Transcriptomic-based bioassays for the detection of type A trichothecenes
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The type A trichothecenes T-2 toxin (T-2) and HT-2 toxin (HT-2) are hazardous Fusarium products that contaminate many field crops growing in cold to temperate regions across the world. Toxicity studies in laboratory and farm animals have been used to derive a temporary tolerable daily intake (t-TDI) for the sum of T-2 and HT-2 of no more than 60 ng/kg body weight. To protect the consumers, it is now necessary to screen a large number of food samples for the presence of these poisonous fungal metabolites. Towards that goal, we discovered that the transcriptional apparatus of a human carcinoma cell line (MCF7) provides a sensitive biological sensor of type A trichothecenes. In fact, exposure of this easy-to-culture cell line to T-2 or HT-2 results in the regulation of >2,000 different transcripts with expression changes ranging from >5,000-fold gene inductions to >40-fold gene repressions. These transcriptional responses have been exploited to develop practical microchip and reverse transcription-polymerase chain reaction (RT-PCR) assays for the detection of type A trichothecenes at parts per billion levels.Trichothecenes, such as deoxynevalenol (DON), nivalenol (NIV) and T-2 toxin are typical immunotoxic mycotoxins. Contamination of trichothecene mycotoxins in wheat is a serious world-wide problem affecting human health. In Japan, in particular, it has been reported that relatively high levels of DON and NIV are frequently found in domestic wheat. I have extensively conducted studies on the toxicity and control of trichothecene mycotoxins in order to assess their risk, and this paper is the summary of those findings.
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The ubiquitous filamentous fungus Fusarium graminearum causes the important disease Fusarium head blight on various species of cereals, leading to contamination of grains with mycotoxins. In a survey of F. graminearum (sensu stricto) on wheat in North America several novel strains were isolated, which produced none of the known trichothecene mycotoxins despite causing normal disease symptoms. In rice cultures, a new trichothecene mycotoxin (named NX-2) was characterized by liquid chromatography-tandem mass spectrometry. Nuclear magnetic resonance measurements identified NX-2 as 3α-acetoxy-7α,15-dihydroxy-12,13-epoxytrichothec-9-ene. Compared with the well-known 3-acetyl-deoxynivalenol (3-ADON), it lacks the keto group at C-8 and hence is a type A trichothecene. Wheat ears inoculated with the isolated strains revealed a 10-fold higher contamination with its deacetylated form, named NX-3, (up to 540 mg kg(-1) ) compared with NX-2. The toxicities of the novel mycotoxins were evaluated utilizing two in vitro translation assays and the alga Chlamydomonas reinhardtii. NX-3 inhibits protein biosynthesis to almost the same extent as the prominent mycotoxin deoxynivalenol, while NX-2 is far less toxic, similar to 3-ADON. Genetic analysis revealed a different TRI1 allele in the N-isolates, which was verified to be responsible for the difference in hydroxylation at C-8.
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The fungal phytopathogen in Fusarium species can cause Fusarium head blight of wheat, barley, oats, and other small cereal grain crops worldwide. Most importantly, these fungi can produce different kinds of mycoxins, and they are harmful to humans and animal health. FAO reported that approximately 25% of the world's grains were contaminated by mycotoxins annually. This chapter will focus on several topics as below: (1) composition of Fusarium graminearum species complex; (2) genotype determination of Fusarium graminearum species complex strains from different hosts and their population structure changes; (3) genetic approaches to genotype determination in type B-trichothecene producing Fusaria fungi; and (4) some newly identified trichothecene mycotoxins, their toxicity, and distribution of the producers.
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Fusarium graminearum is an important pathogen that causes Fusarium head blight (FHB) in several cereal crops worldwide. The potential of this pathogen to contaminate cereals with trichothecene mycotoxins presents a health risk for both humans and animals. This study aimed to evaluate the potential of different trichothecene genotypes of F. graminearum isolated from an alternative host plant to produce mycotoxins under different spring wheat grain incubation conditions. Fourteen F. graminearum strains were isolated from seven alternative host plants and identified as 3-acetyl-deoxynivalenol (3-ADON) and 15-acetyl-deoxynivalenol (15-ADON) genotypes. These strains were cultivated on spring wheat grains at 25 °C and 29 °C for 5 weeks. The mycotoxins produced were analysed with a high-performance liquid chromatograph (HPLC) coupled to a Thermo Scientific TSQ Quantiva MS/MS detector. The obtained results showed that the F. graminearum strains from alternative host plants could produce nivalenol (NIV), deoxynivalenol (DON), fusarenon-X (FUS-X), 3-ADON, deoxynivalenol-3-ß-d-glucoside (D3G), 15-ADON, and zearalenone (ZEA). F. graminearum strains produced DON and ZEA under both temperatures, with the mean concentrations varying from 363 to 112,379 µg kg-1 and from 1452 to 44,816 µg kg-1, respectively. Our results indicated the possible role of dicotyledonous plants, including weeds, as a reservoir of inoculum sources of F. graminearum-induced Fusarium head blight, associated with the risk of mycotoxin contamination in spring wheat.
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Fusarium graminearum (Schwabe) contaminates agricultural crops and commodities with trichothecenes, mostly deoxynivalenol and its acetyl-derivatives. Current techniques available to detect final mycotoxin contamination products usually require an extended time lag between sampling and the corresponding report, and include different clean-up steps and eventually derivatisation. This study was aimed to develop a methodology to detect toxigenic F. graminearum prior to mycotoxin production. Headspace solid-phase microextraction coupled to capillary gas chromatography is shown to be useful to predict the potential of trichothecene mycotoxin formation by detecting the presence of F. graminearum at early stages of fungal growth in wheat cultivars, based on the detection of trichodiene (TRI), the volatile intermediate of trichothecenes. We showed that TRI is a useful marker to detect toxigenic Fusarium in wheat spikes from live plants, regardless of the actual development of Fusarium head blight (FHB). This is the first predictive methodology for FHB and trichothecene occurrence in field-collected samples. It might be a useful tool to help to prevent the risk of mycotoxin contamination.
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Abstract Abstract Trichothecene mycotoxins occur in agricultural commodities and can cause problems from feed refusal to death in animals. This paper describes chromatographic methods for selective analysis for trichothecene mycotoxins. These methods include gas chromatography (GC), thin layer chromatography (TLC), and high pressure liquid chromatography (HPLC). The trichothecene analysis methods by GC and TLC are shown to have a greater sensitivity than in HPLC for the underivatized mycotoxins.
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Fusarium head blight (FHB), or scab, is a destructive disease of small grains caused by members of the Fusarium graminearum species complex, comprised of at least nine distinct, cryptic species. Members of this complex are known to produce mycotoxins including the trichothecenes deoxynivalenol (DON) along with its acetylated derivatives and nivalenol (NIV). In this study, 31 strains, belonging to eight species of this complex and originating from diverse hosts or substrates, were tested for differences in aggressiveness and mycotoxin production. Large variation among strains, both in terms of their aggressiveness and the ability to produce trichothecenes on a susceptible cultivar of wheat was found; variation appears to be a strain-specific rather than species-specific characteristic. While pathogenicity was not influenced by the type of mycotoxin produced, a significant correlation was observed between the amount of the dominant trichothecene (DON and its acetylated forms or NIV) produced by each strain and its level of aggressiveness on wheat. Some isolates also were tested for their ability to infect rice cv. M201, commonly grown in the United States. While tested strains were capable of infecting rice under greenhouse conditions and causing significant amount of disease, no trichothecenes could be detected from the infected rice florets.
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Deoxynivalenol(DON), 3a,7a,15-trihydroxy-12,13-epoxytrichothec-9-en-8-one, is a toxic metabolite such Fusarium spp. as F.graminearuml) and F. culmorum.2)It is commonly found together with nivalenol in wheat and barley grains in Japan,3) and is also one of the most important trichothecene mycotoxins occurring naturally in several cereals and animal feed in other countries.4)As a result of the consumption of these contaminated agricultural products, animals, even man, are suffering from sublethal toxicoses including emesis, diarrhea and feed refusal.5*However, the metabolic fate of the toxin is still unknown.In this paper, we describe the elimination of DONin rats and the structural elucidation of a novel metabolite found in the excreta.DONwas dissolved in 10% aqueous ethanol and intubated to Wistar male rats weighing 240~245g at a single dose of 6mg/kg.Urine and feces samples were collected every 12hr
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