Mechanisms involved in the induction of apoptosis by T-2 and HT-2 toxins in HL-60 human promyelocytic leukemia cells

T-2 and HT-2 toxins belong to a group of mycotoxins that are widely encountered as natural contaminants known to elicit toxic responses in hematopoietic cells. In the present study, HL-60 cells were used to characterize the apoptotic effects of T-2 and a major metabolite, HT-2, and to examine the mechanisms involved. Apoptotic cells were identified microscopically by chromatin condensation and nuclear fragmentation, by flow cytometric analysis, and by DNA gel electrophoresis. T-2 and HT-2 induced concentration-dependent apoptosis after 24 h in HL-60 cells, starting at concentrations of 3.1 and 6.25 ng/ml respectively. An increased number of apoptotic cells could be observed 4–6 h after exposure to 12.5 ng/ml of toxin. Little cytotoxicity (plasma membrane damage) was observed even after exposure to concentrations of toxins (25–50 ng/ml) inducing apoptosis in 60–100% of the cells. The apoptotic process was almost completely blocked in the presence of the general caspase inhibitor zVAD.fmk. In contrast, no or only minor effects were observed with the more specific caspase inhibitors DEVD.CHO, IETD.fmk, and DEVD.fmk. As judged by Western blotting, the levels of several procaspases (-3, -7, -8, -9, but not -12) were reduced 3–6 h after exposure to toxin. Substantial increases in the presumed active form(s) of caspase-8 and -9 were observed. Furthermore, poly(ADP-ribose) polymerase (PARP) was already markedly cleaved 3 h after toxin treatment, indicative of active caspase-3 and -7. No or only minor changes in Bcl-2, Bcl-XL and Bax levels were observed. BAPTA-AM and ZnCl₂ blocked the degradation of procaspases, the fragmentation of PARP, and the induction of apoptosis. In summary, both T-2 and HT-2 induced apoptosis, with T-2 being somewhat more potent than HT-2. The divalent calcium concentration, [Ca²⁺], appears to be involved in the activation of several caspases, resulting in DNA fragmentation, chromosomal condensation, and nuclear fragmentation.     

In vitro metabolism of T-2 toxin in rats.

T-2 toxin was rapidly converted in the 9,000 X g supernatant fraction of rat liver homogenate into HT-2 toxin, T-2 tetraol, and two unknown metabolites designated as TMR-1 and TMR-2. TMR-1 was characterized as 4-deacetylneosolaniol (15-acetoxy-3 alpha, 4 beta, 8 alpha-trihydroxy-12,13-epoxytrichothec-9-ene) by spectroscopic analyses. Since the same metabolites were also obtained from HT-2 toxin used as substrate, it was concluded that T-2 toxin was hydrolyzed preferentially at the C-4 position to give HT-2 toxin, which was then metabolized to T-2 tetraol via 4-deacetylneosolaniol. In addition to HT-2 toxin, 4-deacetylneosolaniol and T-2 tetraol, a trace amount of neosolaniol was transformed from T-2 toxin by rat intestinal strips. In vitro metabolic pathways for T-2 toxin in rats are proposed.     

In vitro formation of 3'-hydroxy T-2 and 3'-hydroxy HT-2 toxins from T-2 toxin by liver homogenates from mice and monkeys.

In vitro metabolism of T-2 toxin was studied in homogenates of mouse and monkey livers. In addition to several hydrolyzed products, including HT-2 toxin, neosolaniol, 4-deacetylneosolaniol, 15-deacetylneosolaniol, and T-2 tetraol, two metabolic products were isolated from the incubation mixture. Their structures were confirmed as 3'-hydroxy T-2 toxin and 3'-hydroxy HT-2 toxin on the basis of mass and nuclear magnetic resonance spectroscopy. The formation of these hydroxylated metabolites was found in the microsomes in the presence of NADPH, and the hydroxylation reaction was enhanced by treating mice with phenobarbital. The results suggest that a cytochrome P-450 is catalyzing the hydroxylation at the C-3' position of T-2 and HT-2 toxins. An in vitro metabolic pathway of T-2 toxin in the hepatic homogenates containing the NADPH-generating system is proposed.     

Metabolism of trichothecene mycotoxins. I. Microsomal deacetylation of T-2 toxin in animal tissues

In an attempt to elucidate the active form of T-2 toxin, one of trichothecene mycotoxins in vivo, the metabolism in animal tissues was studied in vitro by using gas liquid chromatography. T-2 toxin was selectively hydrolysed by the microsomal esterase at C-4, giving rise to HT-2 toxin as the only metabolite. This esterase activity was found mainly in the microsomes of liver, kidney, and spleen of laboratory animals. Since the enzymatic hydrolysis of T-2 toxin was inhibited by eserine, and diisopropylfluorophosphate, it is concluded that non-specific carboxyesterase [EC 3.1.1.1] of microsomal origin participates in this type of selective hydrolysis of T-2 toxin. The microsomal fraction from rabbit liver was proved to be a convinient material for the preparation of HT-2 toxin from T-2 toxin. From the evidence that the toxicity of HT-2 toxin is comparable to that of T-2 toxin and that the microsomal fraction of whole liver possesses the ability to biotransform the total lethal dose of T-2 toxin into HT-2 within a few minutes, T-2 toxin administered to animals is presumed to exhibit its toxicity partly as HT-2 toxin.     

Trichothecene mycotoxins produced byFusarium sporotrichioides strain P-11

A highly toxic strain ofFusarium sporotrichioides Sherb. (P-11) isolated from wheat in Poland produced on rice culture up to 11 trichothecenes, which are: T-2 toxin (750 ppm), neosolaniol (300 ppm), HT-2 toxin (75 ppm), acetyl T-2 toxin (35ppm), 3′-hydroxy-T-2 (20ppm), T-2 triol (12.5ppm), 3′-hydroxy-HT-2 (1.2ppm), 4-acetoxy-T-2 tetraol (1.1 ppm), 15-acetoxy-T-2 tetraol (0.65 ppm), 8-acetoxy-T-2 tetraol (0.45 ppm), and T-2 tetraol (0.2 ppm). The presence of most of these trichothecenes, including the 3′-hydroxy-derivatives, in the excreta of animals treated with T-2 toxin indicates the existence of some correlation between T-2 toxin metabolism in animals and microorganisms, respectively.     

Production and characterization of a generic antibody against group A trichothecenes

An antibody against group A trichothecenes was produced after immunization of rabbits with an immunogen prepared by conjugation of T-2 toxin to bovine albumin at the C-8 position. T-2 toxin was first converted to 3-acetylneosolaniol (3-Ac-NEOS) and then to its hemisuccinate (HS) before conjugation to the protein. The rabbits showed a quick immune response after immunization of the new conjugate. The antibody produced bound with tritiated T-2 toxin, T-2 tetraol tetraacetate, and diacetoxyscirpenol (DAS) and showed good cross-reactivities with most of the group A trichothecenes. The concentrations causing 50% inhibition of binding of 3H-T-2 toxin to the new antibody by unlabeled T-2, acetyl-T-2, 3'-OH-T-2, DAS, 3-Ac-NEOS-HS, 3'-OH-Ac-T-2, T-2 tetraol tetraacetate, iso-T-2, 3-Ac-NEOS, Ac-DAS, and 3,4,15-triacetyl-7-deoxynivalenol were found to be 0.34, 0.34, 0.6, 2.5, 4, 10, 18, 24, 100, 200, and 300 ng/assay, respectively; for HT-2, T-2 triol, and T-2 tetraol, the concentration was greater than 1000 ng/assay. Nivalenol, deoxynivalenol (DON), 15-acetyl-DON, and triacetyl-DON, did not inhibit the binding at 1000 ng/assay. The practical application of using this new antibody for radioimmunoassay (RIA) of trichothecene was tested by spiking T-2 toxin to corn. T-2 toxin was then extracted with acetone, subjected to a simple Sep-Pak C-18 reversed-phase treatment, and analyzed by RIA. The overall recovery for 18 samples spiked with 10 to 50 ppb of T-2 toxin was 94.22%.     

Inhibition of mitogen-induced blastogenesis in human lymphocytes by T-2 toxin and its metabolites.

Concentrations of T-2, HT-2, 3'-OH T-2, 3'-OH HT-2, T-2 triol, and T-2 tetraol toxins which inhibited [3H]thymidine uptake in mitogen-stimulated human peripheral lymphocytes by 50% were 1.5, 3.5, 4.0, 50, 150, and 150 ng/ml, respectively. The results suggested that the initial hydrolysis of T-2 toxin and the hydroxylation of T-2 toxin to 3'-OH T-2 toxin did not significantly decrease the immunotoxicity of the parent molecule, whereas further hydrolysis to T-2 triol and T-2 tetraol toxins or hydroxylation to 3'-OH HT-2 toxin decreased in vitro toxicity for human lymphocytes.     

Inhibition of mitogen-induced blastogenesis in human lymphocytes by T-2 toxin and its metabolites

Concentrations of T-2, HT-2, 3'-OH T-2, 3'-OH HT-2, T-2 triol, and T-2 tetraol toxins which inhibited [3H]thymidine uptake in mitogen-stimulated human peripheral lymphocytes by 50% were 1.5, 3.5, 4.0, 50, 150, and 150 ng/ml, respectively. The results suggested that the initial hydrolysis of T-2 toxin and the hydroxylation of T-2 toxin to 3'-OH T-2 toxin did not significantly decrease the immunotoxicity of the parent molecule, whereas further hydrolysis to T-2 triol and T-2 tetraol toxins or hydroxylation to 3'-OH HT-2 toxin decreased in vitro toxicity for human lymphocytes.     

Influence of T-2 and HT-2 Toxin on the Blood-Brain Barrier In Vitro: New Experimental Hints for Neurotoxic Effects

The trichothecene mycotoxin T-2 toxin is a common contaminant of food and feed and is also present in processed cereal derived products. Cytotoxic effects of T-2 toxin and its main metabolite HT-2 toxin are already well described with apoptosis being a major mechanism of action. However, effects on the central nervous system were until now only reported rarely. In this study we investigated the effects of T-2 and HT-2 toxin on the blood-brain barrier (BBB) in vitro. Besides strong cytotoxic effects on the BBB as determined by the CCK-8 assay, impairment of the barrier function starting at low nanomolar concentrations were observed for T-2 toxin. HT-2 toxin, however, caused barrier disruption at higher concentrations compared to T-2 toxin. Further, the influence on the tight junction protein occludin was studied and permeability of both toxins across the BBB was detected when applied from the apical (blood) or the basolateral (brain) side respectively. These results clearly indicate the ability of both toxins to enter the brain via the BBB.     

Thymocyte apoptosis by T-2 toxin in vivo in mice is independent of Fas/Fas ligand system

To find whether Fas/Fas ligand (FasL) pathway is involved in T-2 toxin (T-2)-mediated thymocyte apoptosis, we used lpr/lpr (lpr) and gld/gld (gld) mice, whose Fas and FasL proteins, respectively, are functionally deficient. Based on the DNA fragmentation profile in gel electrophoresis and measurement of apoptotic cell percent by flow cytometry, the levels of thymocyte apoptosis in lpr and gld mice that had received T-2 showed that both lpr and gld mice had undergone apoptosis essentially to the same magnitude as those of corresponding wild type mice (+/+). These results strongly suggest that T-2-induced thymocyte apoptosis in vivo in mice is independent of the Fas/FasL pathway.     

Large-scale production of selected type A trichothecenes: the use of HT-2 toxin and T-2 triol as precursors for the synthesis of <Emphasis Type="Italic">d</Emphasis><Subscript>3</Subscript>-T-2 and <Emphasis Type="Italic">d</Emphasis><Subscript>3</Subscript>-HT-2 toxin

The type A trichothecenes T-2 and HT-2 toxins are toxic secondary metabolites produced by fungi of the Fusarium genus. Their occurrence in cereals, especially in oats, implies health risks for the consumer. Therefore, it is an important task to develop selective and sensitive methods for the analysis of T-2 and HT-2 toxins, and to undertake further studies on their stability and toxicity. Although most toxins are commercially available, their high prices are the limiting factor on the realization of these experiments. Thus, we developed a method for large-scale production of T-2 and HT-2 toxin as well as T-2 triol and T-2 tetraol. T-2 toxin was obtained in gram quantities by biosynthetic production with cultures of F. sporotrichioides. As HT-2 toxin was only formed as a by-product, and T-2 triol and T-2 tetraol were not generated, these compounds were produced by alkaline hydrolysis of T-2 toxin. Separation and isolation of crude toxins was achieved by fast centrifugal partition chromatography (FCPC), which is an efficient tool for the large-scale purification of natural products. Using this fast and yield effective technique, several hundred milligrams of HT-2 toxin, T-2 triol, and T-2 tetraol were obtained. Subsequent, HT-2 toxin and T-2 triol were used for the large-scale synthesis of isotope-labeled T-2 and HT-2 toxin, respectively. Using these standards, an isotope dilution-(ID)-HPLC-MS/MS method for the quantification of T-2 and HT-2 toxin in different matrices was developed.     

A monoclonal antibody-based enzyme immunoassay for the detection of T-2 toxin at picogram levels

Thirteen monoclonal antibodies reactive with HT-2 were prepared by using a HT-2 hemisuccinate coupled to human serum albumin as antigen for the immunization of BALB/c mice. In a competitive enzyme immunoassay on a double antibody solid phase using HT-2 hemisuccinate coupled to horseradish peroxidase as enzyme linked toxin all antibodies reacted much better with T-2 toxin and acetyl T-2 than with HT-2. Eleven antibodies showed almost the same sensitivity and specificity, and one of these, designated 3E2, is extensively described. Its cross-reactivities with HT-2, T-2 toxin, acetyl T-2, iso T-2, T-2 tetraol tetraacetate and T-2 triol were 1·0, 140·2, 161·2, 0·32, 0·14 and 0·016, respectively. Two other antibodies, designated 2A4 and 2A5, behaved quite differently. The cross-reactivities of antibody 2A4 with these toxins were: 1·0, 113·9, 374·4, 1·35, 0·34 and 0·023, respectively; for antibody 2A5 they were 1·0, 46·1, 155·4, 8·31, 0·9 and 0·08, respectively. All antibodies proved to be IgGl. By using the antibody 3E2 a highly sensitive and very specific enzymc immunoassay for the detection of T-2 toxin was developed. The detection limit for T-2 toxin was 5 pg/ml (0·25 pg/assay).     

Modification of in vitro metabolism of T-2 toxin by esterase inhibitors

In vitro metabolism of T-2 toxin with S-9 fraction obtained from livers of phenobarbital-treated pigs and rats in the presence of different esterase inhibitors, including NaF, p-hydroxymercuribenzoate, phenylmethylsulfonyl fluoride, eserine sulfate, diisopropylfluorophosphate, and diethyl p-nitrophenyl phosphate, was studied. The metabolism was completely shifted to the hydroxylation at the C-3' position in the T-2 toxin molecule when esterase inhibitors were present. Diethyl p-nitrophenyl phosphate was found to be the most potent among six esterase inhibitors tested. In the presence of 10(-4) M diethyl p-nitrophenyl phosphate, 3'-hydroxy-T-2 toxin was the only metabolite detected. Similar results were obtained when other T-2-related metabolites were tested. The yield of conversion of T-2 toxin, acetyl T-2 toxin, HT-2 toxin and T-2 triol to their respective 3'-hydroxyl derivatives were 82, 73, 72, and 75%, respectively.     

Structure-Function Analysis of Porcine Cytochrome P450 3A29 in the Hydroxylation of T-2 Toxin as Revealed by Docking and Mutagenesis Studies

T-2 toxin, one of the type A trichothecenes, presents a potential hazard to human and animal health. Our previous work demonstrated that porcine cytochrome P450 3A29 (CYP3A29) played an important role in the hydroxylation of T-2 toxin. To identify amino acids involved in this metabolic process, T-2 toxin was docked into a homology model of CYP3A29 based on a crystal structure of CYP3A4 using AutoDock 4.0. Nine residues of CYP3A29, Arg105, Arg106, Phe108, Ser119, Lys212, Phe213, Phe215, Arg372 and Glu374, which were found within 5 Å around T-2 toxin were subjected to site-directed mutagenesis. In the oxidation of nifedipine, the CL _int value of R106A was increased by nearly two-folds compared with the wild-type CYP3A29, while the substrate affinities and CL _int values of S119A and K212A were significantly reduced. In the hydroxylation of T-2 toxin, the generation of 3′-OH-T-2 by R105A, S119A and K212A was significantly less than that by the wild-type, whereas R106A slightly increased the generation of 3′-OH-T-2. These results were further confirmed by isothermal titration calorimetry analysis, suggesting that these four residues are important in the hydroxylation of T-2 toxin and Arg105 may be a specific recognition site for the toxin. Our study suggests a possible structure-function relationship of CYP3A29 in the hydroxylation of T-2 toxin, providing with new insights into the mechanism of CYP3A enzymes in the biotransformation of T-2 toxin.     

Production and characterization of antibodies against HT-2 toxin and T-2 tetraol tetraacetate.

Three new immunogens which were prepared by conjugation of the carboxymethyl oxime (CMO) derivatives of HT-2 toxin, T-2 tetraol (T-2 4ol), and T-2 tetraol tetraacetate (T-2 4Ac) to bovine serum albumin (BSA) were tested for the production of antibodies against the major metabolites of T-2 toxin. Antibodies against HT-2 toxin and T-2 4Ac were obtained from rabbits 5 to 10 weeks after immunizing the animals with CMO-HT-2-BSA and CMO-T-2 4Ac-BSA conjugates. Immunization with CMO-T-2 4ol-BSA resulted in no antibody against T-2 4ol. The antibody produced against HT-2 toxin had great affinity for HT-2 toxin as well as good cross-reactivity with T-2 toxin. The relative cross-reactivities of anti-HT-2 toxin antibody with HT-2 toxin, T-2 toxin, iso-T-2 toxin, acetyl-T-2 toxin, 3'-OH HT-2, 3'-OH T-2, T-2 triol, and 3'-OH acetyl-T-2, were 100, 25, 10, 3.3, 0.25, 0.15, 0.12 and 0.08%, respectively. Antibody against CMO-T-2 4Ac was very specific for T-2 4Ac and had less than 0.1% cross-reactivity with T-2 toxin, HT-2 toxin, acetyl-T-2 toxin, diacetoxyscirpenol, deoxynivalenol, and deoxynivalenol triacetate as compared with T-2 4Ac. The detection limits for HT-2 toxin and T-2 4ol by radioimmunoassay were approximately 0.1 and 0.5 ng per assay, respectively.     

Production and characterization of antibodies against HT-2 toxin and T-2 tetraol tetraacetate

Three new immunogens which were prepared by conjugation of the carboxymethyl oxime (CMO) derivatives of HT-2 toxin, T-2 tetraol (T-2 4ol), and T-2 tetraol tetraacetate (T-2 4Ac) to bovine serum albumin (BSA) were tested for the production of antibodies against the major metabolites of T-2 toxin. Antibodies against HT-2 toxin and T-2 4Ac were obtained from rabbits 5 to 10 weeks after immunizing the animals with CMO-HT-2-BSA and CMO-T-2 4Ac-BSA conjugates. Immunization with CMO-T-2 4ol-BSA resulted in no antibody against T-2 4ol. The antibody produced against HT-2 toxin had great affinity for HT-2 toxin as well as good cross-reactivity with T-2 toxin. The relative cross-reactivities of anti-HT-2 toxin antibody with HT-2 toxin, T-2 toxin, iso-T-2 toxin, acetyl-T-2 toxin, 3'-OH HT-2, 3'-OH T-2, T-2 triol, and 3'-OH acetyl-T-2, were 100, 25, 10, 3.3, 0.25, 0.15, 0.12 and 0.08%, respectively. Antibody against CMO-T-2 4Ac was very specific for T-2 4Ac and had less than 0.1% cross-reactivity with T-2 toxin, HT-2 toxin, acetyl-T-2 toxin, diacetoxyscirpenol, deoxynivalenol, and deoxynivalenol triacetate as compared with T-2 4Ac. The detection limits for HT-2 toxin and T-2 4ol by radioimmunoassay were approximately 0.1 and 0.5 ng per assay, respectively.     

Type A-trichothecenes — Quantitative analysis using LC-MS and occurrence in Austrian maize and oats

During this study a method was developed for the quantitative determination of diacetoxyscirpenol (DAS), T-2 toxin and HT-2 toxin using deuterated T-2 toxin (T-2 d3 toxin) as an internal standard. The described method involves a clean up step of maize extract by the use of Mycosep® 227 columns a chromatographic separation on a Zorbax® bonus-RP-column (2,1×150mm) and the detection and quantification step on a mass spectrometer in SIM (Selected Ion Monitoring) mode.Data on the occurrence of three type A trichothecenes in Austrian maize, maize silage and oats were collected. It could be shown that maize and silage samples harvested in 2002 were only contaminated to a small extent with T-2 toxin (8% of the maize, 0% of the silage) and with HT-2 toxin (30% of the maize, 18% of the silage). But most of the analysed oats samples showed significant levels of T-2 toxin (64%) or HT-2 toxin (82%).     

Identification of various T-2 toxin metabolites in chicken excreta and tissues

Gas chromatography-mass spectrometry was used to identify various T-2 toxin metabolites in chicken excreta and organs 18 h after intraperitoneal injection of the toxin. No trichothecenes were detected in the heart and kidneys, and only trace amounts were detected in the lungs. Most of the T-2 metabolites were found in the excreta, although considerable amounts were also found in the liver. In addition to the previously identified T-2 metabolites in chicken excreta (HT-2 toxin, 15 acetoxy T-2 tetraol, and T-2 tetraol), we found 3'-hydroxy HT-2 toxin (the major metabolite in excreta and organs), 3'-hydroxy T-2 toxin, 4-acetoxy T-2 tetraol, and trace amounts of 8-acetoxy T-2 tetraol, 3-acetoxy-3'hydroxy HT-2 toxin, and T-2 triol. Unmetabolized T-2 toxin and an unidentified isomer of T-2 tetraol monoacetate were also detected in the excreta. Most of the metabolites in the chicken are similar to those encountered in cultures of fungal species producing T-2 toxin. A comparison with T-2 toxin metabolism in the cow is also reported.     

Identification of various T-2 toxin metabolites in chicken excreta and tissues.

Gas chromatography-mass spectrometry was used to identify various T-2 toxin metabolites in chicken excreta and organs 18 h after intraperitoneal injection of the toxin. No trichothecenes were detected in the heart and kidneys, and only trace amounts were detected in the lungs. Most of the T-2 metabolites were found in the excreta, although considerable amounts were also found in the liver. In addition to the previously identified T-2 metabolites in chicken excreta (HT-2 toxin, 15 acetoxy T-2 tetraol, and T-2 tetraol), we found 3'-hydroxy HT-2 toxin (the major metabolite in excreta and organs), 3'-hydroxy T-2 toxin, 4-acetoxy T-2 tetraol, and trace amounts of 8-acetoxy T-2 tetraol, 3-acetoxy-3'hydroxy HT-2 toxin, and T-2 triol. Unmetabolized T-2 toxin and an unidentified isomer of T-2 tetraol monoacetate were also detected in the excreta. Most of the metabolites in the chicken are similar to those encountered in cultures of fungal species producing T-2 toxin. A comparison with T-2 toxin metabolism in the cow is also reported.     

Identification of iso-TC-1 as a new T-2 toxin metabolite in cow urine

The structure of a new metabolite T-2 toxin (iso-TC-1) has been established as 3,15-diacetoxy-4-hydroxy-8(3-methyl-3'-hydroxy-butyryloxy)-12, 13-epoxytrichothec-9-ene. The compound is an isomer of TC-1 (a recently isolated T-2 derivative) in which the hydroxy and acetoxy groups at the C-3 and C-4 positions, respectively, are reversed. Direct probe analysis by electron impact (EI) of the underivatized iso-TC-1, as well as EI, positive chemical ionization (CI) in methane, and positive CI in ammonia of its trimethylsilylether or trifluoroacetate provided evidence to support the structure assignment of the new metabolite. The mass spectra of iso-TC-1 were compared with those of TC-1, T-2 toxin and iso-T-2 toxin (the isomer of T-2 toxin having reversed substituents at C-3 and C-4) with regard to molecular weight and fragments involving the substituents at C-3, C-4, C-8 and C-15. Although the two isomers, TC-1 and iso-TC-1, were not easily resolved by thin layer chromatography (TLC), a very good separation of their trimethylsilyl and trifluoroacetate derivatives was obtained by capillary gas chromatography. Acetylation of TC-1 or iso-TC-1 gave the same product. Iso-TC-1 is one of the main products of T-2 metabolism in the cow (more abundant than TC-1) and is found in the urine.