Structure-activity relationships among mycotoxins

Relationships between structural features and biological effects of mycotoxins are reviewed. Structure-activity relationships are characterized at the molecular, subcellular, cellular, or supracellular level. Major chemical and physicochemical factors responsible for bioactivity of mycotoxins are stressed. A variety of chemical families of mycotoxins are then discussed from the point of view of structure-activity relationships. The structurally related families comprise small lactones, macrocyclic lactones, isocoumarin derivatives, aflatoxins and related compounds trichothecenes, anthraquinones, indole-derived tremorgens and selected amino acid-derived mycotoxins such as sporidesmins and cyclosporines. Biological effects of mycotoxins include acute and chronic toxicity, antimicrobial activity, mutagenicity and genotoxicity, carcinogenicity and biochemical modes of action.     

Comparison of cytotoxicity and thin-layer chromatography methods for detection of mycotoxins.

Thirty-three standard mycotoxins were assayed by thin-layer chromatography and by cytotoxicity in HEp-2 and Chang cells. Various levels of detection were found. The cytotoxicity test was significantly more sensitive than thin-layer chromatography for the trichothecenes and should be useful for screening extracts from animal feedstuffs for the presence of unknown mycotoxins.     

Comparison of cytotoxicity and thin-layer chromatography methods for detection of mycotoxins

Thirty-three standard mycotoxins were assayed by thin-layer chromatography and by cytotoxicity in HEp-2 and Chang cells. Various levels of detection were found. The cytotoxicity test was significantly more sensitive than thin-layer chromatography for the trichothecenes and should be useful for screening extracts from animal feedstuffs for the presence of unknown mycotoxins.     

Fusarium mycotoxins: Effects on reproductive function in domestic animals—A review

On a global scale, cereal grains and animal feed may be contaminated with trichothecenes, such as deoxynivalenol and T-2 toxin, zearalenone (ZEA), and fumonisins, the major mycotoxins of Fusarium fungi. Of these mycotoxins, ZEA is unequivocally implicated in reproductive disorders of swine and other domestic animals. Experiments in vivo and in vitro indicate that ZEA and its metabolites exert estrogenic effects resulting in functional and morphological alterations in reproductive organs. Recently, the potential of trichothecenes and fumonisins to cause reproductive disorders in domestic animals has been investigated. The present review summarizes the toxicological data on the effects of Fusarium mycotoxins on ovarian function, testicular function, placenta and fetus, and puberty/sexual maturity of domestic animals. The results of in vivo animal studies and in vitro tests are reported and discussed.     

A colorimetric technique for detecting trichothecenes and assessing relative potencies

We tested a novel colorimetric toxicity test, based on inhibition of beta-galactosidase activity in the yeast Kluyveromyces marxianus, for sensitivity to a range of mycotoxins. A variety of trichothecene mycotoxins could be detected. The order of toxicity established with this bioassay was verrucarin A > roridin A > T-2 toxin > diacetoxyscirpenol > HT-2 toxin > acetyl T-2 toxin > neosolaniol > fusarenon X > T-2 triol > scirpentriol > nivalenol > deoxynivalenol > T-2 tetraol. The sensitivity of detection was high, with the most potent trichothecene tested, verrucarin A, having a 50% effective concentration (concentration of toxin causing 50% inhibition) of 2 ng/ml. Other mycotoxins (cyclopiazonic acid, fumonisin B1, ochratoxin A, patulin, sterigmatocystin, tenuazonic acid, and zearalenone) could not be detected at up to 10 micrograms/ml, nor could aflatoxins B1 and M1 be detected at concentrations up to 25 micrograms/ml. This test should be useful for trichothecene detection and for studies of relevant interactions-both between trichothecenes themselves and between trichothecenes and other food constituents.     

Effects of Fusarium graminearum Metabolites on Wheat Tissue in Relation to Fusarium Head Blight Resistance

Fusarium head blight (FHB) of wheat is caused by Fusarium graminearum which produces many secondary metabolites including the trichothecene mycotoxins deoxynivalenol (vomitoxin) and 3-acetyldeoxynivalenol. Coleoptile tissue segements from 14 spring wheat cultivars were exposed to the F. graminearum metabolites deoxynivalenol, 3-acetyldeoxynivalenol, butenolide (all known mycotoxins), sambucinol, culmorin and dihydroxycalonectrin in a bioassay. The tissue of most cultivars was inhibited, at a concentration of 10^?6M by the trichothecenes tested and up to 10^?3M for the other compounds. Deoxynivalenol and 3-acetyldeoxynivalenol, which affect protein synthesis at the ribosome, are therefore potent phytotoxins in addition to being mycotoxins. The resistance or susceptibility of each cultivar to FHB was established in a field experiment. A comparison of the two sets of data indicated that resistant cultivars could tolerate much higher concentrations of the metabolites tested than susceptible cultivars. Some resistant material can tolerate 10 to 1000 times the concentration of the trichothecenes, compared with susceptible cultivars, with no effect on growth. The data suggest that it may be possible to screen germplasm rapidly for FHB resistance in vitro and a new type of resistance in wheat to this disease is proposed based on the apparent insensitivity to trichothecenes by resistant cultivars, additional to the three types of resistance described in the literature.     

Determination of Gliotoxin in samples associated with cases of intoxication in camels

Suspected cases of mycotoxicoses, characterized by clinical symptoms such as diarrhoea, haemorrhaging and death have been observed in breeding camels in U.A.E. Hay samples, body fluids and intestinal contents were investigated for the presence of mycotoxins using a cell culture bioassay (MTT-test) and/or physicochemical methods such as high performance liquid chromatography (HPLC) and mass spectrometry (MS). Extracts of the hay samples proved to be highly cytotoxic towards the swine kidney monolayers used as target cells in the bioassay. Subsequent analyses of the extracts showed the presence of the epidithiodioxopiperazine mycotoxin gliotoxin up to 0.49 mg/kg in the hay, which is the first proven case on the natural occurrence of this mycotoxin in feed. Trace amounts of ochratoxin A were also detected, while other mycotoxins known to occur naturally (i.e. aflatoxins or trichothecenes) could not be found. Gliotoxin positive HPLC-peaks were also found in some specimens (contents of rumen and intestine, allantois fluid) from camels. Gliotoxin is characterized by a variety of biological activities including antibiotic, antifungal, antiviral, cytotoxic and immunotoxic effects. Based on the results obtained, the involvement of gliotoxin as one causative agent of the intoxications is supposed.     

Free Water Can Leach Mycotoxins from Fusarium‐infected Wheat Heads

The objective of this study was to examine the impact of free environmental moisture, such as from rainfall, on disease development and mycotoxin production and accumulation in planta. In greenhouse experiments in 2009, two single Fusarium graminearum isolates were used to inoculate spikes of three wheat cultivars: ‘Alsen’, ‘2375’ and ‘Wheaton’ at anthesis. On each wetting event/sampling day (7, 14, 21 or 28 days after inoculation), FHB severity was assessed and five pots of each of the two cultivar/isolate treatments were subjected to a wetting event. At the end of the wetting event, the spikes were sampled both from the plants that received the wetting treatment and those that did not and analysed for mycotoxins. Run‐off water samples were also taken 3 h after the start of irrigation and immediately after the wetting treatment concluded and analysed for mycotoxins. The results showed despite statistically similar FHB severity, the levels of DON and other mycotoxins detected were significantly lower in the plants receiving a single wetting event compared to the control. The levels of DON in wetted plants were lower up to 36% in ‘Alsen’, 52% in ‘2375’ and 41% in ‘Wheaton’ compared to that of corresponding controls. DON and 15‐ADON were also detected in run‐off water from the inoculated heads of all cultivars examined. Based on the results of this study, it can be concluded that DON and its derivatives produced in planta can be leached out from the host tissues by free water on contact with plant surfaces.     

Involvement of trichothecenes in fusarioses of wheat, barley and maize evaluated by gene disruption of the trichodiene synthase (Tri5) gene in three field isolates of different chemotype and virulence

Fusarium graminearum is the main causative agent of Fusarium head blight on small grain cereals and of ear rot on maize. The disease leads to dramatic yield losses and to an accumulation of mycotoxins. The most dominant F. graminearum mycotoxins are the trichothecenes, with deoxynivalenol and nivalenol being the most prevalent derivatives. To investigate the involvement of trichothecenes in the virulence of the pathogen, the gene coding for the initial enzyme of the trichothecene pathway was disrupted in three field isolates, differing in chemotype and in virulence. From each isolate three individual disruption mutants were tested for their virulence on wheat, barley and maize. Despite the different initial virulence of the three wild-type progenitor strains on wheat, all disruption mutants caused disease symptoms on the inoculated spikelet, but the symptoms did not spread into other spikelets. On barley, the trichothecene deficient mutants showed no significant difference compared to the wild-type strains: all were equally aggressive. On maize, mutants derived from the NIV-producing strain caused less disease than their wild-type progenitor strain, while mutants derived from DON-producing strains caused the same level of disease as their progenitor strains. These data demonstrate that trichothecenes influence the virulence of F. graminearum in a highly complex manner, which is strongly host as well as moderately chemotype specific.     

Detection of airborne Stachybotrys chartarum macrocyclic trichothecene mycotoxins on particulates smaller than conidia

Highly respirable particles (diameter, <1 microm) constitute the majority of particulate matter found in indoor air. It is hypothesized that these particles serve as carriers for toxic compounds, specifically the compounds produced by molds in water-damaged buildings. The presence of airborne Stachybotrys chartarum trichothecene mycotoxins on particles smaller than conidia (e.g., fungal fragments) was therefore investigated. Cellulose ceiling tiles with confluent Stachybotrys growth were placed in gas-drying containers through which filtered air was passed. Exiting particulates were collected by using a series of polycarbonate membrane filters with decreasing pore sizes. Scanning electron microscopy was employed to determine the presence of conidia on the filters. A competitive enzyme-linked immunosorbent assay (ELISA) specific for macrocyclic trichothecenes was used to analyze filter extracts. Cross-reactivity to various mycotoxins was examined to confirm the specificity. Statistically significant (P < 0.05) ELISA binding was observed primarily for macrocyclic trichothecenes at concentrations of 50 and 5 ng/ml and 500 pg/ml (58.4 to 83.5% inhibition). Of the remaining toxins tested, only verrucarol and diacetylverrucarol (nonmacrocyclic trichothecenes) demonstrated significant binding (18.2 and 51.7% inhibition, respectively) and then only at high concentrations. The results showed that extracts from conidium-free filters demonstrated statistically significant (P < 0.05) antibody binding that increased with sampling time (38.4 to 71.9% inhibition, representing a range of 0.5 to 4.0 ng/ml). High-performance liquid chromatography analysis suggested the presence of satratoxin H in conidium-free filter extracts. These data show that S. chartarum trichothecene mycotoxins can become airborne in association with intact conidia or smaller particles. These findings may have important implications for indoor air quality assessment.     

小麦赤霉病镰孢菌毒素的生物合成及其分子调控

禾谷镰刀菌是小麦赤霉病的主要致病菌,其真菌次生代谢产生的单端孢霉烯类B型毒素,如雪腐镰刀菌烯醇(nivalenol,NIV)、脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)和其它乙酰化衍生物等污染小麦籽粒后对人畜健康构成威胁。综述了近年来国内外对小麦赤霉病镰孢菌单端孢霉烯类B型毒素生物合成的主要途径及分子调控研究进展,对毒素合成过程中的重要调控基因如TRI5、TRI7和TRI13在农业中的应用进行了阐述。     

A multicomponent method for Fusarium toxins in cereal based food and feed samples using HPLC-MS/MS

A reliable, sensitive and selective multicomponent method has been developed to determine 12 differentFusarium mycotoxins (trichothecenes type A and B, zearalenone) simultaneously in cereal and grain samples using liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS). The sample preparation based on a standard extraction step followed by two different kinds of solid phase clean-up (multifunctional MycoSep^® material) for trichothecenes, and an immuno-affinity purification which combined antibodies for aflatoxins, ochratoxin A and zearalenone (AOZ-IAC). For quantification of zearalenone (ZON) an internal standard (zearalanone, ZAN) was used, whereas for trichothecenes a recovery standard (verrucarol, VOL) was applied. The average recoveries for the trichothecenes ranged from 65% for nivalenol (NIV) up to 96% for deoxynivalenol (DON) and 89% for zearalenone. The limit of quantification is different for each of the individual trichothecenes and in the range of 1 ppb to 10 ppb.     

Building-Associated Neurological Damage Modeled in Human Cells: A Mechanism of Neurotoxic Effects by Exposure to Mycotoxins in the Indoor Environment

Damage to human neurological system cells resulting from exposure to mycotoxins confirms a previously controversial public health threat for occupants of water-damaged buildings. Leading scientific organizations disagree about the ability of inhaled mycotoxins in the indoor environment to cause adverse human health effects. Damage to the neurological system can result from exposure to trichothecene mycotoxins in the indoor environment. This study demonstrates that neurological system cell damage can occur from satratoxin H exposure to neurological cells at exposure levels that can be found in water-damaged buildings contaminated with fungal growth. The constant activation of inflammatory and apoptotic pathways at low levels of exposure in human brain capillary endothelial cells, astrocytes, and neural progenitor cells may amplify devastation to neurological tissues and lead to neurological system cell damage from indirect events triggered by the presence of trichothecenes.     

镰刀菌真菌毒素产生与调控机制研究进展

镰刀菌是一种重要的植物病原菌,给世界范围内农作物生产带来巨大破坏。除导致产量下降外,由其产生的镰刀菌真菌毒素能够污染农产品品质,给动物和人类食物安全造成严重隐患。单端孢霉烯族毒素（Trichothecenes）、伏马菌素（Fumonisin）和玉米赤霉烯酮（Zearalenone）是三种最重要的镰刀菌真菌毒素。镰刀菌真菌毒素的生物合成与生产受到体内一系列相关功能基因的调控;此外,pH值、碳氮比等环境条件也能影响真菌毒素的产量。本文简述了镰刀菌真菌毒素在产生机理、主要分类、致病性以及调控因素等方面的研究进展。     

Trichothecene mycotoxins trigger a ribotoxic stress response that activates c-Jun N-terminal kinase and p38 mitogen-activated protein kinase and induces apoptosis.

The trichothecene family of mycotoxins inhibit protein synthesis by binding to the ribosomal peptidyltransferase site. Inhibitors of the peptidyltransferase reaction (e.g. anisomycin) can trigger a ribotoxic stress response that activates c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinases, components of a signaling cascade that regulates cell survival in response to stress. We have found that selected trichothecenes strongly activate JNK/p38 kinases and induce rapid apoptosis in Jurkat T cells. Although the ability of individual trichothecenes to inhibit protein synthesis and activate JNK/p38 kinases are dissociable, both effects contribute to the induction of apoptosis. Among trichothecenes that strongly activate JNK/p38 kinases, induction of apoptosis increases linearly with inhibition of protein synthesis. Among trichothecenes that strongly inhibit protein synthesis, induction of apoptosis increases linearly with activation of JNK/p38 kinases. Trichothecenes that inhibit protein synthesis without activating JNK/p38 kinases inhibit the function (i.e. activation of JNK/p38 kinases and induction of apoptosis) of apoptotic trichothecenes and anisomycin. Harringtonine, a structurally unrelated protein synthesis inhibitor that competes with trichothecenes (and anisomycin) for ribosome binding, also inhibits the activation of JNK/p38 kinases and induction of apoptosis by trichothecenes and anisomycin. Taken together, these results implicate the peptidyltransferase site as a regulator of both JNK/p38 kinase activation and apoptosis.     

Antiviral Activity of Trichothecene Mycotoxins (Deoxynivalenol,Fusarenon-X,and Nivalenol) against Herpes Simplex Virus Types 1 and 2

The effect of trichothecene mycotoxins, deoxynivalenol (DON), fusarenon-X (FX) and nivalenol (NIV), on plaque formation of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) in HEp-2 cells was examined. The 50% effective concentrations (EC₅₀) of DON, FX, and NIV for HSV-1 plaque formation were 160, 56, and 120 ng/ml, respectively. Those for HSV-2 plaque formation were 94, 26, and 50 ng/ml, respectively. These three mycotoxins showed about 2-fold higher selectivity to HSV-2 than to HSV-1. Plaque formation of HSV-1 was not inhibited with trichothecenes at concentrations completely inhibiting plaque formation when cells were treated during virus adsorption period or 15 hr before infection. These results indicate that trichothecenes affect replication of HSV-1 after virus adsorption, but not before or during virus adsorption to the host cells.     

Mycotoxins in developing countries: A case study of maize in Nepal

Maize (Zea mays) is an important food crop in the foothills of the Nepal Himalaya Mountains. Surveys have found that maize in Nepal is contaminated withFusarium species, mainlyF. verticillioides andF. proliferatum, which produce fumonisins, andF. graminearum, which produces trichothecenes, mainly nivalenol and 4-deoxynivalenol. Maize from smallholder farms and markets is often contaminated with fumonisins and trichothecenes above 1000 ng/g, a level of concern for human health. These mycotoxins were not eliminated by traditional fermentation for producing maize beer, but Nepalese women were able to detoxify contaminated maize by hand-sorting visibly disease kernels. An integrated approach to reduce mycotoxins risks in maize in Nepal and other developing countries should include plant breeding to produce ear rot resistant cultivars, along with education in mycotoxins risks and in agricultural and grain storage practices to reduce mycotoxin contamination. Presented at the EU-USA Bilateral Workshop on Toxigenic Fungi & Mycotoxins, New Orleans, USA, July 5–7, 2005     

Comparative Studies on Microbial and Chemical Modifications of Trichothecene Mycotoxins

The microbial modification of several trichothecene mycotoxins by trichothecene-producing strains of Fusarium nivale and F. solani was studied. These results were compared with the corresponding chemical modifications. The growing mycelia of Fusarium spp. did not convert 4beta-acetoxy-3alpha,7alpha, 15-trihydroxy-12, 13-epoxytrichothec-9-en-8-one (fusarenon) into 3alpha,4beta, 7alpha,15-tetrahydroxy-12,13-epoxy-trichothec-9-en-8-one (nivalenol), whereas 3alpha,4beta,7alpha,15-tetracetoxy-12,13-epoxytrichothec-9-en-8-one (tetraacetylnivalenol) was deacetylated to yield 3alpha-hydroxy-4beta,7alpha,15-triacetoxy-12,13-epoxytrichothec-9-en-8-one (4,7,15-triae-tylnivalenol), which was resistant to further deacetylation. T-2 toxin was transformed intoHT-2 toxin, and 8alpha-(3-methylbutyryloxy)-3alpha,4beta,-15-triacetoxy-12,13-epoxytrichothec-9-en-8-one (T-2 acetate) was transformed into HT-2 toxin via T-2 toxin. Chemical modification with ammonium hydroxide converted tetraacetylnivalenol into fusarenon via 4,7,15-triacetylnivalenol. 3alpha-7alpha,15-Triacetoxy-12,13-epoxytrichothec-9-en-8-one (triacetyldeoxynivalenol) gave deacetylation products lacking the C-7 or c-15 acetyl group in addition to 7alpha,15- diacetoxy-3alpha-hydroxy-12, 13-epoxytrichothec-9-en-8-one (7,15-diacetyldeoxynivalenol). These results demonstrate the regio-selectivity in microbial modification of trichothecenes. Based on the results and available knowledge concerning the transformation of trichothecenes, mechanisms for biological modifications of these mycotoxins are postulated.     

Mycotoxins in crude building materials from water-damaged buildings

We analyzed 79 bulk samples of moldy interior finishes from Finnish buildings with moisture problems for 17 mycotoxins, as well as for fungi that could be isolated using one medium and one set of growth conditions. We found the aflatoxin precursor, sterigmatocystin, in 24% of the samples and trichothecenes in 19% of the samples. Trichothecenes found included satratoxin G or H in five samples; diacetoxyscirpenol in five samples; and 3-acetyl-deoxynivalenol, deoxynivalenol, verrucarol, or T-2-tetraol in an additional five samples. Citrinine was found in three samples. Aspergillus versicolor was present in most sterigmatocystin-containing samples, and Stachybotrys spp. were present in the samples where satratoxins were found. In many cases, however, the presence of fungi thought to produce the mycotoxins was not correlated with the presence of the expected compounds. However, when mycotoxins were found, some toxigenic fungi usually were present, even if the species originally responsible for producing the mycotoxin was not isolated. We conclude that the identification and enumeration of fungal species present in bulk materials are important to verify the severity of mold damage but that chemical analyses are necessary if the goal is to establish the presence of mycotoxins in moldy materials.     

Myrotoxins from a plant pathogenic isolate ofMyrothecium roridum

An isolate ofM roridum (ATCC 52485) which is a potent pathogen to muskmelon was shown to produce a series of macrocyclic trichothecenes, the myrotoxins, heretofore found to be produced only by an. isolate ofM roridum which is a pathogen to tomato. The fact that these two isolates are virulent pathogens and both produce the same potent mycotoxins suggests that these novel trichothecenes may play an important role in the pathogenicity of the fungi.