vet Pharmacol Ther. 2015 Feb;38(1):80-5.

T-2 toxin is a trichothecene toxin produced by fungi of the genus Fusarium. It is a common contaminant in cereal foods and animal feed, known to cause a variety of toxic effects in humans and animals. It exhibits multiple toxicities, including mycotoxin, cardiotoxic substance, neurotoxin, environmental contaminant, apoptosis inducer, DNA synthesis inhibitor, and fungal metabolite. It is a trichothecene compound, an acetate compound, and an organic heterotetracyclic compound. It is functionally related to HT-2 toxin. T-2 toxin has been reported in Fusarium heterosporum, Fusarium chlamydosporum, and other organisms with relevant data. T-2 toxin is a type A trichothecene toxin produced by Fusarium langsethiae, Fusarium poae, and Fusarium sporotrichioides, which interferes with membrane phospholipid metabolism by inhibiting protein synthesis and disrupting DNA and RNA synthesis. It frequently contaminates various cereal crops and causes severe inflammatory responses in animals. This is a potent fungal toxin produced in feed by several fungi of the genus Fusarium. It can cause severe inflammatory responses in animals and has teratogenic effects. Mechanism of Action Studies of the whole-cell and cell-free protein synthesis systems and acid-soluble cell components of Tetrahymena pyriformis have shown that T-2 toxin inhibits protein synthesis by damaging the 60S ribosomal subunit and by interfering with cell membrane function to inhibit RNA and DNA synthesis. T2-trihomycete toxins bind to the active sulfhydryl groups (-SH) of creatine phosphokinase, lactate dehydrogenase, and alcohol dehydrogenase in vitro, inhibiting their catalytic activity. The high affinity of T2-trihomycete toxins and higher trihomycete toxins for sulfhydryl compounds provides the molecular basis for their interaction with the spindle mechanism. ...
T-2 toxin can inhibit DNA and RNA synthesis in vivo (0.75 mg/kg body weight, single or multiple doses) and in vitro (>0.1-1 ng/mL).
For more complete data on the mechanism of action of T-2 toxin (14 in total), please visit the HSDB record page.

C24H34O9

466.52136

466.22

C, 61.79; H, 7.35; O, 30.86

21259-20-1

T-2 Toxin-13C24

5284461

White to off-white solid powder

1.3±0.1 g/cm3

544.9±50.0 °C at 760 mmHg

151.5℃

177.0±23.6 °C

0.0±3.3 mmHg at 25°C

1.547

2.25

1

9

9

33

881

8

CC1=C[C@@H]2[C@](C[C@@H]1OC(=O)CC(C)C)([C@]3([C@@H]([C@H]([C@H]([C@@]34CO4)O2)O)OC(=O)C)C)COC(=O)C

BXFOFFBJRFZBQZ-QYWOHJEZSA-N

InChI=1S/C24H34O9/c1-12(2)7-18(27)32-16-9-23(10-29-14(4)25)17(8-13(16)3)33-21-19(28)20(31-15(5)26)22(23,6)24(21)11-30-24/h8,12,16-17,19-21,28H,7,9-11H2,1-6H3/t16-,17+,19+,20+,21+,22+,23+,24-/m0/s1

[(1S,2R,4S,7R,9R,10R,11S,12S)-11-acetyloxy-2-(acetyloxymethyl)-10-hydroxy-1,5-dimethylspiro[8-oxatricyclo[7.2.1.02,7]dodec-5-ene-12,2'-oxirane]-4-yl] 3-methylbutanoate

T-2 TOXIN; T2 Toxin; 21259-20-1; T2-Trichothecene; Mycotoxin T-2; Isaritoxin; Fusariotoxine T2; Fusariotoxin T-2;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 100 mg/mL (~214.35 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.36 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.36 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.36 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)