Toxic Trichothecene Research Articles

The identification of a key gene highlights macrocyclic ring’s role in trichothecene toxicity

Trichothecenes are toxins produced by certain species from several fungal genera, including Aspergillus, Fusarium, Isaria, Paramyrothecium, Stachybotrys, Trichoderma, and Trichothecium. These toxins are of interest because they contribute to the toxigenicity, plant pathogenicity, and/or biological control activities of some fungi. All trichothecenes have the same core (12,13-epoxytrichothec-9-ene or EPT) structure but can differ from one another by the presence or absence of a macrocyclic ring formed from polyketide and isoprenoid substituents esterified to carbon atoms 4 and 15 of EPT, respectively. Genes required for formation and some modifications of EPT have been elucidated, but almost nothing is known about genes specific to the formation of the macrocyclic ring. Therefore, we used genomic, transcriptomic, metabolomic, and gene deletion analyses to identify genes that are required specifically for the formation of the macrocyclic ring. These analyses identified one gene, TRI24, that is predicted to encode an acyltransferase and that is required for macrocyclic ring formation during biosynthesis of macrocyclic trichothecenes by the fungus Paramyrothecium roridum. In addition, a TRI24 deletion mutant of P. roridum caused less severe disease symptoms on common bean and had less antifungal activity than its wild-type progenitor strain. We propose that the reduced aggressiveness and antifungal activity of the mutant resulted from its inability to produce trichothecenes with a macrocyclic ring. To our knowledge, this is the first report of a gene required specifically for the formation of the macrocyclic ring of trichothecenes and that loss of the macrocyclic ring of trichothecenes can alter the biological activities of a fungus.Key points• TRI24 gene is found in all known macrocyclic trichothecene-producing fungi.• A tri24-deletion mutant exhibits a reduction in antifungal and plant disease activities.• TRI24 is the first described gene specific to macrocyclic trichothecene biosynthesis.Graphical abstract

Human next-generation risk assessment of trichothecene toxicity

Trichothecenes are naturally occurring chemicals, produced by fungi, that can be found in contaminated crops. Trichothecenes have the potential to indirectly damage DNA and exacerbate genotoxic effects of genotoxicants. However, genotoxicity data for most trichothecenes are limited and data gaps remain. Here we use the γH2AX/pH3 assay to evaluate DNA damage in vitro of 13 trichothecenes. Three human cell lines (SH-SY5Y, ACHN, and HepG2) were exposed to each trichothecene (0.001–100 μM) to assess toxicity as models for the brain, kidney, and liver, respectively. Concentration-dependent induction of DNA damage, illustrated by γH2AX induction, was observed for all trichothecenes. In vitro-in vivo extrapolation (IVIVE) modeling was employed to support in vivo equivalent potency ranking and screen for risk potential. Diacetoxyscirpenol, T-2, and HT-2 had the highest genotoxic potency, notably in SH-SY5Y cells. Administered equivalent doses (AEDs) derived from IVIVE were compared against exposure data from French total diet studies to assess risk potential. AEDs derived for T-2 and HT-2 from the SH-SY5Y model were within 100-fold of exposure levels for infants aged one year or less. Overall, the potential for trichothecenes to damage DNA and higher exposures in infants highlights the need to investigate the cumulative effects across the broader trichothecene family.

A Role in 15-Deacetylcalonectrin Acetylation in the Non-Enzymatic Cyclization of an Earlier Bicyclic Intermediate in Fusarium Trichothecene Biosynthesis.

The trichothecene biosynthesis in Fusarium begins with the cyclization of farnesyl pyrophosphate to trichodiene, followed by subsequent oxygenation to isotrichotriol. This initial bicyclic intermediate is further cyclized to isotrichodermol (ITDmol), a tricyclic precursor with a toxic trichothecene skeleton. Although the first cyclization and subsequent oxygenation are catalyzed by enzymes encoded by Tri5 and Tri4, the second cyclization occurs non-enzymatically. Following ITDmol formation, the enzymes encoded by Tri101, Tri11, Tri3, and Tri1 catalyze 3-O-acetylation, 15-hydroxylation, 15-O-acetylation, and A-ring oxygenation, respectively. In this study, we extensively analyzed the metabolites of the corresponding pathway-blocked mutants of Fusarium graminearum. The disruption of these Tri genes, except Tri3, led to the accumulation of tricyclic trichothecenes as the main products: ITDmol due to Tri101 disruption; a mixture of isotrichodermin (ITD), 7-hydroxyisotrichodermin (7-HIT), and 8-hydroxyisotrichodermin (8-HIT) due to Tri11 disruption; and a mixture of calonectrin and 3-deacetylcalonectrin due to Tri1 disruption. However, the ΔFgtri3 mutant accumulated substantial amounts of bicyclic metabolites, isotrichotriol and trichotriol, in addition to tricyclic 15-deacetylcalonectrin (15-deCAL). The ΔFgtri5ΔFgtri3 double gene disruptant transformed ITD into 7-HIT, 8-HIT, and 15-deCAL. The deletion of FgTri3 and overexpression of Tri6 and Tri10 trichothecene regulatory genes did not result in the accumulation of 15-deCAL in the transgenic strain. Thus, the absence of Tri3p and/or the presence of a small amount of 15-deCAL adversely affected the non-enzymatic second cyclization and C-15 hydroxylation steps.

Tannic Acid Ameliorates Systemic Glucose and Lipid Metabolic Impairment Induced by Low-Dose T-2 Toxin Exposure.

Subacute mycotoxin exposure in food is commonly overlooked. As one of the most toxic trichothecene mycotoxins, the T-2 toxin severely pollutes human foods and animal feeds. In our study, we investigated the effects of low-dose T-2 toxin on glucose and lipid metabolic function and further investigated the protective effect of tannic acid (TA) in C57BL/6J mice. Results showed that low-dose T-2 toxin significantly impaired blood glucose and lipid homeostasis, promoted ferroptosis in the pancreas and subsequent repression of insulin secretion in β-cells, and impacted hepatic glucose and lipid metabolism by targeted inhibition of the insulin receptor substrate (IRS)/phosphatidylin-ositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway, which induced insulin resistance and steatosis in the liver. TA treatment attenuated pancreatic function and hepatic metabolism by ameliorating oxidative stress and insulin resistance in mice. These findings provide new perspectives on the toxic mechanism and intervention of chronic subacute toxicity of foodborne mycotoxins.

NRF2/PGC-1α-mediated mitochondrial biogenesis contributes to T-2 toxin-induced toxicity in human neuroblastoma SH-SY5Y cells

The T-2 toxin is a highly toxic trichothecene mycotoxin that would cause serious toxicity in humans and animals. Recent studies suggest that the central nervous system (CNS) is susceptible to T-2 toxin, which can easily cross the blood-brain barrier, accumulate in brain tissues, and cause neurotoxicity. The growing evidence indicates that oxidative damage and mitochondrial dysfunction play a critical role in T-2 toxin-induced neurotoxicity, but the mechanisms are still poorly understood. Our present study showed that T-2 toxin decreased cell viability and increased lactate dehydrogenase leakage in human neuroblastoma SH-SY5Y cells in a concentration- and time-dependent manner. T-2 toxin elicited prominent oxidative stress and mitochondrial dysfunction, as evidenced by the promotion of cellular reactive oxygen species generation, disruption of the mitochondrial membrane potential, depletion of glutathione and reduction of the cellular ATP content. T-2 toxin impaired mitochondrial biogenesis, including decreased mitochondrial DNA copy number and affected the nuclear factor erythroid 2 related factor 2 (NRF2) / peroxisome proliferator-activated receptor γ coactivator 1 alpha (PGC-1α) pathway by upregulating NRF2 mRNA and protein expression while inhibiting the expression of PGC-1α, nuclear respiratory factor (NRF1) and mitochondrial transcription factor A (TFAM). NRF2 knockdown was found to significantly exacerbate T-2 toxin-induced cytotoxicity, oxidative stress, and mitochondrial dysfunction, as well as aggravate mitochondrial biogenesis impairment. NRF2 knockdown compromised T-2 toxin-induced upregulation of NRF2, but augmented the inhibition of PGC-1α, NRF1, and TFAM by T-2 toxin. Taken together, these findings suggest that T-2 toxin-induced oxidative stress and mitochondrial dysfunction in SH-SY5Y cells, at least in part by, NRF2/PGC-1α pathway-mediated mitochondrial biogenesis.

Involvement of endoplasmic reticulum stress-activated PERK-eIF2α-ATF4 signaling pathway in T-2 toxin-induced apoptosis of porcine renal epithelial cells

T-2 toxin is a highly toxic trichothecene that can induce toxic effects in a variety of organs and tissues, but the pathogenesis of its nephrotoxicity has not been elucidated. In this study, we assessed the involvement of protein kinase RNA-like ER kinase (PERK)-mediated endoplasmic reticulum (ER) stress and apoptosis in PK-15 cells cultured at different concentrations of T-2 toxin. Cell viability, antioxidant capacity, intracellular calcium (Ca2+) content, apoptotic rate, levels of ER stress, and apoptosis-related proteins were studied. T-2 toxin inhibited cell proliferation; increased the apoptosis rate; and was accompanied by increased cleaved caspase-3 expression, altered intracellular oxidative stress marker levels, and intracellular Ca2+ overloading. The ER stress inhibitor 4-phenylbutyrate (4-PBA) and PERK selective inhibitor GSK2606414 prevented the decrease of cell activity and apoptosis caused by T-2 toxin. The altered expression of glucose regulatory protein 78 (GRP78), C/EBP homologous protein (CHOP), and caspase-12 proved that ER stress was involved in cell injury triggered by T-2 toxin. T-2 toxin activated the phosphorylation of PERK and the alpha subunit of eukaryotic initiation factor 2 (eIF2α) and upregulated the activating transcription factor 4 (ATF4), thereby triggering ER stress via the GRP78/PERK/CHOP signaling pathway. This study provides a new perspective for understanding the nephrotoxicity of T-2 toxin.

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.

An update on T-2 toxin and its modified forms: metabolism, immunotoxicity mechanism, and human exposure assessment.

T-2 toxin is the most toxic trichothecene mycotoxin, and it exerts potent toxic effects, including immunotoxicity, neurotoxicity, and reproductive toxicity. Recently, several novel metabolites, including 3',4'-dihydroxy-T-2 toxin and 4',4'-dihydroxy-T-2 toxin, have been uncovered. The enzymes CYP3A4 and carboxylesterase contribute to T-2 toxin metabolism, with 3'-hydroxy-T-2 toxin and HT-2 toxin as the corresponding primary products. Modified forms of T-2 toxin, including T-2-3-glucoside, exert their immunotoxic effects by signaling through JAK/STAT but not MAPK. T-2-3-glucoside results from hydrolyzation of the corresponding parent mycotoxin and other metabolites by the intestinal microbiota, which leads to enhanced toxicity. Increasing evidence has shown that autophagy, hypoxia-inducible factors, and exosomes are involved in T-2 toxin-induced immunotoxicity. Autophagy promotes the immunosuppression induced by T-2 toxin, and a complex crosstalk between apoptosis and autophagy exists. Very recently, "immune evasion" activity was reported to be associated with this toxin; this activity is initiated inside cells and allows pathogens to escape the host immune response. Moreover, T-2 toxin has the potential to trigger hypoxia in cells, which is related to activation of hypoxia-inducible factor and the release of exosomes, leading to immunotoxicity. Based on the data from a series of human exposure studies, free T-2 toxin, HT-2 toxin, and HT-2-4-glucuronide should be considered human T-2 toxin biomarkers in the urine. The present review focuses on novel findings related to the metabolism, immunotoxicity, and human exposure assessment of T-2 toxin and its modified forms. In particular, the immunotoxicity mechanisms of T-2 toxin and the toxicity mechanism of its modified form, as well as human T-2 toxin biomarkers, are discussed. This work will contribute to an improved understanding of the immunotoxicity mechanism of T-2 toxin and its modified forms.

The Fusarium graminearum t-SNARE Sso2 Is Involved in Growth, Defense, and DON Accumulation and Virulence.

The plant-pathogenic fungus Fusarium graminearum, causal agent of Fusarium head blight (FHB) disease on small grain cereals, produces toxic trichothecenes that require facilitated export for full virulence. Two potential modes of mycotoxin transport are membrane-bound transporters, which move toxins across cellular membranes, and N-ethylmaleimide-sensitive factor attachment receptor (SNARE)-mediated vesicular transport, by which toxins may be packaged as cargo in vesicles bound for organelles or the plasma membrane. In this study, we show that deletion of a gene (Sso2) for a subapically localized t-SNARE protein results in growth alteration, increased sensitivity to xenobiotics, altered gene expression profiles, and reduced deoxynivalenol (DON) accumulation in vitro and in planta as well as reduced FHB symptoms on wheat. A double deletion mutant generated by crossing the ∆sso2 deletion mutant with an ATP-binding cassette transporter deletion mutant (∆abc1) resulted in an additive reduction in DON accumulation and almost complete loss of FHB symptoms in planta. These results suggest an important role of Sso2-mediated subapical exocytosis in FHB progression and xenobiotic defense and are the first report of an additive reduction in F. graminearum DON accumulation upon deletion of two distinct modes of cellular export. This research provides useful information which may aid in formulating novel management plans of FHB or other destructive plant diseases.

ROS: Trichothecenes’ handy weapon?

Trichothecenes are a class of toxic secondary metabolites produced by filamentous fungi such as Fusarium, which pose a threat to human and animal health. Among them, T-2 toxin and deoxynivalenol (DON) have attracted the most attention because of their extensive pollution and strong toxicity. Accumulated studies have found that an increase of reactive oxygen species (ROS) during oxidative stress produces a wide range of secondary consequences by modifying cell signaling proteins, such as DNA RNA damage, inflammatory damage, cell cycle arrest, and apoptosis. Although there has been a comprehensive review of antioxidant agents against trichothecenes, there is no systematic summary of the mechanism of trichothecenes inducing ROS production and then regulating downstream signaling molecules of cells to produce the corresponding effects. Therefore, from the perspective of ROS regulation of downstream signaling pathways, this review mainly explores the mechanisms by which ROS, as trichothecenes’ handy weapon, affect an organism. It may provide more ideas and new targets for studies of antagonizing the toxicity of trichothecenes from the aspect of antioxidation.

ОПТИМИЗАЦИЯ ПРОТОКОЛА ИММУНОФЕРМЕНТНОГО АНАЛИЗА ДЛЯ ИНДИКАЦИИ Т-2 ТОКСИНА

T-2 toxin is one of the most common and toxic trichothecene mycotoxins – secondary metabolites of molds that develop on cereals and some other crops. This article discusses the development stage of a test system for the determination of T-2 toxin based on enzyme-linked immunosorbent assay (ELISA), which uses an enzyme label – a conjugate of anti-rabbit goat antibodies with peroxidase. An important step in the creation of any ELISA-based test system is the preliminary titration of reaction components. The aim of the work was to determine the optimal concentration of the conjugate of antispecies antibodies in an indirectly competitive enzyme-linked immunosorbent enzyme immunoassay with the indication of T-2 toxin. A number of dilutions of the antivirus conjugate were examined: 1 : 1000; 1 : 2000; 1 : 3000; 1 : 4000; 1 : 5000; 1 : 7500; 1 : 10000; 1 : 12 500; 1 : 15,000; 1 : 17,500; 1 : 20,000; 1 : 30 000. In the ELISA staging protocol, calibration solutions of T-2 toxin at concentrations of 0.0 were used; 2.5; 5, 10, 20, 40, 80, 160 ng/ml and specific polyclonal rabbit antibodies to the T-2-BSA conjugate. Based on the average optical density values, calibration plots were constructed using the percentage of signal absorption from the zero standard. When evaluating the results of the study, the criterion for choosing the dilution of the conjugate of anti-species antibodies was considered the greatest, at which the best linearity of the grading plot is achieved and the level of its non-specific reaction with the zero standard would be the lowest. It was established that the optimal variants of dilutions of the conjugate of anti-species antibodies under the same experimental conditions tested were 1: 4000, 1 : 5000 and 1 : 12 500. Dose-dependent signal absorption was observed in all concentrations of anti-species antibodies. Dilutions of the conjugate of anti-species antibodies 1 : 1000–1 : 3000 and 1 : 17 500–1 : 30 000 were not taken into account, since the optical density of most wells was higher than the optimal boundaries in the first case (> 3.9) and lower in the second (< 0.4). Based on the foregoing, optimal dilution of the conjugate of anti-species antibodies was selected 1 : 12 500.

T-2 toxin cytotoxicity mediated by directly perturbing mitochondria in human gastric epithelium GES-1 cells.

T-2 toxin is one of the most toxic trichothecenes and harmful to human health and animal husbandry. The mechanism underlying its growth suppression remains unclear, especially for mitochondrial damage in human gastric epithelial cells. In the present study, we investigated cell death caused by T-2 toxin in a human gastric epithelial cell line (GES-1) and the possible mechanism of T-2-induced cytotoxicity. T-2 strongly reduced the viability of GES-1 cells in a time- and dose-dependent manner within a small range of concentrations. However, when the concentrations of T-2 were >40 nM, there was no concentration dependence, only time dependence. Moreover, T-2 induced apoptosis, with the activation of caspase-3 in GES-1 and mitochondrial membrane potential (MMP) decrease and cytochrome c release. T-2 also resulted in the accumulation of reactive oxygen species (ROS) and DNA damage with a positive signal of p-H2A.X in GES-1 cells. While T-2 caused a MMP decrease, DNA damage and cell death were not blocked by pretreatment with 3 mM glutathione (GSH), a typical scavenger of ROS. The induction of mitochondrial permeability transition pore (mPTP) regulators voltage-dependent anion channel (VDAC1) and cyclophilin D (CypD) were also observed in T-2-treated cells. Interestingly, cyclosporine A (CsA), a CypD inhibitor, significantly reversed the drop in MMP and the DNA damage, as well as ROS accumulation caused by T-2. Additionally, GES-1 cell death could also be protected to some extent by 4, 4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS), an inhibitor of VDAC1, especially the combination of CsA and DIDS, and 3 mM GSH could further enhance the effect of CsA + DIDS on cell viability. In conclusion, our present findings indicate that the T-2 induced MMP decrease, DNA damage and cell death, as well as ROS accumulation in GES-1 cells, starts with T-2 directly perturbing the mitochondria triggering ROS generation by acting on CypD and VDAC1. This study presents a new viewpoint for evaluating the toxicity of T-2 toxin.

Reduced Toxicity of Trichothecenes, Isotrichodermol, and Deoxynivalenol, by Transgenic Expression of the Tri101 3-O-Acetyltransferase Gene in Cultured Mammalian FM3A Cells.

In trichothecene-producing fusaria, isotrichodermol (ITDol) is the first intermediate with a trichothecene skeleton. In the biosynthetic pathway of trichothecene, a 3-O-acetyltransferase, encoded by Tri101, acetylates ITDol to a less-toxic intermediate, isotrichodermin (ITD). Although trichothecene resistance has been conferred to microbes and plants transformed with Tri101, there are no reports of resistance in cultured mammalian cells. In this study, we found that a 3-O-acetyl group of trichothecenes is liable to hydrolysis by esterases in fetal bovine serum and FM3A cells. We transfected the cells with Tri101 under the control of the MMTV-LTR promoter and obtained a cell line G3 with the highest level of C-3 acetylase activity. While the wild-type FM3A cells hardly grew in the medium containing 0.40 μM ITDol, many G3 cells survived at this concentration. The IC50 values of ITDol and ITD in G3 cells were 1.0 and 9.6 μM, respectively, which were higher than the values of 0.23 and 3.0 μM in the wild-type FM3A cells. A similar, but more modest, tendency was observed in deoxynivalenol and 3-acetyldeoxynivalenol. Our findings indicate that the expression of Tri101 conferred trichothecene resistance in cultured mammalian cells.

Trichothecenes in Cereal Grains - An Update.

Trichothecenes are sesquiterpenoid mycotoxins produced by fungi from the order Hypocreales, including members of the Fusarium genus that infect cereal grain crops. Different trichothecene-producing Fusarium species and strains have different trichothecene chemotypes belonging to the Type A and B class. These fungi cause a disease of small grain cereals, called Fusarium head blight, and their toxins contaminate host tissues. As potent inhibitors of eukaryotic protein synthesis, trichothecenes pose a health risk to human and animal consumers of infected cereal grains. In 2009, Foroud and Eudes published a review of trichothecenes in cereal grains for human consumption. As an update to this review, the work herein provides a comprehensive and multi-disciplinary review of the Fusarium trichothecenes covering topics in chemistry and biochemistry, pathogen biology, trichothecene toxicity, molecular mechanisms of resistance or detoxification, genetics of resistance and breeding strategies to reduce their contamination of wheat and barley.

T-2 toxin neurotoxicity: role of oxidative stress and mitochondrial dysfunction.

Mycotoxins are highly diverse secondary metabolites produced in nature by a wide variety of fungi. Mycotoxins cause animal feed and food contamination, resulting in mycotoxicosis. T-2 toxin is one of the most common and toxic trichothecene mycotoxins. For the last decade, it has garnered considerable attention due to its potent neurotoxicity. Worryingly, T-2 toxin can cross the blood-brain barrier and accumulate in the central nervous system (CNS) to cause neurotoxicity. This review covers the current knowledge base on the molecular mechanisms of T-2 toxin-induced oxidative stress and mitochondrial dysfunction in the CNS. In vitro and animal data have shown that induction of reactive oxygen species (ROS) and oxidative stress plays a critical role during T-2 toxin-induced neurotoxicity. Mitochondrial dysfunction and cascade signaling pathways including p53, MAPK, Akt/mTOR, PKA/CREB and NF-κB contribute to T-2 toxin-induced neuronal cell death. T-2 toxin exposure can also result in perturbations of mitochondrial respiratory chain complex and mitochondrial biogenesis. T-2 toxin exposure decreases the mitochondria unfolded protein response and dampens mitochondrial energy metabolism. Antioxidants such as N-acetylcysteine (NAC), activation of Nrf2/HO-1 and autophagy have been shown to provide a protective effect against these detrimental effects. Clearly, translational research and the discovery of effective treatment strategies are urgently required against this common food-borne threat to human health and livestock.

Functional characterization of a soluble NADPH-cytochrome P450 reductase from Fusarium graminearum

Fusarium head blight is a devastating disease in wheat caused by some fungal pathogens of the Fusarium genus mainly F. graminearum, due to accumulation of toxic trichothecenes. Most of the trichothecene biosynthetic pathway has been mapped, although some proteins of the pathway remain uncharacterized, including an NADPH-cytochrome P450 reductase. We subcloned a F. graminearum cytochrome P450 reductase that might be involved in the trichothecene biosynthesis. It was expressed heterologously in E. coli as N-terminal truncated form with an octahistidine tag for purification. The construct yielded a soluble apoprotein and its incubation with flavins yielded the corresponding monomeric holoprotein. It was characterized for activity in the pH range 5.5–9.5, using thiazolyl blue tetrazolium bromide (MTT) or cytochrome c as substrates. Binding of the small molecule MTT was weaker than for cytochrome c, however, the rate of MTT reduction was faster. Contrary to other studies of cytochrome reductase proteins, MTT reduction proceeded in a cooperative manner in our studies. Optimum kinetic activity was found at pH 7.5–8.5 for bothMTT and cytochrome c. This is the first paper presenting characterization of a cytochrome P450 reductase from F. graminearum which most likely is involved in mycotoxin biosynthesis or some primary metabolic pathway such as sterol biosynthesis in F. graminearum.

Preparation of T-2-glucoronide with Rat Hepatic Microsomes and Its Use along with T-2 for Activation of the JAK/STAT Signaling Pathway in RAW264.7 Cells.

T-2 toxin (T-2), one of the most toxic trichothecene A-type mycotoxins, is biotransformed in animal tissues to modified T-2s (mT-2s) including T-2-glucuronide (T-2-GlcA). In this study, the optimal conditions for T-2-GlcA synthesis were established, and the JAK/STAT pathway in RAW264.7 cells was used to study the toxicity of T-2-GlcA. Because many mT-2 standards are not readily available, optimal conditions for T-2-GlcA synthesis in vitro were established by incubating T-2 with rat liver microsomes, UDPGA, and 0.2% Triton X-100 for 90 min. qRT-PCR and Western blot results showed 21- and 760-fold increases in IL-6 mRNA expression induced by T-2-GlcA and T-2, respectively. Similar differences were observed in JAK3, SOCS2/3, and CIS mRNA expression. T-2-GlcA induced a dose-responsive decrease in STAT1 mRNA expression, whereas the result with T-2 was the opposite. Moreover, the phosphorylation of STAT3 induced by T-2-GlcA was higher than that by T-2, whereas the phosphorylation of STAT1 was to the contrary. Overall, the results show that T-2-GlcA was somewhat toxic, but activation of the JAK/STAT pathway in RAW264.7 was higher by T-2.

Solvent and Water Mediated Structural Variations in Deoxynivalenol and Their Potential Implications on the Disruption of Ribosomal Function.

Fusarium head blight (FHB) is a disease of cereal crops caused by trichothecene producing Fusarium species. Trichothecenes, macrocylicic fungal metabolites composed of three fused rings (A–C) with one epoxide functionality, are a class of mycotoxins known to inhibit protein synthesis in eukaryotic ribosomes. These toxins accumulate in the kernels of infected plants rendering them unsuitable for human and animal consumption. Among the four classes of trichothecenes (A–D) A and B are associated with FHB, where the type B trichothecene deoxynivalenol (DON) is most relevant. While it is known that these toxins inhibit protein synthesis by disrupting peptidyl transferase activity, the exact mechanism of this inhibition is poorly understood. The three-dimensional structures and H-bonding behavior of DON were evaluated using one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy techniques. Comparisons of the NMR structure presented here with the recently reported crystal structure of DON bound in the yeast ribosome reveal insights into the possible toxicity mechanism of this compound. The work described herein identifies a water binding pocket in the core structure of DON, where the 3OH plays an important role in this interaction. These results provide preliminary insights into how substitution at C3 reduces trichothecene toxicity. Further investigations along these lines will provide opportunities to develop trichothecene remediation strategies based on the disruption of water binding interactions with 3OH.

Acetylated Deoxynivalenol Generates Differences of Gene Expression that Discriminate Trichothecene Toxicity.

Deoxynivalenol (DON), which is a toxic secondary metabolite generated by Fusarium species, is synthesized through two separate acetylation pathways. Both acetylation derivatives, 3-acetyl-DON (3ADON) and 15-acetyl-DON (15ADON), also contaminate grain and corn widely. These derivatives are deacetylated via a variety of processes after ingestion, so it has been suggested that they have the same toxicity as DON. However, in the intestinal entry region such as the duodenum, the derivatives might come into contact with intestinal epithelium cells because metabolism by microflora or import into the body has not progressed. Therefore, the differences of toxicity between DON and these derivatives need to be investigated. Here, we observed gene expression changes in the yeast pdr5Δ mutant strain under concentration-dependent mycotoxin exposure conditions. 15ADON exposure induced significant gene expression changes and DON exposure generally had a similar but smaller effect. However, the glucose transporter genes HXT2 and HXT4 showed converse trends. 3ADON also induced a different expression trend in these genes than DON and 15ADON. These differences in gene expression suggest that DON and its derivatives have different effects on cells.

2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) inhibits trichothecene production by Fusarium graminearum through suppression of Tri6 expression

Fusarium head blight (FHB) is a devastating disease of wheat (Triticum aestivum L.) caused by a mycotoxigenic fungus Fusarium graminearum resulting in significantly decreased yields and accumulation of toxic trichothecenes in grains. We tested 7 major secondary metabolites from wheat for their effect on trichothecene production in liquid cultures of F. graminearum producing trichothecene 15-acetyldeoxynivalenol (15-ADON). 2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) benzoxazinoid completely abolished toxin production without any apparent effect on fungal growth. DIMBOA strongly affected the expression of Tri6, encoding a major transcriptional regulator of several genes of the trichothecene biosynthesis pathway. DIMBOA also repressed expression of Tri5, encoding trichodiene synthase, the first enzyme in the trichothecene biosynthesis pathway. Thus, DIMBOA could play an important role against the accumulation of trichothecenes in wheat grain. Breeding or engineering of wheat with increased levels of benzoxazinoids could provide varieties with increased resistance against trichothecene contamination of grain and lower susceptibility to FHB.