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.     

Determination of cytotoxic trichothecenes in corn by cell culture toxicity assay

The great sensitivity of some cell species to toxins has been adapted to a direct biological determination of trichothecene contamination of food and feeds. The murine spleen lymphocyte stimulated by PHA (Phaseolus vulgaris phytohaemagglutinin) appeared to be the most convenient cells because of their particular sensitivity to cytotoxic trichothecenes and the opportunity to translate this cytotoxicity to immunosuppressive hazard, one of the most important concerns for trichothecenes. In this paper, the use of cell cultures was adapted for a survey of corn. The toxins were extracted by aqueous methanol, and the extract was defatted with hexane and purified on a silica gel/Florisil column. This extract was then used for a gas chromatographic (GC) determination and the biological test. The growth of cells was measured by the incorporation of tritiated thymidine (3H Tdr), and the inhibition was expressed by the IC50: concentration of corn extract inhibiting by half the 3H Tdr incorporation. We have tested pure toxins, control corn, corn spiked with T-2 toxin, corn experimentally inoculated with toxigenic Fusarium strains, and naturally contaminated corn. A good correlation exists between IC50 and the T-2 toxin concentration as determined by GC analysis. The response is not affected by the presence of zearalenone or by small amounts of deoxynivalenol. A quantitative evaluation of cytotoxic trichothecenes in corn is valuable in the range of 100 ppb to 10 ppm, expressed as T-2 toxin equivalents. The result is obtained in 48 h.     

Use of solid-phase extraction systems to improve the sensitivity of Artemia bioassays for trichothecene mycotoxins

Solid-phase extraction was used to preconcentrate trichothecene mycotoxins from rivers and streams in order to develop and improve a rapid and sensitive bioassay using the brine shrimp Artemia salina. For T-2 toxin, HT-2 toxin, and 4,15-diacetoxyscirpenol, LC50 values obtained were 172, 600, and 700 micrograms/L, respectively. The LC50 for 4-deoxynivalenol was 21 mg/L. A more than 5-fold increase in sensitivity was observed when solid-phase extraction (SPE) was used in conjunction with the Artemia bioassay. For T-2 toxin, HT-2 toxin, and 4,15-diacetoxyscirpenol, LC50/SPE values after solid-phase extraction were 21, 83, and 130 micrograms/L. The use of river and stream waters and chlorinated water did not seem to interfere with the bioassay.     

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.     

Enzyme‐Linked Immunosorbent Assay Detection of Trichothecenes Produced by the Bioherbicide Myrothecium verrucaria in Cell Cultures,Extracts, and Plant Tissues

A rapid technique for trichothecene detection was needed in screening tests of the potential bioherbicide Myrothecium verrucaria (MV), in order to select strains, mutants, or formulations that were void of or that possessed low amounts of these undesirable mycotoxin compounds. Commercially available enzyme‐linked immunosorbent assay (ELISA) plates for trichothecene detection, possessing cross‐reactivity with several trichothecene mycotoxins (e.g., verrucarin A, and J, roridin A, L‐2, E, and H), were tested for their ability to detect trichothecenes produced by a strain of Myrothecium verrucaria (MV) in cell cultures, in plant tissues (hemp sesbania and kudzu) treated with purified roridin A, or ethyl acetate fractions of MV cultures. Evaluations of ELISA assays showed linear responses for standards of verrucarin A and roridin A over a concentration range of 0.2 to 20 ppb. Ethyl acetate or aqueous extractions were used to obtain samples from MV cultures and plant tissues for testing. Trichothecenes were detected in conidia and mycelia of MV, and in agar upon which wild‐type MV was grown, indicating secretion into the growth media. Two MV sectors (morphological variants of wild type) also tested positive for trichothecenes. Purified roridin A and concentrated extracts containing trichothecenes from MV spore cultures exhibited phytotoxicity (growth inhibition or necrosis) when applied to excised shoots of hemp sesbania seedlings and intact kudzu leaf tissues. Evidence of some translocation of trichothecenes from the application point in kudzu was found, but translocation to the upper shoot portion of hemp sesbania was not detected at the lowest limit of detection in this assay (0.14 ppb). This assay is also being employed to identify induced mutants and/or other naturally occurring sectors deficient in trichothecene mycotoxin production. Results indicated that ELISA is a sensitive and rapid assay method to quantify trichothecenes produced by this bioherbicidal fungus and in certain plant tissues treated with trichothecenes.     

Fusarium graminearum TRI14 is required for high virulence and DON production on wheat but not for DON synthesis in vitro

Fusarium head blight (FHB) of wheat (Triticum aestivum L.), caused by the fungus Fusarium graminearum, is a major concern worldwide. FHB grain is reduced in yield, may fail to germinate, and is often contaminated with deoxynivalenol, a trichothecene mycotoxin linked to a variety of animal diseases and feed refusals. Annual losses in the tens of millions of dollars due to FHB underscore the need to develop improved methods of disease control and prevention. Previous research has identified deoxynivalenol biosynthesis as a virulence factor on wheat. Recently, we found that the TRI14 gene of F. sporotrichioides, closely related to F. graminearum, was not required for synthesis of a related trichothecene, T-2 toxin. TRI14 does not share similarity with any previously described genes in the databases. In this study, we examined the role that F. graminearum TRI14 may play in both deoxynivalenol synthesis and in virulence on wheat. TRI14 deletion mutants synthesize deoxynivalenol on cracked maize kernel medium and exhibit wild-type colony morphology and growth rate on complex and minimal agar media. However, FHB assays on greenhouse-grown wheat indicate that FgDeltaTri14 mutants cause 50-80% less disease than wild type and do not produce a detectable quantity of deoxynivalenol on plants. We discuss a number of possible roles that TRI14 may play in the disease process.     

Identification and apoptotic potential of T-2 toxin metabolites in human cells

The mycotoxin T-2 toxin, produced by various Fusarium species, is a widespread contaminant of grain and grain products. Knowledge about its toxicity and metabolism in the human body is crucial for any risk assessment as T-2 toxin can be detected in processed and unprocessed food samples. Cell culture studies using cells of human origin represent a potent model system to study the metabolic fate of T-2 toxin as well as the cytotoxicity in vitro. In this study the metabolism of T-2 toxin was analyzed in a cell line derived from human colon carcinoma cells (HT-29) and primary human renal proximal tubule epithelial cells (RPTEC) using high-performance liquid chromatography coupled with Fourier transformation mass spectrometry (HPLC-FTMS). Both cell types metabolized T-2 toxin to a variety of compounds. Furthermore, cell cycle analysis in RPTEC proved the apoptotic effect of T-2 toxin and its metabolites HT-2 toxin and neosolaniol in micromolar concentrations.     

Criteria for determining purity of Fusarium mycotoxins

Physical and chemical properties that may be used to determine the purity of several Fusarium mycotoxins have been investigated. A combination of analytical procedures, which include high performance thin-layer chromatography (HPTLC), liquid chromatography (LC), gas chromatography (GC), gas chromatography/mass spectrometry (GC/MS), ultraviolet spectrometry (UV), and nuclear magnetic resonance (NMR) spectrometry have been used to examine mycotoxin standards obtained from commercial sources and from laboratory fermentations. Results of this investigation indicate that commercially available standards are greater than 90% pure, but the label weight of purchased reference standards in individual containers should be verified. Mycotoxin standards, determined to be greater than 98% pure by HPTLC, LC, and GC/MS, were examined by UV spectrometry and the coefficients of extinction were determined. An interlaboratory study, involving 5 collaborators who determined coefficients of extinction (in methanol) for identical samples, gave the following results: alpha-zearalenol (lambda 236 = 28 538 +/- 558); beta-zearalenol (lambda 238 = 24 963 +/- 747); deoxynivalenol (lambda 219 = 6395 +/- 349, lot 1), (6020 +/- 228, lot 2); and T-2 toxin (lambda 202 = 3681 +/- 255). UV maxima and coefficients of extinction are also reported for HT-2 toxin (lambda 202 = 1959), diacetoxyscirpenol (lambda 203 = 2487), neosolaniol (lambda 203 = 2644), nivalenol (lambda 220 = 5142), and fusarenon-X (lambda 217 = 5997).     

Antioxidizing potency of phenol compounds in olive oil mill wastewater

The antioxidizing potency of phenol compounds contained in olive oil mill wastewater (OOMWW) has been elucidated. Commercially available phenol standards at varying concentrations and the Rancimat oxidation test have been used. Refined purified olive oil was utilized as an oxidation lipid substrate. Synthetic antioxidants, such as 2,3-tert-butyl-4-hydroxyanisole (BHA), 3,5-di-tert-butyl-4-hydroxytoluene (BHT), l-ascorbic acid, and gallates (commonly used as food preservatives), and other known chemicals endowed with antioxidizing properties have been employed as reference compounds. The OOMWW phenol compounds have been classified into different groups depending on their antioxidizing potency. This was significantly affected by the tested concentrations of the standards. Mixtures of phenol standards and other antioxidants (l-proline, chlorophyll a, chlorophyll b, and alpha-, gamma-, and delta-tocopherol) have also been tested. Many phenol compounds present in OOMWW showed antioxidizing potency higher compared to that of the less safe synthetic antioxidants and could therefore replace these in the industrial preservation of food items. They could also be used in combination with other natural antioxidants (e.g., tocopherols). In fact, some mixtures of antioxidants, owing also to the synergistic phenomena, showed strong antioxidizing potency.     

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.     

Metabolites inhibiting germination of Orobanche ramosa seeds produced by Myrothecium verrucaria and Fusarium compactum

Myrothecium verrucaria and Fusarium compactum were isolated from diseased Orobanche ramosa plants collected in southern Italy to find potential biocontrol agents of this parasitic weed. Both fungi grown in liquid culture produced metabolites that inhibited the germination of O. ramosa seeds at 1-10 muM. Eight metabolites were isolated from M. verrucaria culture extracts. The main metabolite was identified as verrucarin E, a disubstituted pyrrole not belonging to the trichothecene group. Seven compounds were identified by spectroscopic methods as macrocyclic trichothecenes, namely, verrucarins A, B, M, and L acetate, roridin A, isotrichoverrin B, and trichoverrol B. The main metabolite produced by F. compactum was neosoloaniol monoacetate, a trichothecene. All the trichothecenes proved to be potent inhibitors of O. ramosa seed germination and possess strong zootoxic activity when assayed on Artemia salina brine shrimps. Verrucarin E is inactive on both seed germination and zootoxic assay.     

Gas-liquid chromatographic/mass spectrometric screening method for T-2 toxin in milk

A method for the analysis of T-2 toxin in milk is presented. Ethyl acetate extracts of milk samples which had been spiked with T-2 toxin were purified by thin layer chromatography and derivatized with N,O-bis(trimethylsilyl)acetamide to produce the T-2 toxin trimethylsilyl ether (T-2 toxin-TMS). N,O-bis(trimethylsilyl-d9)acetamide was used to make T-2 toxin d9-trimethylsilyl ether (T-2 toxin-d9 TMS) which was added to the derivatized milk extract as an internal standard. Samples were analyzed by combined gas-liquid chromatography/mass spectrometry using either electron impact ionization or chemical ionization mass spectrometry. In electron impact ionization analyses, simultaneous monitoring of the T-2 toxin-TMS fragment ion at m/z 436 and the T-2 toxin-d9TMS fragment ion at m/z 445 gave a T-2 toxin-TMS detectability estimated at 6 microgram/kg. In chemical ionization analyses, the T-2 toxin-TMS fragment ion at m/z 377 and the T-2 toxin-d9TMS fragment ion at m/z 386 were simultaneously monitored to give a T-2 toxin-TMS detectability estimated at 3 microgram/kg. Average recovery was 85% at 200 microgram/kg and 65% at 20 microgram/kg.     

Patterns of trichothecene production, genetic variability, and virulence to wheat of Fusarium graminearum from smallholder farms in Nepal

Fusarium graminearum causes wheat head blight and contaminates grain with the trichothecenes 4-deoxynivalenol and nivalenol. Sequence analysis of trichothecene genes indicates that nivalenol production is the ancestral trait; however, deoxynivalenol producers occur worldwide and predominate in North and South America and in Europe. Analysis of a large field population (>500 strains) from Nepal identified three groups that were both genetically distinct and polymorphic for trichothecene production: SCAR1 comprising 95% deoxynivalenol producers, SCAR2 comprising 94% nivalenol producers, and SCAR3/5 comprising 34% deoxynivalenol producers/63% nivalenol producers. The ability to cause wheat head blight differed between SCAR groups and trichothecene chemotypes: deoxynivalenol producers were more virulent than nivalenol producers across all three SCAR groups and within the SCAR3/5 genetic background. These data support the hypothesis that production of deoxynivalenol rather than nivalenol confers a selective advantage to this important wheat pathogen.     

Antiproliferative lactams and spiroenone from adlay bran in human breast cancer cell lines

Two new lactams, coixspirolactam D (1) and coixspirolactam E (2), and a new spiroenone, coixspiroenone (3), together with seven known compounds, coixspirolactam A (4), coixspirolactam B (5), coixspirolactam C (6), coixlactam (7), coixol (8), ethyl dioxindole-3-acetate (9), and isoindol-1-one (10), and two neolignans, zhepiresionol (11) and ficusal (12), were isolated from the bioactive subfraction of adlay bran ethanolic extract (ABE). Compounds 9 and 10 are the first isolates from natural resources. The structures of new compounds were identified by spectroscopic methods, including infrared (IR) spectrum, 1D and 2D nuclear magnetic resonance (NMR), and mass spectrum (MS). All of the isolated compounds were tested for antiproliferative effects on MCF-7, MDA-MB-231, and T-47D cells. Results showed that compounds 1, 3, 4, 6, and 7 at 50 μM significantly inhibited MCF-7 cell proliferation by 30.2, 19.2, 21.0, 13.5, and 32.4%, respectively; compounds 2, 4, and 7 significantly inhibited T-47D cells at 50 μM by 20.7, 24.8, and 28.9%; and compounds 1, 2, and 12 significantly inhibited MDA-MB-231 cells at 50 μM by 47.4, 25.3, and 69.3%, respectively. In conclusion, ABE has antiproliferative activities, and this effect is partially related to the presence of lactams and spiroenone.     

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.     

The Occurrence of Type A and B Trichothecenes in Lithuanian Cereals

Samples of winter wheat (n =84), winter rye (46) and barley (29) were collected from the larger family farms and from partnerships in Lithuania just after the 1998 harvest. The number of samples collected from each region was proportional to the amount of grain produced in it. The levels of the Fusarium toxins deoxynivalenol (DON), 3-acetyl-DON, 15-acetyl-DON, nivalenol (NIV), fusarenon-X (4-acetyl-NIV), T-2 toxin, HT-2 toxin, 4,5-diacetoxyscirpenol (DAS), 1,5-monoacetoxyscirpenol (MAS) and scirpentriol in the grain were determined by gas chromatography with mass-selective detection (GC-MS). DON was most often detected in the wheat and rye samples and NIV in the barley samples. The concentrations found were lower than those causing acute or chronic toxic effects in livestock or humans. No fusarenon-X or 15-acetyl-DON was detected, and only small amounts of other trichothecenes were present. Climatic conditions in Lithuania in the summer of 1998 were slightly cooler and wetter than the average for the 1992-1996 but were close to the norm. Because the samples analysed were representative of grain produced for the market in seasons with normal weather, trichothecene contamination of grain from large family farms and partnerships would not be expected to be a problem in most years.     

Structure-activity relationships of trichothecene toxins in an Arabidopsis thaliana leaf assay

Many Fusarium species produce trichothecenes, sesquiterpene epoxides that differ in patterns of oxygenation and esterification at carbon positions C-3, C-4, C-7, C-8, and C-15. For the first comprehensive and quantitative comparison of the effects of oxygenation and esterification on trichothecene phytotoxicity, we tested 24 precursors, intermediates, and end products of the trichothecene biosynthetic pathway in an Arabidopsis thaliana detached leaf assay. At 100 microM, the highest concentration tested, only the trichothecene precursor trichodiene was nontoxic. Among trichothecenes, toxicity varied more than 200-fold. Oxygenation at C-4, C-8, C-7/8, or C-15 was, on average, as likely to decrease as to increase toxicity. Esterification at C-4, C-8, or C-15 generally increased toxicity. Esterification at C-3 increased toxicity in one case and decreased toxicity in three of eight cases tested. Thus, the increase in structural complexity along the trichothecene biosynthetic pathway in Fusarium is not necessarily associated with an increase in phytotoxicity.     

Enhancement of trichothecene production in Fusarium graminearum by cobalt chloride

The effects of cobalt chloride on the production of trichothecene and ergosterol in Fusarium graminearum were examined. Incorporation experiments with (13)C-labeled acetate and leucine confirmed that both 3-acetyldeoxynivalenol and ergosterol were biosynthesized via a mevalonate pathway by the fungus, although hydroxymethyl-glutaryl CoA (HMG-CoA) from intact leucine was able to be partially used for ergosterol production. Addition of cobalt chloride at concentrations of 3-30 μM into liquid culture strongly enhanced 3-acetyldeoxynivalenol production by the fungus, whereas the amount of ergosterol and the mycelial weight of the fungus did not change. The mRNA levels of genes encoding trichothecene biosynthetic proteins (TRI4 and TRI6), ergosterol biosynthetic enzymes (ERG3 and ERG25), and enzymes involved in the mevalonate pathway (HMG-CoA synthase (HMGS) and HMG-CoA reductase (HMGR)) were all strongly up-regulated in the presence of cobalt chloride. Precocene II, a specific trichothecene production inhibitor, suppressed the effects of cobalt chloride on Tri4, Tri6, HMGS, and HMGR, but did not affect erg3 and erg25. These results indicate that cobalt chloride is useful for investigating regulatory mechanisms of trichothecene and ergosterol production in F. graminearum.     

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.     

Enhanced surveillance of foodborne mycotoxins by immunochemical assay

Mycotoxins are a chemically diverse group of fungal secondary metabolites with a wide range of toxic effects. Conventional thin-layer and instrumental methods of mycotoxin analysis are time-consuming and make routine safety and quality control screening of these compounds in agricultural commodities difficult. As an alternative, specific polyclonal and monoclonal antibodies have been raised against mycotoxin-protein conjugates and used in sensitive radioimmunoassays (RIAs) and enzyme-linked immunosorbent assays (ELISAs). One of the simplest ELISA approaches involves competition for a solid-phase antibody between a mycotoxin-enzyme conjugate and an unconjugated mycotoxin in the sample extract. ELISAs have been developed for aflatoxins B1 and M1, zearalenone, T-2 toxin, and deoxynivalenol, which are highly specific, rapid (10 min), easily adaptable for analyzing large numbers of samples, and directly applicable to assaying methanol-water extracts of a wide range of foods. Several commercial mycotoxin ELISAs using this approach (most typically for aflatoxin B1) are currently being marketed. Since ELISAs will be used in large part by personnel with limited technical expertise, individual kits must be critically evaluated by analytical chemists for suggested sampling procedures, efficiency of extraction, cross-reactivity, mycotoxin recovery, assay reproducibility, and product shelf-life prior to routine use in food safety and quality control screening.