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Plant-derived mycotoxin conjugates like deoxynivalenol-3-glucoside can be partly hydrolyzed to their aglycones in vivo, albeit to different extent depending on the mycotoxin conjugate and on the animal species. The aim of this work was to investigate the metabolization of the trichothecene mycotoxin nivalenol (NIV) and the fate of its modified form NIV-3-glucoside (NIV3G) in rats. To that end, 350 μg/kg body weight of NIV and the equimolar dose of NIV3G were administered to six rats by gavage in a 5 × 6 design and excreta were collected for 2 days after each treatment. For further analysis of NIV and NIV3G metabolites in rat urine and feces, seven novel NIV- and NIV3G metabolites including NIV sulfonates (NIVS) 1, 2 and 3, deepoxy-NIV (DNIV), DNIV sulfonate 2, NIV3G sulfonate (NIV3GS) 2 and NIV-3-glucuronide were produced, isolated and characterized. Subsequently, LC–MS/MS based methods for determination of NIV, NIV3G and their metabolites in excreta samples were developed, validated and applied. The biological recoveries of administered toxins in the form of their fecal and urinary metabolites were 57 ± 21% for NIV and 94 ± 36% for NIV3G. The majority of NIV and NIV3G metabolites was excreted into feces, with DNIV and NIVS 2 as major NIV metabolites and NIV3GS 2 and DNIV as major metabolites of NIV3G. Only 1.5% of the administered NIV3G was recovered in urine, with NIV3G itself as major urinary metabolite. The biological recovery of free NIV in urine was approximately 30 times lower after treatment with NIV3G than after administration of NIV, indicating that exposure of rats to NIV3G results in lower toxicity than exposure to NIV.

The sulfated forms of the Fusarium toxin deoxynivalenol (DON), deoxynivalenol-3-sulfate (DON-3-Sulf) and deoxynivalenol-15-sulfate (DON-15-Sulf) were recently described, however little is known about their mechanism of action in mammalian cells. DON-3-Sulf and DON-15-Sulf were taken up by HT-29 colon carcinoma cells, although to a lesser extent compared to DON. All three compounds were found to enhance the intracellular ROS level in the dichlorofluorescein assay (≥ 1μM), even though substantial differences were observed in their cytotoxic potential. In silico modelling highlighted that DON-sulfates do not share the classical mechanism of action of DON, being unable to fit into the ribosomal pocket and trigger the classical ribotoxic stress response. However, DON-3-Sulf and DON-15-Sulf sustained a distinctive proliferative stimulus in HT-29 and activated autophagy. The mechanisms of action of DON-3-Sulf and DON-15-Sulf suggest a potential interplay between the onset of ribosomal inhibition and autophagy activation as an alternative and/or complementary mode of action for DON and its sulfated analogues.

A new type A trichothecene mycotoxin, NX-2, was previously reported to be produced by North American isolates of the cereal pathogen Fusarium graminearum. Here we describe the isolation and structural characterization of a rearrangement product, called NX2-M1, and related compounds with different acetylation patterns (NX3-M1 and NX4-M1). In the NX-M1 derivatives, the epoxide ring is opened, and a covalent bridge between C-10 and C-12 of the trichothecene backbone is formed. In vitro translation assays showed that NX3-M1 is less toxic for eukaryotic ribosomes than NX-3. NX3-M1 also has a greatly reduced cytotoxic potential on two tested human colon cell lines. Formation of NX3-M1 can therefore be regarded as a detoxification reaction. The related F. graminearum mycotoxin deoxynivalenol (DON), which is frequently occurring worldwide, is very stable during food processing. Testing NX-3 at different pH-values and temperature conditions, as well as under conditions that simulate the storage of infected grains and bread-making process, revealed a strongly reduced stability of NX-3 and concurrent formation of NX3-M1. Although the NX-3 formed in planta is as toxic as DON, the extensive formation of the non-toxic rearrangement product should be taken into account for risk assessment of this emerging food contaminant.

Deoxynivalenol is a food borne mycotoxin belonging to the trichothecenes family that may cause severe injuries in human and animals. The inhibition of protein synthesis via the interaction with the ribosome has been identified as a crucial mechanism underlying toxic action. However, it is not still fully understood how and to what extent compounds belonging to trichothecenes family affect human and animal health. In turn, this scenario causes delay in managing the related health risk. Aimed at supporting the hazard identification process, the in silico analysis may be a straightforward tool to investigate the structure-activity relationship of trichothecenes, finding out molecules of possible concern to carry forth in the risk assessment process. In this framework, this work investigated through a molecular modeling approach the structural basis underlying the interaction with the ribosome under a structure-activity relationship perspective. To identify further forms possibly involved in the total trichothecenes-dependent ribotoxic load, the model was challenged with a set of 16 trichothecene modified forms found in plants, fungi and animals, including also compounds never tested before for the capability to bind and inhibit the ribosome. Among them, only the regiospecific glycosylation in the position 3 of the sesquiterpenoid scaffold (i.e. T-2 toxin-3-glucuronide, α and β isomers of T-2 toxin-3-glucoside and deoxynivalenol-3-glucuronide) was found impairing the interaction with the ribosome, while the other compounds tested (i.e. neosolaniol, nivalenol, fusarenon-X, diacetoxyscirpenol, NT-1 toxin, HT-2 toxin, 19- and 20-hydroxy-T-2 toxin, T-2 toxin triol and tetraol, and 15-deacetyl-T-2 toxin), were found potentially able to inhibit the ribosome. Accordingly, they should be included with high priority in further risk assessment studies in order to better characterize the trichothecenes-related hazard.

The trichothecene deoxynivalenol (DON), a potent ribotoxic mycotoxin produced by the cereal blight fungus Fusarium graminearum, commonly contaminates grain-based foods. Oral exposure to DON causes decreased food intake, reduced weight gain and body weight loss in experimental animals - effects that have been linked to dysregulation of hormones responsible for mediating satiety at the central nervous system level. When diet-induced obese (DIO) mice are fed DON, they consume less food, eventually achieving body weights of control diet-fed mice. Here, we extended these findings by characterizing: (1) reversibility of DON-induced body weight loss and anorexia in DIO mice and (2) the role of double-stranded RNA-activated protein kinase (PKR) which has been previously linked to initiation of the ribotoxic stress response. The results demonstrated that DON-induced weight loss was reversible in DIO mice and this effect corresponded to initiation of a robust hyperphagic response. When DIO mice deficient in PKR were exposed to DON, they exhibited weight suppression similar to DIO wild-type fed the toxin, suggesting the toxin's weight effects were not dependent on PKR. Taken together, DON's effects on food consumption and body weight are not permanent and, furthermore, PKR is not an essential signaling molecule for DON's anorectic and weight effects.

Trichothecenes are sesquiterpenoid mycotoxins commonly found as contaminants in cereal grains and are a major health and food safety concern due to their toxicity to humans and farm animals. Trichothecenes are predominantly produced by the phytopathogenic Fusarium fungus, and in plants they act as a virulence factor aiding the spread of the fungus during disease development. Known for their inhibitory effect on eukaryotic protein synthesis, trichothecenes also induce oxidative stress, DNA damage and cell cycle arrest and affect cell membrane integrity and function in eukaryotic cells. In animals, trichothecenes can be either immunostimulatory or immunosuppressive and induce apoptosis via mitochondria-mediated or -independent pathway. In plants, trichothecenes induce programmed cell death via production of reactive oxygen species. Recent advances in molecular techniques have led to the elucidation of signal transduction pathways that manifest trichothecene toxicity in eukaryotes. In animals, trichothecenes induce mitogen-activated protein kinase (MAPK) signalling cascades via ribotoxic stress response and/or endoplasmic reticulum stress response. The upstream signalling events that lead to the activation trichothecene-induced ribotoxic stress response are discussed. In plants, trichothecenes exhibit elicitor-like activity leading to the inductions MAPKs and genes involved in oxidative stress, cell death and plant defence response. Trichothecenes might also modulate hormone-mediated defence signalling and abiotic stress signalling in plants.

Deoxynivalenol (DON) and nivalenol (NIV), trichothecene mycotoxins, are secondary metabolites produced by Fusarium fungi. Trichothecene mycotoxins cause immune dysfunction, thus leading to diverse responses to infection. The present study evaluated the effect of DON and NIV on nitric oxide (NO) production by RAW264 cells stimulated with lipopolysaccharide (LPS). LPS-induced NO production was reduced in the presence of these toxins. The transcriptional activation and expression of inducible NO synthase (iNOS) by LPS were also repressed by these toxins. DON or NIV inhibited LPS-induced expression of interferon-beta (IFN-beta), which plays an indispensable role in LPS-induced iNOS expression. These results indicate that DON and NIV inhibit the LPS-induced NO and IFN-beta production, which both play an important role for host protection against invading pathogens, and suggests that the inhibition of these factors may be involved in the immunotoxic effects of these mycotoxins.

Deoxynivalenol (DON or vomitoxin) is a trichothecene mycotoxin commonly found in cereal grains that adversely affects growth and immune function in experimental animals. A competitive enzyme-linked immunosorbent assay (ELISA) was used to monitor the kinetics of distribution and clearance of DON in tissues of young adult B6C3F1 male mice that were orally administered 25mg/kg bw of the toxin. DON was detectable from 5 min to 24h in plasma, liver, spleen and brain and from 5 min to 8h in heart and kidney. The highest DON plasma concentrations were observed within 5-15 min (12 microg/mL) after dosing. There was rapid clearance following two-compartment kinetics (t(1/2)alpha=20.4 min, t 1/2 beta=11.8h) with 5% and 2% maximum plasma DON concentrations remaining after 8 and 24h, respectively. DON distribution and clearance kinetics in other tissues were similar to that of plasma. At 5 min, DON concentrations in mug/g were 19.5+/-1.9 in liver, 7.6+/-0.5 in kidney, 7.3+/-0.8 in spleen, 6.8+/-0.9 in heart and 0.8+/-0.1 in the brain. DON recoveries in tissues by ELISA were comparable to a previous study that employed (3)H-DON and 25mg/kg bw DON dose. The ELISA was further applicable to the detection of DON in plasma of mice exposed to the toxin via diet. This approach provides a simple strategy that can be used to answer relevant questions in rodents of how dose, species, age, gender, genetic background and route/duration of exposure impact DON uptake and clearance.

International audience; The genotoxic risk associated with deoxynivalenol (DON), a prevalent trichothecene mycotoxin which contaminates cereal-based products has not yet been deeply explored. In this work, the alkaline version of the Comet assay was used to evaluate DNA damage stemming from DON exposure in both dividing and differentiated Caco-2 cells, an epithelial intestinal cell line. To avoid false positive results, cytotoxic and apoptotic thresholds were firstly established using the MTS and neutral red assays and the Hoestch staining method, respectively. Dividing cells were found to be more sensitive to DON than differentiated cells and the lowest IC10 (0.5 mu M) obtained for dividing cells exposed for 72 It was used as the highest working concentration in the genotoxicity study. Both differentiated and dividing cells responded with a dose-dependant relationship to DON in terms of DNA damage in the 0.01-0.5 mu M range. These results demonstrated the existence of a genotoxic potential for DON at low concentrations compatible with actual exposure situations and calls for additional studies to determine the functional consequences which could be taken into account for the risk assessment of this food contaminant

Methods to analyze trichothecenes should be fast, reliable and economical. The known methods can be divided into two categories: the 'instrumental methods' as gas chromatography (GC) and high performance liquid chromatography (HPLC) and the so called 'fast methods' like thin layer chromatography (TLC), enzyme-linked immunosorbant analyses (ELISA) and flow through immunoassays. The most frequently used instrumental methods for the determination of deoxynivalenol (DON), HT-2- and T2-toxin in cereals and cereal products are based on gas chromatography with electron capture detectors or mass spectrometric (MS) detectors. More than 70% of the literature published in the last decade focuses on these techniques. Recently, high performance liquid chromatography with fluorescence detection and with mass spectrometric detection have gained importance. Up to now, a method which is able to detect types A and B trichothecenes simultaneously with a sufficiently low limit of detection is lacking, even though methods are published which have at least the same clean up step.

Fusarium head blight (FHB) of small grain cereals and ear rot in maize are significant diseases across the world. Infection can not only result in reduced yield as a result of shrunken grains but also result in reduced milling and malting quality and the contamination of grains with mycotoxins. Mycotoxins are hazardous to animal and human health. Therefore, guidelines and legislation are in place, or under consideration, in most countries to protect consumers and animal welfare. As fusarium mycotoxins are produced within the growing crop, it is important to understand how agricultural practices affect mycotoxin contamination of grain. Such information could then be used to determine guidelines on "Good Agricultural Practice" (GAP) to minimise the mycotoxin contamination of cereal products. Evidence is provided to show the importance of choice of cultivar, crop rotation, soil cultivation, fertiliser and the chemical and biological control of insects, weeds and fungi.

Trichothecene mycotoxins frequently occur in cereal grains that are intended for food production. The process of converting grains into food and drinks for consumers has significant effects on the levels of toxins in the final food. Surveillance of retail food and drinks of cereal origin demonstrates that trichothecenes do survive the production processes employed. Trichothecenes are relatively heat stable chemicals, with high water solubility, properties that affect their processing fate. It is known that the extent of transmission into final food products is dependent on the pattern of Fusarium infection in the grains. For dry-milled products, the most highly contaminated fractions are those that contain the whole or the outer portions of the grain. For wet milling, the trichothecenes primarily transfer to the aqueous fractions, the most contaminated streams enter the animal food chain, although there is no evidence of significant trichothecenes transmission into animal products. However, anomalies remain in the understanding of the processing effects of several major cereal processes used in European food production (e.g. baking and brewing).

The present paper summarizes toxicity data relevant for hazard characterization for the trichothecene mycotoxins deoxynivalenol (DON), nivalenol (NIV), T-2 and HT-2 from recent opinions prepared by the European Commission Scientific Committee on Food (SCF) and the Joint FAO/WHO Expert Committee on Food Additives (JECFA). Details on immunotoxicity, cardiovascular toxicity and co-occurrence of different trichothecenes and other mycotoxins and their possible interactions are considered in separate papers in the present issue as well as other aspects such as mould growth, trichothecenes formation, storage, processing, sampling, analytical measurements, exposure assessment and surveillance. The toxicological profiles of DON, NIV, T-2 and HT-2 are similar. The general toxicity and immunotoxicity in experimental animals, and for NIV also haematotoxicity, are considered to be the critical effects. Tolerable Daily Intakes of 1, 0.7 and 0.06 microg/kg b.w. were established for DON, NIV and the sum of T-2 and HT-2, respectively. The TDI's for NIV, T-2 and HT-2 were made temporary because of deficiencies the database.

A number of mycotoxins of the class of trichothecenes are produced by a variety of Fusarium fungi commonly found on cereals. Unfavourable weather conditions may lead to a high level of Fusarium infections in crops such as wheat and correspondingly high trichothecene contents. The ILSI Europe Natural Toxin Task Force therefore organised a workshop on trichothecenes with a special focus on deoxynivalenol (DON). A number of experts reviewed the current knowledge on trichothecenes with respect to occurrence, including aspects of mould growth, toxin formation, storage and effects of processing; prevention; analytical methodologies, including sampling; surveillance and exposure assessments; and toxicology and risk assessment. A number of recommendations were given under the headings: prevention, sampling and analytical methods, exposure assessment, and toxicology. Gaps in knowledge were also identified.

In 2001 the SCOOP (SCOOP: Scientific Co-operation on Questions relating to Food) task 3.2.10 "Collection of occurrence data of Fusarium toxins in food and assessment of dietary intake by the population of EU Member States" was established. The task was divided in three subtasks (zearalenone, fumonisins and trichothecenes). Results of the subtask trichothecenes, which is co-ordinated by The Netherlands, will be presented. About 35,000 results were received about occurrence of 12 different trichothecenes (deoxynivalenol (DON), nivalenol (NIV), 3 and 15 acetyl-deoxynivalenol (3/15-AcDON), fusarenon-X (FUS-X), T-2 and HT-2 toxin, T2-triol, diacetoxyscirpenol (DAS), neosolaniol (NEOSOL, monoacetoxyscirpenol (MAS) and verrucarol (VOL)) in various food and food raw materials from 12 countries. Only the results of DON, NIV, T-2 and HT-2 toxin are included in this paper, because most of the data refer to these toxins and only for these trichothecenes the Scientific Committee for Food sets (temporary)-Tolerable Daily Intakes (t-TDIs). Occurrence data: By far most of the occurrence data were obtained for DON in wheat. Among cereals, corn showed the highest level of contamination with trichothecenes. Consumption data: There is a significant lack of consumption data in some countries. In particular information on baby's and children's food is generally not available. Intake data: Wheat and wheat containing products (like bread and pasta) represent the major source of intake for the four trichothecenes. The mean intakes for DON are below the TDI, however for the young children groups the mean intakes are sometimes (very) close to the TDI. By comparing the high intake levels for DON with the TDI, it is clear that especially for the young children groups most of the intakes are above the TDI. For NIV, the (mean and high level) intakes are far below the TDI. The summarised T-2 and HT-2 toxin intakes are in most cases (for the mean as well as the high level intake) above the t-TDI.

The frequent contamination of food and feed with trichothecene mycotoxins, the high consumption of these products, and the potential risk associated herewith, has led to an increasing public awareness and therefore to the establishment of measures to control trichothecene contamination. The analytical difficulty and the economic importance of controlling trichothecenes in food and feed support the need for certified reference materials (CRMs) and validated methods. They form invaluable tools to ensure comparability and traceability in analytical measurements and are very useful for the implementation of written standards, legislation/regulations and laboratory accreditation. The present paper provides an overview of previous work, current strategies and prospectives for the production of CRMs and validation of analytical methods in the field of trichothecene analysis. Additional information is given on methodological demands, normative frameworks and commonly accepted procedures.

Mycotoxins induce diverse and powerful biological effects in test systems; some are carcinogenic, mutagenic, teratogenic, estrogenic, hemorrhagic, immunotoxic, nephrotoxic, hepatotoxic, dermotoxic, and neurotoxic. Mycotoxins have been unambiguously linked to the etiology of several diseases in animals. The discovery of aflatoxins in the early 1960s led to the resurgence of interest in human mycotoxicoses; mycotoxins are now recognized as causal factors of primary liver cancer, ergotism and alimentary toxic aleukia. The fumonisins and ochratoxins are suspected of playing a role in the etiology of esophageal cancer and Balkan endemic nephrotoxicity, respectively.

The ex vivo biophysical response of isolated rat aorta to T-2 toxin, diacetoxyscirpenol (DAS) and Roridin-A were determined. Roridin-A caused a dose-dependent relaxation of precontracted (30 mM KCl) aorta, while T-2 toxin and DAS were without effect. On the other hand, the relaxation response of aorta to isoproterenol and sodium fluoride is impaired in the presence of T-2 toxin or DAS. Roridin-A did not alter the relaxation response to isoproterenol or sodium fluoride. These experiments suggest that macrocyclic trichothecenes (e.g., Roridin-A) are capable of altering vascular smooth muscle tone directly, while simple trichothecenes (e.g., T-2 toxin, DAS) interfere with beta-adrenergic responses.

Type B 8-keto trichothecenes are muco-active mycotoxins that are notable contaminants in cereal-based food stuffs. Epithelial responses are of primary concern during gastrointestinal exposure in human and animal intoxications. Therefore, optimized biomarkers to assess the specific action of trichothecenes on the human epithelial barrier are needed. In the present study, 8-keto trichothecenes were tested to trigger endoplasmic reticulum (ER) stresses that are evident as alteration of eukaryotic initiation factors 2α phosphorylation and other ER stress markers in human intestinal epithelial cells. Based on the ER stress-inducing activity of 8-keto trichothecenes, we developed a bio-monitoring method using intestinal epithelial cells constitutively expressing secretory alkaline phosphatase (SEAP) reporter, which can be used as a sensitive and selective indicator for monitoring ER stress in the epithelial barrier. Type B 8-keto trichothecenes suppressed the release of SEAP in a dose-dependent manner, showing a negative correlation between logarithm of toxin dose and SEAP activity. However, tested toxins did not affect SEAP enzymatic activity and mRNA levels, but reduced the cellular release of SEAP. Moreover, in order to correct the monitoring of 8-keto trichothecene in crop matrix, the bioassay was proven to work in standard cereal extract of corn and rice. The mechanism-based monitoring of 8-keto trichothecenes is promising as a supplementary analysis means of the presence of bioactive 8-keto trichothecenes, which are potentially exposed in human gut epithelia.

Contamination of cereal commodities with mycotoxins represents a significant hazard to consumer health and has thus received increasing attention from food safety authorities and legislators. For trichothecenes, and deoxynivalenol (DON) in particular, the imminent implementation of legislative limits has focused attention on ways to prevent entry of such mycotoxin contaminants into the food and feed chains. Knowledge of the pre- and post-harvest stages in the cereal production chain and in particular information on where prevention strategies can be implemented is being used to develop quality assurance systems for improving food safety. Information on the ecology of Fusarium species, breeding for resistance, more effective fungicides, potential for biological control and effective drying and storage and preservation systems, are all helping to develop effective preventative strategies for minimising consumer exposure to trichothecenes and other mycotoxins.

The state of the art in Fusarium head blight resistance research is reviewed with reference to breeding for genetic resistance to Fusarium in wheat in practice. Fusarium graminearum and F. culmorum produce the trichothecene mycotoxin deoxynivalenol (DON). DON has phytotoxic properties and is an important aggressiveness factor in head blight. Head blight resistance in wheat is not specific for either F. graminearum or F. culmorum. Resistance components include resistance to penetration, resistance to colonization and mechanisms that influence kernel DON content. The resistance to Fusarium in wheat is a quantitative trait with relative high heritability and controlled by a few genes with major effect. A major quantitative trait locus (QTL) for head blight resistance from the Chinese variety Sumai 3 has been identified and verified by several research groups via molecular marker analysis. Research is now directed at identifying additional QTLs to make accumulation of resistance genes in elite wheat lines possible. The policy of official variety list trials may affect the head blight resistant level of future wheat varieties by excluding candidate varieties that are a too susceptible to Fusarium. A higher level of Fusarium head blight will guarantee lower risks for the farmer of crop loss due to reduced grain yield, low quality and mycotoxin contamination.

Human toxicosis induced by consumption of foodstuffs contaminated with trichothecenes presents one common major symptom: a haematological perturbation manifesting principally as thrombocytopenia and leukopenia. The patients have rapidly progressing coagulation problems, and compromised resistance to infections. Consequently, they are subject to septicaemia and massive haemorrhages. In horses, cattle, poultry, cats, mice and guinea pigs, subacute and subchronic ingestion of trichothecenes causes a decrease of circulating blood cells frequently associated with bone marrow failure. The origins of haematological effects observed in Fusarium toxin intoxications have been elucidated using in vitro tests. Haematopoietic progenitors are the main target of trichothecenes. Deoxynivalenol (DON) is the least myelotoxic and T-2 the most. As circulating blood cells present a less important sensitivity to these toxins, haematological troubles observed in toxicosis are due to myelotoxicity of these toxins.

Grain should be harvested at lowest possible humidity, or immediately dried to safe storage humidity to prevent the growth of fungi. Critical steps of grain harvest like conditions of used combine harvester, temperatures and aeration are discussed on their impact on mycotoxin formation.

Method development for deoxynivalenol (DON) and other trichothecenes in recent years was driven by the analytical necessities arising from its widespread (and increasing) occurrence in foods and feeds. This has resulted in the establishment of guideline levels for animal feed, tolerable daily intake (TDI) levels for humans, and most importantly, in the prospect of low-tolerance levels for these toxins in foods in the near future. In order to ensure reliable determination of the toxin content at the tolerance levels, routine analytical methods must have detection limits of less than the tolerance level. This paper intends to give an overview of current analytical developments of rapid testing for deoxynivalenol and other trichothecene mycotoxins, with a special focus on antibody-based techniques. This includes high-throughput instrumental analysis for the laboratory environment, as well as rapid visual tests for on-site testing. The applicability of rapid tests within an integrated detection system for mycotoxins in foods is discussed.

Trichothecenes are agriculturally important mycotoxins of relevance to human health. Fungi capable of producing trichothecenes can be found throughout the world, and include certain species of Fusarium, Myrothecium, and Stachybotrys. The production of mycotoxins by these toxigenic species is determined by genetic factors and the environmental conditions of their growth. The environmental fate of trichothecenes may be affected by other microorganisms that can detoxify them. Deoxynivalenol and T-2 toxin are examples of trichothecenes that are detectable as natural and unavoidable contaminants of certain agricultural commodities as well as commercial foods. Current estimates of dietary exposure to deoxynivalenol and T-2 toxin are below thresholds for adverse effects that have been reported in experimental animal studies, although historical epidemics of human illness have rarely been described in association with consumption of food derived from heavily contaminated grains. The toxicodynamic properties of trichothecenes include inhibition of protein synthesis and immunomodulatory effects. Very little information is available relating to their toxicokinetics and toxicodynamics in humans. While there is general agreement that the diet represents an important source of human exposure to trichothecenes, risk assessment from non-dietary routes of exposure is complicated by the limited epidemiological data that are currently available.

The effects of several congeners of the macrocyclic class of trichothecene mycptoxins on murine splenic cells in vitro were investigated. The mycotoxins were roritoxin B, myrotoxin B, roridins A, D and E, baccharinoids B4, B5 and B12, 16-hydroxyverrucarin A, and verrucarins A and J. Lymphocytes from CD-1 mice were cultured with each of the mycotoxins for 48 h to assess cytotoxicity. The maximum effect of various trichothecenes produced on cells occurred at concentrations ranging from 10 −6 to 10 −4 M. Mycotoxins had no effect at concentrations ranging from 10 −12 to 10 −7 M. The mitogenic stimulants concanavalin A, lipopolysaccharide, phytohemagglutinin, and pokeweed mitogen were added to splenic lymphocyte cultures along with varying concentrations of selected mycotoxins. Blastogenesis was inhibited at concentrations 2–5 orders of magnitude lower than those which produced lethality on resting lymphocytes.

Antidotic effects of radioprotective and anti-inflammatory drugs against the acute toxicity of T-2 toxin and fusarenon-X, the trichothecene mycotoxins of Fusarium species, were examined in mice and rats with two toxicological parameters, the lethal toxicity and the increment of intestinal fluid volume. The s.c. and i.v. LD50 values of T-2 in male 6-week-old mice were 2.1 and 3.8 mg/kg, respectively, and the s.c. LD50 in 4-week-old mice was 1.6 mg/kg. Pretreatment of mice with steroidal anti-inflammatory agents such as prednisolone and hydrocortisone resulted in a marked reduction in the lethal toxicity of T-2 toxin. However, pretreatment of mice with several radioprotective agents exhibited neither a prolongation of survival time nor a decrease in lethal toxicity in the mice. Enteropooling assay revealed that the steroidal anti-inflammatory drugs also depressed the trichothecene-induced increment of intestinal fluid volume in rats, while no significant effects were observed with non-steroidal inhibitors of the arachidonate cascade. In rats given fusarenon-X, the content of cyclic AMP in the mucosa of ileum was dose- and time-dependently increased, but its maximal increment was only 1.5-fold of the control value. These findings indicate an involvement of the action site of steroidal anti-inflammatory drugs in the development of acute toxicity of trichothecenes in rodents.

T-2 toxin has been found previously to be markedly more toxic upon intracerebral than upon systemic administration. In order to study the generality of this difference, we have applied to brain and liver of albino rats two different types of trichothecenes: T-2 toxin as a representative of ‘simple’ and myrotoxin B as a representative of ‘macrocyclic’ members of this series. Myrotoxin B, when applied intracerebrally, was about 100 times more poisonous than T-2 toxin. In contrast, upon intrahepatic injection both compounds exhibited similar degrees of toxicity. The different behavior of the two trichothecenes in the organs tested may be due to local metabolic factors.

In mice, administration of pure T-2 toxin caused a rapid decrease of lymphocyte counts, which was linear with respect to dose, whereas granulocyte counts showed a delayed decrease. The blood cell counts of both cell types attained normal values after 4–7 days. Similar results were obtained for crude A-, B-and macrocyclic type trichothecene. Intoxication of rats with T-2 toxin or crude A-type trichothecene caused changes in white blood cells, which differed quantitatively from those in the mouse: lymphocyte counts decreased less and a rapid transient increase of granulocytes was more obvious. Results of this study show that lymphocyte and granulocyte blood cell counts of small rodents respond sensitively to acute intoxication with various trichothecenes.

(1) 3α-Hydroxy-4β,15-diacetoxy-8α-(3-methylbutyryloxy)-12,13-epoxytrichothec-9-ene (T-2 toxin) and HT-2 toxin both inhibited the incorporation of tritiated thymidine into the DNA of human fibroblasts; the inhibition was dose-related. 1. (2) The presence of hydroxyurea (HU) did not affect unscheduled DNA synthesis in human fibroblasts damaged by T-2 toxin (0.006 μg/ml to 20.0 μg/ml), or by HT-2 toxin (0.032 μg/ml to 100.0 μg/ml) as with cycloheximide. 2. (3) In the presence of the rat-liver microsomal fraction, S-9 and of hydroxyurea, there was an increase in unscheduled DNA synthesis in human fibroblasts exposed to 100 μg/ml of HT-2 toxin. 3. (4) Thus in the presence of the microsomal S-9 fraction and of hydroxyurea the effect of HT-2 toxin (at 100 μg/ml) appeared to resemble that of benzo(a)pyrene: further studies are needed to clarify the role of its derivatives in the mechanism of action of T-2 toxin.

T-2 mycotoxin, given to mice 4 days prior to an intraperitoneal inoculation with Listeria monocytogenes EGD, increases the acute phase response as determined by measurements of serum amyloid protein-P (SAP), and decreases the severity of the infection. Conversely, when T-2 toxin is given simultaneously with L. monocytogenes the mice become more susceptible to the infection, and the SAP levels attained are diminished relative to the non-toxin-treated Listeria-infected controls. T-2 toxin given 4 days prior to intraperitoneal inoculation with Salmonella typhimurium had no effect on either the resultant infection or SAP levels. These results indicate that T-2 toxin modulates the acute phase response to infection, and are consistent with an in vivo role for SAP as a nonspecific host resistance factor.

White weanling rats were given intragastrically 4 doses of T-2 toxin (3 alpha-hydroxy-4 beta-1,5-diacetoxy-8 alpha-(3-methylbutyryloxy)-12,13-epoxytrichothec-9-ene) a secondary metabolite of certain Fusarium species, 6 out of 9 treated rats developed increased blood pressure; these animals showed microscopically severe cardiovascular abnormalities. Occasional contamination of foodstuffs wh Fusarium may contribute to hypertension and cardiovascular lesions in animals and man.

The capacity of the trichothecene T-2 toxin to alter resistance to bacterial lipopolysaccharide (LPS) was examined in the mouse. Both LPS-susceptible (C3H/HeN) and LPS-resistant (C3H/HeJ) mouse strains exhibited markedly enhanced mortality when a single oral dose of T-2 toxin (1 mg/kg) was coadministered with a subacute i.p. dose of Salmonella typhimurium LPS. In the absence of LPS, T-2 toxin did not cause lethal effects when administered at this level. LD50 values for LPS decreased by 14-fold and 4.5-fold upon co-administration with T-2 toxin (1 mg/kg) in C3H/HeN and C3H/HeJ mice, respectively. Increased mortality was accompanied by an impaired splenic response to LPS in C3H/HeN mice. C3H/HeN mice pretreated with a sublethal dose of LPS 24 h prior to T-2 toxin administration also exhibited significantly increased susceptibility to T-2 toxin. Histopathological assessment revealed that the liver and spleen of mice exposed to T-2 toxin and LPS exhibited extensive cell death as compared to control mice treated with T-2 toxin or LPS only. The results suggest that bacterial LPS and trichothecenes such as T-2 toxin interact synergistically. This interaction may contribute to increased mortality that has been observed previously in animals challenged with Salmonella and T-2 toxin.

Deoxynivalenol (DON), 3-acetyldeoxynivalenol (acetylDON) and zearalenone (Ze) were examined for their in vitro effect on mitogen-induced lymphocyte blastogenesis using rat or human peripheral blood lymphocytes (PBL). A dose-dependent reduction of lymphocyte proliferation was demonstrated for each mycotoxin. However, the inhibitory effect of DON was significantly higher than that of the acetylated compound. Concentrations of 90 ng/ml and 220 ng/ml inhibited rat and human lymphocyte blastogenesis by 50%, respectively, whereas 450 ng/ml and 1060 ng/ml acetylDON were required to produce the same effect. The amount of Ze necessary to inhibit blastogenesis by 50% was 250 times greater than required for DON. There was no evidence of cell death and combinations of DON and Ze did not alter the expected response.

Trichothecenes are mycotoxins which produce lethal toxicosis in humans and animals, yet no adequate therapeutic regimen has been developed. This study provides evidence that the selective platelet activating factor (PAF) antagonist, BN 52021 (5–15 mg kg i.v.) can prolong the survival of conscious rats exposed to a highly lethal T-2 toxicosis. These data also suggest that PAF is an important mediator of this unique toxicosis.