Trichothecenes and their analysis

The analysis of foods and feeds for the naturally occurringFusarium produced trichothecenes is reviewed. Each major step (extraction, purification and detection-quantitation) is discussed. Although none of the extraction solvents has been thoroughly evaluated, aqueous metanol is the preferred system in most of the published procedures. The sample extracts are generally purified by liquid-liquid partitions followed by silica gel column and/or preparative thin layer chromatography (TLC). Both thin layer and gas liquid chromatography (GLC) are used for the detection and quantitation of the trichothecenes. The detection limit for the GLC procedures has not been determined in most of the reported methods; however, T-2 toxin, diacetoxyscirpenol, deoxynivalenol, and neosolaniol have been detected in the 25–100 ng/g range.     

Isolation and purification of deoxynivalenol and a new trichothecene by high pressure liquid chromatography

Deoxynivalenol (3,7,15-trihydroxy-12,l3-epoxytrichothec-9-ene-8-one) was extracted from corn with methanol/water (80:20, v/v) and purified by liquid:liquid partitioning and by preparative high pressure liquid chromatography (HPLC). This procedure was used to prepare mg quantities of toxin from field-inoculated corn for reference standards. Analysis of the isolated deoxynivalenol by analytical HPLC, gas liquid chromatography (GLC) and gas liquid chromatography/mass spectroscopy (GLC/MS) indicated the presence of a second compound similar to deoxynivalenol. This compound comigrates with deoxynivalenol on thin layer chromatography plates in chloroform/methanol (90:10, v/v), but can be separated by HPLC on a reverse-phase C₈ column with methanol/water (10:90, v/v). GC/MS of the compound and the trimethylsilyl ether derivative gave parent ions of m/e 280 and 424, respectively. These data and NMR data indicate that the compound is 3,15-dihydroxy-12,13-epoxytrichothec-9-ene-8-one, a previously unreported trichothecene.     

Microbial Detoxification of Mycotoxins

Mycotoxins are fungal natural products that are toxic to vertebrate animals including humans. Microbes have been identified that enzymatically convert aflatoxin, zearalenone, ochratoxin, patulin, fumonisin, deoxynivalenol, and T-2 toxin to less toxic products. Mycotoxin-degrading fungi and bacteria have been isolated from agricultural soil, infested plant material, and animal digestive tracts. Biotransformation reactions include acetylation, glucosylation, ring cleavage, hydrolysis, deamination, and decarboxylation. Microbial mycotoxin degrading enzymes can be used as feed additives or to decontaminate agricultural commodities. Some detoxification genes have been expressed in plants to limit the pre-harvest mycotoxin production and to protect crop plants from the phytotoxic effects of mycotoxins. Toxin-deficient mutants may be useful in assessing the role of mycotoxins in the ecology of the microorganisms.     

T-2 Toxin-induced Toxicity in Pregnant Mice and Rats

T-2 toxin is a cytotoxic secondary fungal metabolite that belongs to the trichothecene mycotoxin family. This mycotoxin is a well known inhibitor of protein synthesis through its high binding affinity to peptidyl transferase, which is an integral part of the ribosomal 60s subunit, and it also inhibits the synthesis of DNA and RNA, probably secondary to the inhibition of protein synthesis. In addition, T-2 toxin is said to induce apoptosis in many types of cells bearing high proliferating activity. T-2 toxin readily passes the placenta and is distributed to embryo/fetal tissues, which include many component cells bearing high proliferating activity. This paper reviews the reported data related to T-2 toxin-induced maternal and fetal toxicities in pregnant mice and rats. The mechanisms of T-2 toxin-induced apoptosis in maternal and fetal tissues are also discussed in this paper.     

Chicken Cytochrome P450 1A5 Is the Key Enzyme for Metabolizing T-2 Toxin to 3′OH-T-2

The transmission of T-2 toxin and its metabolites into the edible tissues of poultry has potential effects on human health. We report that T-2 toxin significantly induces CYP1A4 and CYP1A5 expression in chicken embryonic hepatocyte cells. The enzyme activity assays of CYP1A4 and CYP1A5 heterologously expressed in HeLa cells indicate that only CYP1A5 metabolizes T-2 to 3′OH-T-2 by the 3′-hydroxylation of isovaleryl groups. In vitro enzyme assays of recombinant CYP1A5 expressed in DH5α further confirm that CYP1A5 can convert T-2 into TC-1 (3′OH-T-2). Therefore, CYP1A5 is critical for the metabolism of trichothecene mycotoxin in chickens.     

Direct T-2 Toxicity on Human Skin—Fibroblast Hs68 Cell Line—In Vitro Study

T-2 toxin is produced by different Fusarium species, and it can infect crops such as wheat, barley, and corn. It is known that the T-2 toxin induces various forms of toxicity such as hepatotoxicity, nephrotoxicity, immunotoxicity, and neurotoxicity. In addition, T-2 toxin possesses a strong dermal irritation effect and can be absorbed even through intact skin. As a dermal irritant agent, it is estimated to be 400 times more toxic than sulfur mustard. Toxic effects can include redness, blistering, and necrosis, but the molecular mechanism of these effects still remains unknown. This in vitro study focused on the direct toxicity of T-2 toxin on human skin—fibroblast Hs68 cell line. As a result, the level of toxicity of T-2 toxin and its cytotoxic mechanism of action was determined. In cytotoxicity assays, the dose and time-dependent cytotoxic effect of T-2 on a cell line was observed. Bioluminometry results showed that relative levels of ATP in treated cells were decreased. Further analysis of the toxin’s impact on the induction of apoptosis and necrosis processes showed the significant predominance of PI-stained cells, lack of caspase 3/7 activity, and increased concentration of released Human Cytokeratin 18 in treated cells, which indicates the necrosis process. In conclusion, the results of an in vitro human skin fibroblast model revealed for the first time that the T-2 toxin induces necrosis as a toxicity effect. These results provide new insight into the toxic T-2 mechanism on the skin.     

A national survey of mycotoxins in canada

Mycotoxin contamination of animal feedstuffs continues to be an area of concern in various regions of Canada. A survey of research institutions across Canada revealed that the mycotoxins, zearalenone, ochratoxin A, tricothecenes and aflatoxin, have been detected in suspected feed samples. These mycotoxins have produced mycotoxicoses in poultry, hogs and cattle. Estrogenic syndromes in hogs may be caused by the ingestion of zearalenone produced by Fusarium molds on grain com grown in southern Ontario. Overwintered barley contaminated with T-2 toxin was suspected in an outbreak of fusariotoxicoses in swine in northern Alberta. Central and eastern Canada, which seem to be the major focus of mycotoxin occurrence, will be the object of further field level examination to discover patterns of occurrence and economic impact.     

The Ribosome-Binding Mode of Trichothecene Mycotoxins Rationalizes Their Structure—Activity Relationships

Trichothecenes are the most prevalent mycotoxins contaminating cereal grains. Some of them are also considered as the virulence factors of Fusarium head blight disease. However, the mechanism behind the structure-activity relationship for trichothecenes remains unexplained. Filling this information gap is a crucial step for developing strategies to manage this large family of mycotoxins in food and feed. Here, we perform an in-depth re-examination of the existing structures of Saccharomyces cerevisiae ribosome complexed with three different trichothecenes. Multiple binding interactions between trichothecenes and 25S rRNA, including hydrogen bonds, nonpolar pi stacking interactions and metal ion coordination interactions, are identified as important binding determinants. These interactions are mainly contributed by the key structural elements to the toxicity of trichothecenes, including the oxygen in the 12,13-epoxide ring and a double bond between C⁹ and C¹⁰. In addition, the C³-OH group also participates in binding. The comparison of three trichothecenes binding to the ribosome, along with their binding pocket architecture, suggests that the substitutions at different positions impact trichothecenes binding in two different patterns. Moreover, the binding of trichothecenes induced conformation changes of several nucleotide bases in 25S rRNA. This then provides a structural framework for understanding the structure-activity relationships apparent in trichothecenes. This study will facilitate the development of strategies aimed at detoxifying mycotoxins in food and feed and at improving the resistance of cereal crops to Fusarium fungal diseases.     

1H and 13C NMR Study of the Trichothecenes T-2 toxin and T-2 triol

T-2 toxin dissolved in CDCl₃ and C₆D₆ and T-2 triol in CDCl₃ were studied by 1D and 2D n.m.r. spectrometric methods. Comprehensive n.m.r. spectral parameters are reported. Iterative analysis of the isovaleroxy proton resonances of T-2 toxin in C₆D₆ and T-2 triol in CDCl₃ was carried out. The assignments of the proton and carbon resonances of the ace-toxy groups and of the H-13 and H-15 resonances of T-2 toxin are given. The known conformation of the trichothecene ring system was confirmed by NOESY.     

Enzyme-linked immunosorbent assay for T-2 toxin

An enzyme-linked immunosorbent assay (ELISA) was developed for the rapid quantitation of T-2 toxin, a tricothecene mycotoxin produced by members of the genusFusarium. T-2 toxin was first converted to the T-2 hemisuccinate (T-2 HS) and then conjugated by the water-soluble carbodiimide method to either bovine serum albumin for use as an immunogen or to horseradish peroxidase for use as an enzyme marker. T-2 antiserum was air-dried onto polystyrene microtissue culture plates and the ELISA conducted by simultaneously incubating standards of T-2 toxin and the T-2 HS-peroxidase conjugate. Competition curves were prepared by determining total bound enzyme. The ELISA took about 2 hr to complete and allowed minimal detection of T-2 at levels of 2.5 pg/assay. Average recoveries from samples of wheat flour spiked with T-2 toxin in the 1.0-30 ppb range were 95 ± 25% and those for corn meal spiked in the 5.0-30 ppb range were 98 ± 19%. The results suggested the ELISA is a simple and convenient alternative for the screening of T-2 toxin in food and feeds.     

Determination of deoxynivalenol (DON) in wheat, barley and corn and its relationship with the levels of total molds, Fusarium spp., colonization percentage and water activity

Fifty samples of cereals including 30 of wheat (10 of wheat hard red spring), 10 of wheat soft red winter and 10 of wheat durum ámber), 10 of barley and 10 of corn (5 of white corn and 5 of yellow corn) were analyzed to detect and determine by the TLC method, the quantity of deoxynivalenol levels, which is a toxic secondary metabolite produced by Fusarium species. The aw of samples and the internal and external micoflora and Fusarium spp. levels were also investigated. Results showed that the highest grade of infection (12-80%), and the highest count of total molds (3.9 Log UFC/g) were detected in wheat samples, while the highest levels of Fusarium spp. (2.3 Log UFC/g) were detected in white corn. Deoxynivalenol was found in the wheat and barley samples but not in corn. The wheat red winter soft samples showed the highest levels of deoxynivalenol (3.2 ug/g) which is over the limit levels accepted by the FDA. Correlation was not found among count of total molds, Fusarium spp., infestation grade, aw, and deoxynivalenol levels. These results suggest that it is necessary to exert measures to avoid and to control the importation of contaminated cereals with DON levels higher to those allowed.     

Mechanisms of Mycotoxin-Induced Neurotoxicity through Oxidative Stress-Associated Pathways

Among many mycotoxins, T-2 toxin, macrocyclic trichothecenes, fumonisin B(1) (FB(1)) and ochratochin A (OTA) are known to have the potential to induce neurotoxicity in rodent models. T-2 toxin induces neuronal cell apoptosis in the fetal and adult brain. Macrocyclic trichothecenes bring about neuronal cell apoptosis and inflammation in the olfactory epithelium and olfactory bulb. FB(1) induces neuronal degeneration in the cerebral cortex, concurrent with disruption of de novo ceramide synthesis. OTA causes acute depletion of striatal dopamine and its metabolites, accompanying evidence of neuronal cell apoptosis in the substantia nigra, striatum and hippocampus. This paper reviews the mechanisms of neurotoxicity induced by these mycotoxins especially from the viewpoint of oxidative stress-associated pathways.     

T-2 Toxin Induces Oxidative Stress at Low Doses via Atf3ΔZip2a/2b-Mediated Ubiquitination and Degradation of Nrf2

T-2 toxin is mainly produced by Fusarium species, which is an extremely toxic mycotoxin to humans and animals. It is well known that T-2 toxin induces oxidative stress, but the molecular mechanism is still unknown. In this study, we found that T-2 toxin significantly promoted reactive oxygen species (ROS) accumulation in MCF-7 cells at low doses which maintains cell viability at least 80%. Further analysis showed that T-2 toxin downregulated the expression of the master regulator of antioxidant defense gene, nuclear factor erythroid 2-related factor (Nrf2), and its targeted antioxidant genes. Overexpression of Nrf2 or its target gene heme oxygenase 1 (HO1) significantly blocked the ROS accumulation in MCF-7 cells under T-2 toxin treatment. Moreover, we found that T-2 toxin downregulated the antioxidant genes via inducing the expression of ATF3ΔZip2a/2b. Importantly, overexpression of ATF3ΔZip2a/2b promoted the ubiquitination and degradation of Nrf2. Altogether, our results demonstrated that T-2 toxin-induced ROS accumulation via ATF3ΔZip2a/2b mediated ubiquitination and degradation of Nrf2, which provided a new insight into the mechanism of T-2 toxin-induced oxidative stress.     

Immune Response among Patients Exposed to Molds

Macrocyclic trichothecenes, mycotoxins produced by Stachybotrys chartarum, have been implicated in adverse reactions in individuals exposed to mold-contaminated environments. Cellular and humoral immune responses and the presence of trichothecenes were evaluated in patients with mold-related health complaints. Patients underwent history, physical examination, skin prick/puncture tests with mold extracts, immunological evaluations and their sera were analyzed for trichothecenes. T-cell proliferation, macrocyclic trichothecenes, and mold specific IgG and IgA levels were not significantly different than controls; however 70% of the patients had positive skin tests to molds. Thus, IgE mediated or other non-immune mechanisms could be the cause of their symptoms.     

Zearalenone in cereal grains

Zearalenone, a secondary metabolite with estrogenic properties, is produced by severalFusarium species that colonize cereal grains in the field and in storage. Recently, there have been reports of zearalenone contamination in corn, oats, barley, wheat, and grain sorghum. Corn and grain sorghum were examined for contamination due to obvious mold damage. Wheat, corn, and sorghum have been examined to determine the incidence of zearalenone in grains moving through commercial channels and stored on farms and at country elevators. Other grains such as oats and barley were analyzed because of associated estrogenic disturbances in farm animals. Stepsin procedures for the determination of zearalenone are extraction of a representative sample, partial purification of the extract by column chromatography, alkali treatment, or liquid-liquid partitioning, and subsequent measurement of the isolated toxin. Zearalenone is measured in partially purified extracts by thin layer chromatography (TLC), gas liquid chromatography (GLC), and high pressure liquid chromatography (HPLC). Confirmation of zearalenone contamination can be accomplished by gas chromatography-mass spectroscopy (GC-MS). Multitoxin screening procedures have been deveoped for zearalenone in combination with one or more of the following mycotoxins: aflatoxin, T-2 toxin, diacetoxyscirpenol, patulin, ochratoxin, penicillic acid, citrinin, penitrem A, and sterigmatocystin.     

Development of a Generic PCR Detection of 3-Acetyldeoxy-nivalenol-, 15-Acetyldeoxynivalenol- and Nivalenol-Chemotypes of Fusarium graminearum Clade

Fusarium graminearum clade pathogens cause Fusarium head blight (FHB) or scab of wheat and other small cereal grains, producing different kinds of trichothecene mycotoxins that are detrimental to human and domestic animals. Type B trichothecene mycotoxins such as deoxynivalenol, 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON) and nivalenol (NIV) are the principal Fusarium mycotoxins reported in China, as well as in other countries. A genomic polymerase chain reaction (PCR) to predict chemotypes was developed based on the structural gene sequences of Tri13 genes involved in trichothecene mycotoxin biosynthesis pathways. A single pair of primers derived from the Tri13 genes detected a 583 bp fragment from 15-AcDON-chemotypes, a 644 bp fragment from 3-AcDON-chemotypes and an 859 bp fragment from NIV-producing strains. Fusarium strains from China, Nepal, USA and Europe were identified by this method, revealing their mycotoxin chemotypes identical to that obtained by chemical analyses of HPLC or GC/MS and other PCR assays. The mycotoxin chemotype-specific fragments were amplified from a highly variable region located in Tri13 genes with three deletions for 15-AcDON-chemotypes, two deletions for 3-AcDON-chemotypes and no deletion for NIV-producers. This PCR assay generated a single amplicon and thus should be more reliable than other PCR-based assays that showed the absence or presence of a PCR fragment since these assays may generate false-negative results. The results with strains from several different countries as well as from different hosts further indicated that this method should be globally applicable. This is a rapid, reliable and cost-effective method for the identification of type B trichothecene mycotoxin chemotypes in Fusarium species and food safety controls.     

Method of analysis for deoxynivalenol and zearalenone from cereal grains

A method was developed to determine deoxynivalenol and zearalenone in corn, wheat, oats, rice and barley. The toxins are extracted with methanol/water (50:50, v/v) (2×) and partially purified by partitioning into ethyl acetate and then defatting with acetonitrile-petroleum ether. Toxins are isolated by silica gel column chromatography. Interfering materials are removed from the column with benzene; zearalenone is eluted with benzene/acetone (95:5, v/v), and after a column wash of chloroform/methanol (95:2, v/v), deoxynivalenol is eluted with chloroform/methanol (95:5, v/v). Zearalenone is quantitated by thin-layer chromatography and deoxynivalenol by gas-liquid chromatography of the trimethylsilyl derivative. The detection limit is about 0.02 μg/g for each toxin. Recoveries of added toxins varied with substrate and level of toxins. Recovery of deoxynivalenol ranged from 58% for 1 ppm in rice to 108% for 1 ppm in corn. Average recoveries for all levels (1, 2 and 5 ppm) ranged from 69% for barley to 89% for oats. Recovered zearalenone ranged from 40% for 5 ppm in wheat to 100% for 1 ppm in barley. Average recoveries for zearalenone at 1, 2 and 5 ppm varied from 53% for wheat to 87% for rice.     

Allelopathic potential of corn pollen

The effects of corn pollen and aqueous leachates of pollen upon the radicle growth ofBidens pilosa, Cassia jalapensis, andRumex crispus are shown. Extractions of pollen with various solvents and methods were carried out so as to assess its active principle and its effect uponC. jalapensis. The preliminary steps to separate and identify the allelopathic compounds of the sonicated and macerated pollen extracted with methylene chloride are described. The strongest inhibitory effect was produced by the hexane fraction. The allelopathic effect of corn pollen upon the growth ofC. jalapensis in several substrates is shown. The possible structure of some of the active fractions is discussed as well as the possibility that the allelopathic potential of pollen might actually occur in nature.     

Fumonisins,Trichothecenes and Zearalenone in Cereals

Fumonisins are phytotoxic mycotoxins which are synthesized by various species of the fungal genus Fusarium such as Fusarium verticillioides (Sacc.) Nirenberg (ex F.moniliforme Sheldon) and Fusarium proliferatum. The trichothecene (TC) mycotoxins are secondary metabolites produce by species that belong to several fungal genera, especially Fusarium, Stachybotrys, Trichothecium, Trichoderma, Memnoniella and Myrothecium. Fusarium mycotoxins are widely dispersed in cereals and their products. Zearalenone (ZEA) is an estrogenic compound produced by Fusarium spp. such as F. graminearum and F. culmorum. Fumonisins, the TCs and ZEA are hazardous for human and animal health. Contamination with TCs causes a number of illnesses in human and animal such as decrease in food consumption (anorexia), depression or inhibition on immune system function and haematoxicity. The purpose of this paper is to give a review of the papers published on the field of fumonisin, TC and ZEA mycotoxins in cereals consumed in the world.     

Double Gamers—Can Modified Natural Regulators of Higher Plants Act as Antagonists against Phytopathogens? The Case of Jasmonic Acid Derivatives

As key players in biotic stress response of plants, jasmonic acid (JA) and its derivatives cover a specific and prominent role in pathogens-mediated signaling and hence are promising candidates for a sustainable management of phytopathogenic fungi. Recently, JA directed antimicrobial effects on plant pathogens has been suggested, supporting the theory of oxylipins as double gamers in plant-pathogen interaction. Based on these premises, six derivatives (dihydrojasmone and cis-jasmone, two thiosemicarbazonic derivatives and their corresponding complexes with copper) have been evaluated against 13 fungal species affecting various economically important herbaceous and woody crops, such as cereals, grapes and horticultural crops: Phaeoacremonium minimum, Neofusicoccum parvum, Phaeomoniella chlamydospora, Fomitiporia mediterranea, Fusarium poae, F. culmorum, F. graminearum, F. oxysporum f. sp. lactucae, F. sporotrichioides, Aspergillus flavus, Rhizoctonia solani, Sclerotinia spp. and Verticillium dahliae. The biological activity of these compounds was assessed in terms of growth inhibition and, for the two mycotoxigenic species A. flavus and F. sporotrichioides, also in terms of toxin containment. As expected, the inhibitory effect of molecules greatly varied amongst both genera and species; cis-jasmone thiosemicarbazone in particular has shown the wider range of effectiveness. However, our results show that thiosemicarbazones derivatives are more effective than the parent ketones in limiting fungal growth and mycotoxins production, supporting possible applications for the control of pathogenic fungi.