T-2 toxin, a trichothecene mycotoxin: review of toxicity, metabolism, and analytical methods

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.     

Determination of 12 type A and B trichothecenes in cereals by liquid chromatography-electrospray ionization tandem mass spectrometry

A new sensitive method for the simultaneous determination of 12 trichothecenes (deoxynivalenol, nivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, fusarenon X, T-2 toxin, HT-2 toxin, neosolaniol, monoacetoxyscirpenol, diacetoxyscirpenol, T-2 triol, and T-2 tetraol) by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is presented. The development of the method and investigations on the matrix influence on the MS signal are described in particular. The matrix effect was thereby minimized by using an internal standard, a special mobile phase, and specific fragmentation parameters. The sample was extracted with acetonitrile/water (84:16, v/v), and the extract was cleaned up with a MycoSep 227 column. Quantification was based on the internal standard de-epoxy-deoxynivalenol. Calibration curves were linear between 16 and 1600 ng/g, and the limits of detection ranged from 0.18 to 5.0 ng/g. The developed method was applied for the determination of trichothecenes in 120 naturally contaminated wheat and oat samples.     

Fast and sensitive screening method for detection of trichothecenes in maize by using protein synthesis inhibition in cultured fibroblasts

A fast and sensitive bioassay with hamster (BHK-21 C13) fibroblasts for the detection of toxic trichothecenes in maize is described. Cells are exposed to pure toxins or crude maize extracts for 30 min. The mixture is then incubated with [1-14C]-leucine for an additional 60 min and the radioactivity incorporated into the protein of the washed cells is determined. The sensitivity of the assay was in the range 1-10 ng/mL (or 50 ppb in maize) for T-2, HT-2, and diacetoxyscirpenol. At least 1000-fold higher concentrations of non-trichothecene mycotoxins and plant toxins were necessary to cause an inhibition of protein synthesis in the cells. Of 24 maize samples tested, 14 gave a positive response in this assay and the presence of trichothecenes could be confirmed chemically in 11 samples. Therefore, the described bioassay is proposed as a useful screening method for cytotoxic trichothecenes in maize.     

Detection of twelve mycotoxins in mixed animal feedstuffs, using a novel membrane cleanup procedure.

A multimycotoxin thin layer chromatographic screening method is described which is applicable to most animal feedstuffs. Interference from nonspecific lipid, pigment, and other components of simple and mixed feeds is reduced to a minimum by using a membrane cleanup step. Aflatoxins B1, B2, G1, and G2, citrinin, diacetoxyscirpenol, ochratoxin A, patulin, penitrem A, sterigmatocystin, T-2 toxin, and zearalenone may be reliably detected. The sensitivity of the method is generally low for mixed feeds but even so aflatoxin B1 can be detected at a level of 3 ppb and ochratoxin A at 80 ppb. While the basic method is less sensitive for sterigmatocystin (330 ppb), patulin (600 ppb), zearalenone (1000 ppb), and the trichothecenes (1000-4000 ppb), it may be adapted so as to reduce the above detection limits when the presence of these toxins is suspected. Lower levels may be detected in extracts of simple feeds.     

Methods for the detection of trichothecenes.

The trichothecenes are a group of fungal metabolites with a tetracyclic, sesquiterpenoid ring system and include a number of compounds which are highly toxic. These compounds are produced by various species of the imperfect fungi including members of the following genera: Calonectria, Fusarium, Myrothecium, Stachybotrys, Trichoderma, and Trichothecium. Both biological and chemical methods for detection of various trichothecenes are reviewed. Some of the bioassay techniques in use for the detection of the various trichothecenes include the rabbit dermal toxicity tests, cytotoxicity, and inhibition of protein synthesis tests; these are highly sensitive but lace specificity. The sensitivity of these tests for the T-2 toxin are 0.005 mug for rabbit skin toxicity, 0.03 mug/ml for inhibition of protein synthesis in rabbit reticulocytes, and less than 1 mug/ml for cytotoxicity to human karyoblast cells. In the present state of development of the chemical assay methods, thin layer and gas-liquid chromatography are specific for various trichothecenes but lack sensitivity. The lower detection limit of the trichothecenes on thin layer plates varies from 0.2 to 2-3 mug/spot while gas-liquid chrommatography has a reported sensitivity of approximately 0.05 mug/injection.     

Enzyme-linked immunosorbent assay for T-2 toxin metabolites in urine

A direct competitive enzyme-linked immunosorbent assay (ELISA) for determination of total T-2 toxin metabolites in urine was developed. The assay involves coating anti-3-acetyl-neosolaniol-hemisuccinate-bovine serum albumin conjugate (anti-3-Ac-NEOS-HS-BSA) antibody to the ELISA plate and using 3-Ac-NEOS-HS-peroxidase as the enzyme marker. Competitive ELISA revealed that the antibody had good cross-reactivity with acetyldiacetoxyscirpenol (Ac-DAS), T-2 tetraol tetraacetate, 3'-OH-Ac-T-2, 3-Ac-NEOS, and 3,4,15-triacetyl-12,13-epoxytrichothec-9-en-8-one (Ac-T-2-8-one), but less cross-reactivity with Ac-T-2 toxin and T-2 toxin. All metabolites of T-2 toxin in urine were converted to T-2 tetraol tetraacetate (T-2-4ol-4Ac) by acetylation of the sample extract before ELISA. To test the ELISA accuracy, a radioimmunoassay (RIA) was performed simultaneously. The linear portion of the standard curve of this direct ELISA for T-2-4ol-4Ac was 0.2-2.0 ng/mL, which was 10 times more sensitive than RIA. The minimum detection level for T-2-4ol-4Ac was 0.02 ng/mL (0.4 pg/assay) in the absence of urine sample. The overall analytical recoveries for T-2 toxin, HT-2, T-2-4ol, 3'-OH-HT-2, NEOS, and a mixture of these 5 toxins added to the urine samples in the ELISA at concentrations of 0.05 and 0.2 ng/mL were 87 and 94%, respectively.     

Simultaneous extraction and fractionation and thin layer chromatographic determination of 14 mycotoxins in grains.

A simple, systematic analytical method for multiple mycotoxins was developed for detecting 14 mycotoxins; aflatoxins B1, B2, G1, and G2, sterigmatocystin, T-2 toxin, diacetoxyscirpenol, neosolaniol, fusarenon X, zearalenone, ochratoxin A, citrinin, luteoskyrin, and rugulosin. These mycotoxins were extracted with 20% H2SO4-4% KCl-acetonitrile (2 + 20 + 178), defatted with isooctane, and transferred to chloroform. The chloroform extract was cleaned up by silica gel column chromatography; the first 10 toxins were eluted with chloroform-methanol (97 + 3) and the remaining 4 toxins with benzene-acetone-acetic acid (75 + 20 + 5). Each fraction was analyzed by thin layer chromatography for the final determination. The method has been applied to polished rice, rough rice, corn, wheat, and peanuts as an analytical screening procedure. The detection limits in these commodities ranged from 10.00 to 800.0 microgram/kg, depending on the mycotoxin, but all limits were superior to those obtained for the individual mycotoxins by using other methods.     

Gas chromatographic screening method for T-2 toxin, diacetoxyscirpenol, deoxynivalenol, and related trichothecenes in feeds

A gas chromatographic method for screening trichothecene mycotoxins in feeds is described. Feed is extracted with acetonitrile-water, and the toxins are purified with charcoal-alumina-Celite, Florisil, and silica mini-columns. Deoxynivalenol (DON), nivalenol (NIV), diacetoxyscirpenol (DAS), T-2 toxin, and their fungal metabolites are hydrolyzed to their corresponding parent alcohols (DON, NIV, scirpentriol, or T-2 tetraol) by alkaline hydrolysis. After derivatization to their pentafluoropropionyl analogs, they are quantitated by capillary gas chromatography with electron capture detection. Identity can be confirmed and sensitivity can be increased by using negative chemical ionization mass spectrometry with no additional sample workup. Recoveries of DAS, DON, and T-2 toxin averaged, respectively, 80, 65, and 85% in corn; 84, 65, and 88% in soybeans; and 70, 57, and 96% in mixed feeds at concentrations ranging from 0.1 to 2.0 ppm. Recoveries of 15-monoacetoxyscirpenol (MAS), HT-2, NIV, and T-2 tetraol were 97, 97, 86, and 56%, respectively, in corn at a concentration of 0.25 ppm: A detection limit of 0.02 ppm in corn, soybeans, and mixed feeds, and 0.05 ppm in silages is estimated.     

Analysis of the Fusarium mycotoxins fusaproliferin and trichothecenes in grains using gas chromatography-mass spectrometry

A method is described using gas chromatography-mass spectrometry (GC-MS) for the simultaneous detection of the Fusarium mycotoxins fusaproliferin and seven trichothecenes from grains. Sample purification of the raw extract was carried out with commercial solid phase extraction columns, and the recovery of the more polar analytes was increased by rinsing the column with acetonitrile. A significant matrix effect was found for the analysis of fusaproliferin and trichothecenes; thus, the calibrants should be prepared in a blank matrix. The response was linear in the range used. The mean recovery for fusaproliferin was 60.4 or 62.9%, depending on the spiking level. With respect to the trichothecenes, the recovery was generally higher (70.2-125.3%). The method proved to be repeatable for the analysis of fusaproliferin and trichothecenes. The limit of detection for fusaproliferin in the blank matrix mixture was 50 microg/kg, and that for trichothecenes was 5-15 microg/kg. Thirty-eight Finnish grain samples were analyzed for fusaproliferin and trichothecenes with the method developed. Fusaproliferin was not detected in any of the samples. The mean levels of deoxynivalenol, 3-acetyldeoxynivalenol, nivalenol, HT-2 toxin, and T-2 toxin in Finnish grain samples were 272, 17, 150, 40, and <20 microg/kg, respectively.     

Simultaneous analysis of T-2 toxin and HT-2 toxin by an indirect enzyme-linked immunosorbent assay

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of HT-2 toxin in the presence or absence of T-2 toxin is described. In the indirect ELISA, the relative cross-reactivities of antibodies against T-2 toxin (anti-T-2) with T-2 toxin and HT-2 toxin were 1 and 0.1, whereas anti-HT-2 cross-reactivities with T-2 toxin and HT-2 toxin were 0.33 and 1, respectively. Using such relationships, a formula was established that could be used to calculate the individual toxin concentration in a mixed sample after experimentally analyzing for T-2 and HT-2 toxins in the 2 indirect ELISAs. This method was tested by analyzing urine samples spiked with HT-2 toxin alone and samples spiked with both T-2 toxin and HT-2 toxin. A cleanup protocol for treatment of urine samples before ELISA was also established. The overall analytical recovery of HT-2 toxin when it was added at concentrations of 0.1-10 parts per billion (ppb) to the urine samples was ca 89%. When both T-2 and HT-2 toxins were added to the urine samples at equal concentrations of 0.5 to 5.0 ppb, their recoveries were 112 and 109%, respectively.     

Use of small charcoal/alumina cleanup columns in determination of trichothecene mycotoxins in foods and feeds

Small charcoal/alumina cleanup columns have been effectively used to remove interfering materials from grain, feed, and food extracts prior to chromatographic determination of trichothecene mycotoxins. A thin layer chromatographic method has been developed that can simultaneously detect part per billion concentrations of deoxynivalenol, fusarenon X, nivalenol, T-2 toxin, HT-2 toxin, neosolaniol, and diacetoxyscirpenol in food and feed samples. Recoveries of 90-99% can be obtained. The use of charcoal/alumina cleanup columns in conjunction with liquid chromatography and gas chromatography of trichothecenes is also discussed.     

Characterization of a fusarium 2-gene cluster involved in trichothecene C-8 modification

The Fusarium trichothecenes T-2 toxin and deoxynivalenol (DON) are potent inhibitors of eukaryotic protein synthesis and are a significant agricultural problem. Three coregulated loci are required for T-2 toxin synthesis by Fusarium sporotrichioides. The core-trichothecene gene cluster consists of 12 genes (Tri3-Tri14) while the second locus consists of a single gene (Tri101). The third locus was recently partially described and encodes 1-2 biosynthetic enzymes and a putative regulatory gene. Here, we describe a detailed characterization of this locus. Located adjacent to Tri1 is Tri16, which is required for esterification of the C-8 hydroxyl. A putative regulatory gene, also adjacent to Tri1, is not required for T-2 toxin synthesis. The genomic sequence of Fusarium graminearum (a DON producer) contains a putative functional Tri1 and a nonfunctional Tri16. The presence of the Tri16 pseudogene is consistent with the chemical structure of DON, which has a C-8 keto group rather than the C-8 ester of T-2 toxin.     

Determination of T-2 and HT-2 toxins in cereals including oats after immunoaffinity cleanup by liquid chromatography and fluorescence detection

A reliable method for the determination of T-2 toxin and HT-2 toxin in different cereals, including oats, as well as in cereal products was developed. After extraction with methanol/water (90/10, v/v) and dilution with a 4% NaCl solution, the toxins were purified with immunoaffinity columns, derivatized with 1-anthroylnitrile, separated by HPLC, and determined using fluorescence detection. Due to the unspecific derivatization reagents, validation parameters were matrix dependent: in the range 10-200 microg/kg, recovery rates of 74-120% with relative standard deviations between 0.5 and 20.3% were obtained. On average, the limit of quantitation was shown to be 8 microg/kg for each toxin. For naturally contaminated samples, comparable results were obtained when analysis was performed according to this method without derivatization as well as according to a method based on a SPE cleanup utilizing tandem mass spectrometric detection in both cases. Using aqueous acetonitrile as extractant resulted in incorrectly high toxin concentrations due to water absorption of dry samples and toxin accumulation in the organic phase in the subsequent phase separation of the extractant. Furthermore, when comparing the commercially available immunoaffinity columns for T-2 and HT-2 toxins, significant differences regarding capacity and cleanup performance were observed.     

Quantitative determination and structure elucidation of type A- and B-trichothecenes by HPLC/ion trap multiple mass spectrometry.

A method is presented for the quantification and structure confirmation of trichothecenes in wheat by high-performance liquid chromatography combined with multiple mass spectrometry (MS(n)()). Nine type A- and B-trichothecenes were determined (nivalenol, deoxynivalenol, fusarenon-X, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, neosolaniol, diacetoxyscirpenol, HT-2 toxin, and T-2 toxin). Extraction was carried out with acetonitrile/water. The extract was purified on a MycoSep column. Quantification was based on an internal standard and atmospheric pressure chemical ionization in the positive ion mode. Recoveries from spiked wheat were in the range of 80-106% at levels of 500 ppb. The limits of quantification for the whole method were between 10 and 100 ppb. Ion adduct formation with ammonium and acetate ions and MS(n) experiments provided information about substitution and fragmentation behavior of the mycotoxins. A scheme has been established for the partial structure elucidation of type A- and B-trichothecenes in fungal cultures.     

Detection and quantitation of T-2 mycotoxin in rat organs by radioimmunoassay

A standard radioimmunoassay was compared with radiochromatography for the ability to detect unlabeled T-2 mycotoxin in organs from exposed animals. When 10% of HT-2, the only known metabolite that cross-reacts with T-2, was included and expressed as T-2 equivalents in the radiochromatographic detection, correlation between toxin detection in liver, spleen, and kidney by the 2 techniques was r = 0.98. An unknown metabolite was detected in heart extract by radiochromatography. Inclusion of this material in the T-2 equivalents detected by radiochromatography indicated a near-perfect correlation (r = 0.95; p greater than 0.05; slope = 0.82; y = intercept = 72) among all 4 tissues.     

Worldwide occurrence of mycotoxins in foods and feeds--an update

In a review presented at the first FAO/WHO/UNEP Conference on Mycotoxins in 1977, the occurrence of aflatoxins, zearalenone, ochratoxin A, citrinin, trichothecenes, patulin, penicillic acid, and the ergot alkaloids was indicated to be significant in naturally contaminated foods and feeds. The information presented on aflatoxin contamination greatly exceeded that for all other mycotoxins combined. This study reviews the worldwide levels and occurrence of mycotoxins in various commodities since 1976. Comparatively few countries have lowered the acceptable levels for aflatoxins in susceptible commodities. However, intensified efforts are needed to establish control of aflatoxin levels in the global food supply, particularly in peanuts, tree nuts, corn, and animal feeds. Extensive deoxynivalenol (DON) contamination of grains, especially wheat, was demonstrated. Co-contamination of grains by Fusarium toxins, especially DON and nivalenol, with zearalenone to a lesser extent, was reported. However, more information on co-occurrence of Fusarium toxins in cereals should be developed. When contamination of feeds by ochratoxin A was significant, this toxin occurred in swine kidney and smoked meats in high levels. On the basis of occurrence and/or toxicity, patulin and penicillic acid contamination of foods does not appear to be of real concern. More recent developments suggest, however, that expanded monitoring studies of Alternaria toxins, moniliformin, citrinin, cyclopiazonic acid, penitrem A, and ergot alkaloids are indicated.     

Redistribution of 16 Fusarium Toxins During Commercial Dry Milling of Maize

The content of 13 A‐ and B‐type trichothecenes, zearalenone, as well as α‐ and β‐zearalenol was determined in products processed from raw maize by dry milling in an industrial plant. Two batches of samples were investigated derived from different lots of raw maize. Each of the toxins investigated was found in at least one of the samples analyzed, with up to 13 toxins co‐occurring within one sample. For both batches, toxins were either not detected or their content was low in raw and tempered maize, grits, and two types of flour. Markedly higher concentrations were found in screenings, bran, germ, or germ meal. The results suggest a similar redistribution during dry milling of maize for the whole spectrum of Fusarium toxins analyzed. In germ oil, only 15‐acetyldeoxynivalenol, zearalenone, HT‐2 toxin, and T‐2 toxin were detected due to the higher lipophilic properties of these substances compared with the other toxins found in the basing germ. This is the first time that the redistribution of a spectrum of 16 Fusarium toxins has been measured in a single dry‐milling study.     

Optimization of chick embryotoxicity bioassay for testing toxicity potential of fungal metabolites

The optimization of a simple, sensitive procedure using a chick embryotoxicity screening test (CHEST) bioassay for detection of toxic compounds is presented. Dosing protocols of eggs, using several mycotoxins (aflatoxin B1, deoxynivalenol, T-2 toxin) and appropriate controls, were evaluated for embryonic sensitivity, overall practicality of the procedure, and consistency of results. It was found that both type of carrier solvent and volume injected could significantly affect overall embryonic mortality. The chick embryo was most sensitive to the effects of toxins and solvents after 1 or 2 days of incubation; a rapid decrease in response was observed as the age of the embryo at dosing increased. Following administration of the toxins just below the shell membrane by way of a small hole (less than 0.5 mm diameter) punched in the shell, a good dose-response (% mortality) could be obtained regardless of the site of injection (except directly into the yolk), although dosing via the air sac position resulted in a slightly better statistical outcome. Although some variations in calculated LD50 values were found among repeated assays, statistical analyses showed that the differences were not due to dosing protocol but to the variations in embryo sensitivities among batches of eggs. Thus, if standard reference toxins for comparison are run concurrently, the CHEST assay can prove to be a very satisfactory model, as well as having considerable flexibility to be adapted to the needs and resources of many laboratories.     

Mass spectral investigations on trichothecene mycotoxins. II. Detection and quantitation of macrocyclic trichothecenes by gas chromatography/negative ion chemical ionization mass spectrometry

A general, sensitive gas chromatographic/negative ion chemical ionization mass spectrometric (GC/NICIMS) method of analysis was developed for the detection and quantitation of several polar, thermally labile, toxic macrocyclic trichothecenes. The procedure involves the conversion of the molecules to their corresponding alcohols (verrucarols) by alkaline hydrolysis, followed by derivatization of the hydrolysate with heptafluorobutyrylimidazole and analysis by GC/MS technique under negative ion chemical ionization conditions. Nanogram (250 ng) quantities of several macrocyclic trichothecenes with different verrucarol and ester moieties were analyzed successfully with good precision by this procedure. The method was applicable for the accurate determination of at least low ppb levels of these macrocyclic trichothecenes in environmental samples, such as fungal products, fermentation broths, and plant samples. This is the first reported, well developed, sensitive, and applicable method for the detection and quantitation of these compounds in naturally occurring samples.     

Development of qualitative and semiquantitative immunoassay-based rapid strip tests for the detection of T-2 toxin in wheat and oat

Novel qualitative as well as semiquantitative rapid strip tests for screening of T-2 mycotoxin in agricultural commodities were developed. Colloidal gold particles were coated with monoclonal anti-T-2 antibodies and used as detector reagent, indicating the strip test results by formation of up to two colored lines in a competitive assay format. The test line comprises a protein conjugate of the T-2 mycotoxin and the control line an antispecies-specific antibody to confirm the correct test development. To perform the test, 5 g of sample was extracted in a ratio of 1:5 with methanol/water (70:30) by shaking for 3 min and the extract directly used without further cleanup steps. The T-2 toxin lateral flow device (LFD) presented has a cutoff level around 100 microg/kg for naturally contaminated wheat and oat. The semiquantitative test may be used in the lower micrograms per kilogram range and allows for rapid semiquantitative photometric classification of the level of sample contamination. For both tests, results were obtained within 4 min. The developed LFDs therefore allow for the first time fast and on-site screening for the determination of T-2 toxin in cereals.