Gut satiety hormones cholecystokinin and glucagon-like Peptide-17-36 amide mediate anorexia induction by trichothecenes T-2 toxin, HT-2 toxin, diacetoxyscirpenol and neosolaniol.

The food-borne trichothecene mycotoxins have been documented to cause human and animal food poisoning. Anorexia is a hallmark of the trichothecene mycotoxins-induced adverse effects. Type B trichothecenes have been previously demonstrated to elicit robust anorectic responses, and this response has been directly linked to secretion of the gut satiety hormones cholecystokinin (CCK) and glucagon-like peptide-17-36 amide (GLP-1). However, less is known about the anorectic effects and underlying mechanisms of the type A trichothecenes, including T-2 toxin (T-2), HT-2 toxin (HT-2), diacetoxyscirpenol (DAS), neosolaniol (NEO). The purpose of this study was to relate type A trichothecenes T-2, HT-2, DAS and NEO-induced anorectic response to changes plasma concentrations of CCK and GLP-1. Following both oral gavage and intraperitoneal (IP) administration of 1mg/kg bw T-2, HT-2, DAS and NEO evoked robust anorectic response and secretion of CCK and GLP-1. Elevations of plasma CCK markedly corresponded to anorexia induction by T-2, HT-2, DAS and NEO. Following oral exposure, plasma CCK was peaked at 6h, 6h, 2h, 2h and lasted up to 24h, 24h, > 6h, > 6h for T-2, HT-2, DAS and NEO, respectively. IP exposed to four toxins all induced elevation of CCK with peak point and duration at 6h and >24h, respectively. In contrast to CCK, GLP-1 was moderately elevated by these toxins. Following both oral and IP exposure, T-2 and HT-2 evoked plasma GLP-1 elevation with peak point and duration at 2h and 6h, respectively. Plasma GLP-1 was peaked at 2h and still increased at 6h for IP and oral administration with DAS and NEO, respectively. In conclusion, CCK plays a contributory role in anorexia induction but GLP-1 might play a lesser role in this response.

Potential Role of Individual and Combined Effects of T-2 Toxin, HT-2 Toxin and Neosolaniol on the Apoptosis of Porcine Leydig Cells.

T-2 toxin usually co-occurs with HT-2 toxin and neosolaniol (NEO) in the grains and feed. Our previous studies found that T-2 toxin and its metabolites' binary or ternary combination exposure to porcine Leydig cells (LCs) displayed synergism in certain range of dosage and cannot be predicted based on individual toxicity. However, the possible mechanism of these mycotoxins' combined exposure to cell lesions remains unknown. Based on 50% cell viability, the mechanism of apoptosis in porcine Leydig cells was investigated after exposure to T-2, HT-2, NEO individual and binary or ternary combinations. Compared with control, the adenosine triphosphate (ATP) content decreased, reactive oxygen species (ROS) level increased, and mitochondrial membrane potential (MMP) decreased in all treated groups. Additionally, the cell apoptosis rates were significantly increased in test groups (p < 0.05), and the B-cell lymphoma 2 (Bcl-2) Associated X (Bax)/Bcl-2 ratio and the expression of caspase 3, caspase 8, cytochrome c (Cytc) in the treated group are all significantly higher than the control group. Moreover, the expression of Cytc and caspase 8 gene in NEO and T-2+NEO groups was significantly higher than that in other individual and combined groups. It can be concluded that the toxicities of T-2, HT-2, and NEO individually and in combination can induce apoptosis related to the oxidative stress and mitochondrial damage, and the synergistic effect between toxins may be greater than a single toxin effect, which is beneficial for assessing the possible risk of the co-occurrences in foodstuffs to human and animal health.

Fusarium chaquense, sp. nov, a novel type A trichothecene-producing species from native grasses in a wetland ecosystem in Argentina.

The Chaco wetland is among the most biologically diverse regions in Argentina. In collections of fungi from asymptomatic native grasses (Poaceae) from the wetlands, we identified isolates of Fusarium that were morphologically similar to F. armeniacum, but distinct from it by their production of abundant microconidia. All the isolates had identical, or nearly identical, partial sequences of TEF1 and RPB2. But they were distinct from reference sequences from F. armeniacum and Fusarium species closely related to it. Phylogenetic analysis of 34 full-length housekeeping gene sequences retrieved from whole genome sequences of three Chaco wetland isolates, 29 genes resolved the isolates as an exclusive clade within the F. sambucinum species complex. Based on results of the morphological and phylogenetic analysis, we concluded that the Chaco wetland isolates are a distinct and novel species, herein described as Fusarium chaquense, sp. nov., which is closely related to F. armeniacum. F. chaquense in culture can produce the trichothecenes T-2 and HT-2 toxin, neosolaniol, diacetoxyscirpenol, and monoacetoxyscirpenol, as well as beauvericin and the pigment aurofusarin. Genome sequence analysis also revealed the presence of three previously described loci required for trichothecene biosynthesis. This research represents the first study of Fusarium in a natural ecosystem in Argentina.

Free and conjugated Alternaria and Fusarium mycotoxins during Pilsner malt production and double-mash brewing.

Malting and brewing have previously been demonstrated to be risky procedures in terms of mycotoxins contamination. The goal of the study was to describe the fate of less investigated Fusarium and Alternaria mycotoxins, together with their conjugates, during these processes. The Pilsner malt producing process, together with double-mash brewing, were performed in a pilot-scale malting and brewery plants to simulate production of lager - the most popular type of central European beer. In addition, changes in temperature during barley germination were investigated to assess the influence of this critical step. QuEChERS-like extraction followed by UHPLC-HRMS/MS were utilized to quantify the mass balance of 13 mycotoxins and four of their conjugates. The results confirmed germination as the most determining malting step, followed by mashing of malt during brewing. Occurrence of type A trichothecenes, Alternaria mycotoxins and their conjugates in the final beer product indicates the need to take mitigation measures.

The trichothecene neosolaniol stimulates an emetic response through neuropeptide Y2 and serotonin 3 receptors in mink.

Type A trichothecene neosolaniol (NEO) is considered a potential risk to human and animal health by the European Food Safety Authority (EFSA). To date, available data do not allow making conclusions about the toxicological properties of this toxin. Trichothecenes have been previously demonstrated to induce emetic responses in mink, and this response has been associated with neurotransmitter peptide YY (PYY) and serotonin (5-hydroxytryptamine, 5-HT). The goal of this study was to compare emetic effects of NEO administered by intraperitoneal and oral routes and relate these effects to PYY and 5-HT. The effective doses resulting in emetic events in 50% of the animals following intraperitoneal and oral exposure to NEO were 0.4 and 0.09 mg/kg bw, respectively. This emetic response corresponded to elevated PYY and 5-HT levels. Blocking the neuropeptide Y2 receptor diminished emesis induction by PYY and NEO. The 5-HT3 receptor inhibitor granisetron completely restrained the induction of emesis by 5-HT and NEO. To summarize, our findings demonstrate that PYY and 5-HT play important roles in the NEO-induced emetic response.

Comparison of Anorectic Potencies of Type A Trichothecenes T-2 Toxin, HT-2 Toxin, Diacetoxyscirpenol, and Neosolaniol.

Trichothecene mycotoxins are common contaminants in cereal grains and negatively impact human and animal health. Although anorexia is a common hallmark of type B trichothecenes-induced toxicity, less is known about the anorectic potencies of type A trichothecenes. The purpose of this study was to compare the anorectic potencies of four type A trichothecenes (T-2 toxin (T-2), HT-2 toxin (HT-2), diacetoxyscirpenol (DAS), and neosolaniol (NEO)) in mice. Following oral exposure to T-2, HT-2, DAS, and NEO, the no observed adverse effect levels (NOAELs) and lowest observed adverse effect levels (LOAELs) were 0.01, 0.01, 0.1, and 0.01 mg/kg body weight (BW), and 0.1, 0.1, 0.5, and 0.1 mg/kg BW, respectively. Following intraperitoneal (IP) exposure to T-2, HT-2, DAS, and NEO, the NOAELs were 0.01 mg/kg BW, except for DAS (less than 0.01 mg/kg BW), and the LOAELs were 0.1, 0.1, 0.01, and 0.1 mg/kg BW, respectively. Taken together, the results suggest that (1) type A trichothecenes could dose-dependently elicit anorectic responses following both oral gavage and IP exposure in mice; (2) the anorectic responses follow an approximate rank order of T-2 = HT-2 = NEO > DAS for oral exposure, and DAS > T-2 = HT-2 = NEO for IP administration; (3) IP exposure to T-2, HT-2, DAS, and NEO evoked stronger anorectic effects than oral exposure. From a public health perspective, comparative anorectic potency data should be useful for establishing toxic equivalency factors for type A trichothecenes.

First study on trichothecene and zearalenone exposure of the Romanian population through wheat-based products consumption.

In this study, a dietary exposure assessment of mycotoxins was conducted for the Romanian population using the contamination data of a various categories of wheat-based products for direct human consumption. Wheat-based foods (n = 181) commercialized in Romania, including flour, bread, biscuits, breakfast cereals and pasta, were evaluated by GC-QqQ-MS/MS for the occurrence of deoxynivalenol (DON), 3-acetyldeoxynivalenol (3AcDON), 15-acetyldeoxynivalenol (15AcDON), fusarenon-X, nivalenol, HT-2 and T-2 toxins, diacetoxyscirpenol, neosolaniol and zearalenone (ZEA). DON and 15AcDON were detected in 63 and 5% of all the analyzed samples, whereas 13-AcDON, HT-2, T-2, NIV and ZEA were not detected. Exposure of Romanian adult population was assessed, the EDIs for the sum of DON+3AcDON+15AcDON were 669 ng kg-1 bw day-1 at low-bound estimation, and 690 ng kg-1 bw day-1 at upper-bound estimation, being lower than the TDI set (1000 ng kg-1 bw day-1).

Role of Peptide YY3-36 and Glucose-Dependent Insulinotropic Polypeptide in Anorexia Induction by Trichothecences T-2 Toxin, HT-2 Toxin, Diacetoxyscirpenol, and Neosolaniol.

Trichothecences, secondary metabolites produced by Fusarium, are serious health risks to humans and animals worldwide. Although type A trichothecence-induced food refusal has been observed, the mechanism underlying the anorexia caused by these compounds is not fully understood. In this study, we hypothesized that anorexia induced by type A trichothecenes, including T-2 toxin (T-2), HT-2 toxin (HT-2), diacetoxyscirpenol (DAS), and neosolaniol (NEO), in mice corresponds to the changes in the gut satiety hormones peptide YY3-36 (PYY3-36) and glucose-dependent insulinotropic polypeptide (GIP) in plasma. A well-characterized mouse food refusal model was used in this assay. Oral exposure to or intraperitoneal (ip) injection of 1 mg/kg bw T-2, HT-2, DAS, or NEO resulted in dramatically decreased food intake, and PYY3-36 and GIP concentrations were elevated accordingly. Specifically, the PYY3-36 and GIP concentrations peaked at 2 h following oral exposure to these 4 toxins individually, although the durations were not identical. After ip administration of T-2 or HT-2, PYY3-36 significantly increased within 6 h. However, no significant difference was found in the DAS and NEO groups. The GIP levels peaked within 2, 2, 0.5, and 0.5 h, respectively, and remained increased up to 6, 6, 2, and 6 h, respectively, following T-2, HT-2, DAS, or NEO ip exposure. The increase in GIP was greater than that of PYY3-36 after exposure to the 4 toxins using 2 administration routes. Together, these findings suggest that PYY3-36 and GIP play a role in T-2-, HT-2-, DAS-, and NEO-induced anorexia.

A method of analysis for T-2 toxin and neosolaniol by UPLC-MS/MS in apple fruit inoculated with Trichothecium roseum.

Trichothecenes are one of the most important groups of mycotoxins produced by Trichothecium roseum, which causes core rot of apple. A reliable and sensitive method was developed and successfully applied for the rapid detection of trichothecenes including T-2 toxin and neosolaniol in harvested apple using UPLC-MS/MS. After the extraction of the two mycotoxins from the apple matrix with methanol/water (80/20, v/v), the concentrated extracts were cleaned-up by PriboFast M270 columns and then analysed by UPLC-MS/MS. T-2 toxin and neosolaniol were effectively separated as unique peaks. The validity of this method was established by its linearity (R(2) ≥ 0.9995), precision (relative standard deviation ≤ 3.6%), accuracy, selectivity, limit of detection of 2-5 µg kg(-1), limit of quantification of 5-10 µg kg(-1) and average recovery of 73-96%. Levels of T-2 toxin were found in the range 7.1-128.4 µg kg(-1) in the core rot lesion of three cultivars apple (cvs. Red Delicious, Fuji and Ralls). T-2 was detected not only in the lesion, but also in the tissue without any disease symptoms. However, neosolaniol was only detected in the lesion on 'Red Delicious' apples. In addition, the concentration of T-2 toxin in the susceptible cultivar (cv. Fuji) was significantly higher than that in the resistant one (cv. Ralls). This method proved to be suitable at detecting T-2 and neosolaniol simultaneously in apples infected with T. roseum.

Study of the natural occurrence of T-2 and HT-2 toxins and their glucosyl derivatives from field barley to malt by high-resolution Orbitrap mass spectrometry.

This paper reports a new method for the determination of T-2 and HT-2 toxins and their glucosylated derivatives in cereals, and some survey data aimed at obtaining more comprehensive information on the co-occurrence of T-2 and HT-2 toxins and their glucosylated derivatives in naturally contaminated cereal samples. For these purposes, barley samples originating from a Northern Italian area were analysed by LC-HRMS for the presence of T-2, HT-2 and relevant glucosyl derivatives. Quantitative analysis of T-2 and HT-2 glucosides was performed for the first time using a recently made available standard of T-2 glucoside. The glucosyl derivative of HT-2 was detected at levels up to 163 µg kg(-1) in 17 of the 18 analysed unprocessed barley grains, whereas the monoglucosyl derivative of T-2 toxin was detected in only a few samples and at low µg kg(-1) levels. The ratio between glucosylated toxins (sum of T-2 and HT-2 glucosides) and native toxins (sum of T-2 and HT-2) ranged from 2% to 283%. Moreover, taking advantage of the possibility of retrospective analysis of full-scan HRMS chromatograms, samples were also screened for the presence of other type-A trichothecenes, namely neosolaniol, diacetoxyscirpenol and their monoglucosyl derivatives, which were detected at trace levels. A subset of nine different samples was subjected to micro-maltation in order to carry out a preliminary investigation on the fate of T-2, HT-2 and relevant glucosides along the malting process. Mycotoxin reduction from cleaned barley to malt was observed at rates ranging from 4% to 87%.

Bottled water: analysis of mycotoxins by LC-MS/MS.

The presence of mycotoxins in food samples has been widely studied as well as its impact in human health, however, information about its distribution in the environment is scarce. An analytical method comprising a solid phase extraction procedure followed by liquid chromatography tandem mass spectrometry analysis was implemented and validated for the trace analysis of mycotoxins in drinking bottled waters. Limits of quantification achieved for the method were between 0.2ngL(-1) for aflatoxins and ochratoxin, and 2.0ngL(-1) for fumonisins and neosolaniol. The method was applied to real samples. Aflatoxin B2 was the most frequently detected mycotoxin in water samples, with a maximum concentration of 0.48±0.05ngL(-1) followed by aflatoxin B1, aflatoxin G1 and ochratoxin A. The genera Cladosporium, Fusarium and Penicillium were the fungi more frequently detected. These results show that the consumption of these waters does not represent a toxicological risk for an adult.

Effect of cultivars, Fusarium strains and storage temperature on trichothecenes production in inoculated potato tubers.

Four trichothecenes (Fus-X, 3ADON, DAS and T-2) were detected in potato tubers inoculated with Fusarium spp. by UPLC-MS/MS. The influence of cultivars, Fusarium strains and storage temperature on trichothecenes production was evaluated. The concentration of trichothecenecs was much higher in susceptible cultivar (Longshu No. 3) than in resistant one (Longshu No. 6). The susceptible cultivar infected with Fusarium sulphureum had the maximum concentration of Fus-X, 3ADON and DAS. Among the three Fusarium strains, Fusarium solani had the strongest ability to produce T-2 in both susceptible and resistant cultivars. Room temperature storage was more likely to accumulate trichothecenes than low temperature storage. Meanwhile, the trichothecenes were found not only in the lesion but also in the adjacent asymptomatic tissue. Trichothecenes concentration showed a strong trend of decline with increase in distance from the infection point.

T-2 toxin is hydroxylated by chicken CYP3A37.

T-2 toxin (T-2) is an acute toxic trichothecene mycotoxin produced mainly by Fusarium species, detected in many crops including oats, wheat and barley, in animal feed and food. It is important to know the metabolic pathway and kinetics of T-2 in food animals given that T-2 can cause serious adverse effects on human health. In this study, we investigated the metabolic capacity of chicken CYP3A37 in the metabolism of T-2 using reconstituted bacteria produced enzymes. Our results showed that chicken CYP3A37 is able to convert T-2 to 3'-OH T-2 with an apparent Km of 15.29 µM, and T-2 hydroxylation activity of CYP3A37 is strongly inhibited by ketoconazole (IC50=0.11 µM). We also observed that chicken CYP3A37 can catalyze erythromycin N-demethylation, another CYP3A-specific activity. These findings imply that chicken CYP3A37 may have a broad substrate spectrum, similar to its human homologue CYP3A4.