Microbial Detoxification of Deoxynivalenol (DON), Assessed via a Lemna minor L. Bioassay, through Biotransformation to 3-epi-DON and 3-epi-DOM-1.
ABSTRACT: Mycotoxins are toxic metabolites produced by fungi. To mitigate mycotoxins in food or feed, biotransformation is an emerging technology in which microorganisms degrade toxins into non-toxic metabolites. To monitor deoxynivalenol (DON) biotransformation, analytical tools such as ELISA and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) are typically used. However, these techniques do not give a decisive answer about the remaining toxicity of possible biotransformation products. Hence, a bioassay using Lemna minor L. was developed. A dose-response analysis revealed significant inhibition in the growth of L. minor exposed to DON concentrations of 0.25 mg/L and higher. Concentrations above 1 mg/L were lethal for the plant. This bioassay is far more sensitive than previously described systems. The bioassay was implemented to screen microbial enrichment cultures, originating from rumen fluid, soil, digestate and activated sludge, on their biotransformation and detoxification capability of DON. The enrichment cultures originating from soil and activated sludge were capable of detoxifying and degrading 5 and 50 mg/L DON. In addition, the metabolites 3-epi-DON and the epimer of de-epoxy-DON (3-epi-DOM-1) were found as biotransformation products of both consortia. Our work provides a new valuable tool to screen microbial cultures for their detoxification capacity.
Project description:Bacteria are able to de-epoxidize or epimerize deoxynivalenol (DON), a mycotoxin, to deepoxy-deoxynivalenol (deepoxy-DON or DOM-1) or 3-epi-deoxynivalenol (3-epi-DON), respectively. Using different approaches, the intestinal toxicity of 3 molecules was compared and the molecular basis for the reduced toxicity investigated. In human intestinal epithelial cells, deepoxy-DON and 3-epi-DON were not cytotoxic, did not change the oxygen consumption or impair the barrier function. In intestinal explants, exposure for 4?hours to 10??M DON induced intestinal lesions not seen in explants treated with deepoxy-DON and 3-epi-DON. A pan-genomic transcriptomic analysis was performed on intestinal explants. 747 probes, representing 323 genes, were differentially expressed, between DON-treated and control explants. By contrast, no differentially expressed genes were observed between control, deepoxy-DON and 3-epi-DON treated explants. Both DON and its biotransformation products were able to fit into the pockets of the A-site of the ribosome peptidyl transferase center. DON forms three hydrogen bonds with the A site and activates MAPKinases (mitogen-activated protein kinases). By contrast deepoxy-DON and 3-epi-DON only form two hydrogen bonds and do not activate MAPKinases. Our data demonstrate that bacterial de-epoxidation or epimerization of DON altered their interaction with the ribosome, leading to an absence of MAPKinase activation and a reduced toxicity.
Project description:Deoxynivalenol (DON) is a frequent mycotoxin in grains, produced by Fusarium fungi, which demonstre multiple side effects such as modulation of immune responses, reduced feed intake and weight gain or impairment of the intestinal barrier function. Among animal species, pigs are the best model for humans and are very sensitive to DON. In wheat, DON can be conjugated to glucose to form DON-3-β-D-glucoside (D3G). Some bacteria isolated from digestive tracts or soil, are also able to de-epoxydize or epimerize DON to metabolites such as deepoxy-deoxynivalenol (DOM-1) or 3-epi-deoxynivalenol (epi-DON). The toxicity of these DON metabolites is poorly documented. By the way of ingestion, the intestine is the first organ exposed to these molecules and so constitute a relevant model. The aim of this study was to compare the intestinal toxicity of three DON metabolites (D3G, DOM-1 and epi-DON) with the one of DON. Intestinal explants from 6 pigs were treated with 10mM DON, D3G, DOM-1 or epi-DON for 4 hours and transcriptomic analysis was performed using an “Agilent Porcinet 60K”. Jejunal explants from 4 piglets aged of 5 weeks were sampled and exposed in vitro to differents molecules (DON, D3G, DOM-1 & 3-epi-DON) at 10µM during 4h. Then RNA was extracted.
Project description:Food and feed can be naturally contaminated by several mycotoxins, and concern about the hazard of exposure to mycotoxin mixtures is increasing. In this study, more than 800 metabolites were analyzed in 524 finished pig feed samples collected worldwide. Eighty-eight percent of the samples were co-contaminated with deoxynivalenol (DON) and other regulated/emerging mycotoxins. The Top 60 emerging/regulated mycotoxins co-occurring with DON in pig feed shows that 48%, 13%, 8% and 12% are produced by Fusarium, Aspergillus, Penicillium and Alternaria species, respectively. Then, the individual and combined toxicity of DON and the 10 most prevalent emerging mycotoxins (brevianamide F, cyclo-(L-Pro-L-Tyr), tryptophol, enniatins A1, B, B1, emodin, aurofusarin, beauvericin and apicidin) was measured at three ratios corresponding to pig feed contamination. Toxicity was assessed by measuring the viability of intestinal porcine epithelial cells, IPEC-1, at 48-h. BRV-F, Cyclo and TRPT did not alter cell viability. The other metabolites were ranked in the following order of toxicity: apicidin > enniatin A1 > DON > beauvericin > enniatin B > enniatin B1 > emodin > aurofusarin. In most of the mixtures, combined toxicity was similar to the toxicity of DON alone. In terms of pig health, these results demonstrate that the co-occurrence of emerging mycotoxins that we tested with DON does not exacerbate toxicity.
Project description:The biological detoxification of mycotoxins, including deoxynivalenol (DON), represents a very promising approach to address the challenging problem of cereal grain contamination. The recent discovery of Devosia mutans 17-2-E-8 (Devosia spp. 17-2-E-8), a bacterial isolate capable of transforming DON to the non-toxic stereoisomer 3-epi-deoxynivalenol, along with earlier reports of bacterial species capable of oxidizing DON to 3-keto-DON, has generated interest in the possible mechanism and enzyme(s) involved. An understanding of these details could pave the way for novel strategies to manage this widely present toxin. It was previously shown that DON epimerization proceeds through a two-step biocatalysis. Significantly, this report describes the identification of the first enzymatic step in this pathway. The enzyme, a dehydrogenase responsible for the selective oxidation of DON at the C3 position, was shown to readily convert DON to 3-keto-DON, a less toxic intermediate in the DON epimerization pathway. Furthermore, this study provides insights into the PQQ dependence of the enzyme. This enzyme may be part of a feasible strategy for DON mitigation within the near future.
Project description:The trichothecene mycotoxin deoxynivalenol (DON) is a common contaminant of wheat, barley, and maize. New strategies are needed to reduce or eliminate DON in feed and food products. Microorganisms from plant and soil samples collected in Blacksburg, VA, USA, were screened by incubation in a mineral salt media containing 100 ?g/mL DON and analysis by gas chromatography mass spectrometry (GC/MS). Two mixed cultures derived from soil samples consistently decreased DON levels in assays using DON as the sole carbon source. Nuclear magnetic resonance (NMR) analysis indicated that 3-keto-4-deoxynivalenol was the major by-product of DON. Via 16S rRNA sequencing, these mixed cultures, including mostly members of the genera Acinetobacter, Leadbetterella, and Gemmata, were revealed. Incubation of one of these mixed cultures with wheat samples naturally contaminated with 7.1 ?g/mL DON indicated nearly complete conversion of DON to the less toxic 3-epimer-DON (3-epi-DON). Our work extends previous studies that have demonstrated the potential for bioprospecting for microorganisms from the environment to remediate or modify mycotoxins for commercial applications, such as the reduction of mycotoxins in fuel ethanol co-products.
Project description:Biotransformation of mycotoxins in animals comprises phase I and phase II metabolisation reactions. For the trichothecene deoxynivalenol (DON), several phase II biotransformation reactions have been described resulting in DON-glutathiones, DON-glucuronides and DON-sulfates made by glutathione-S-transferases, uridine-diphosphoglucuronyl transferases and sulfotransferases, respectively. These metabolites can be easily excreted and are less toxic than their free compounds. Here, we demonstrate for the first time in the animal kingdom the conversion of DON to DON-3-glucoside (DON-3G) via a model system with plant pathogenic aphids. This phase II biotransformation mechanism has only been reported in plants. As the DON-3G metabolite was less toxic for aphids than DON, this conversion is considered a detoxification reaction. Remarkably, English grain aphids (Sitobion avenae) which co-occur with the DON producer Fusarium graminearum on wheat during the development of fusarium symptoms, tolerate DON much better and convert DON to DON-3G more efficiently than pea aphids (Acyrthosiphon pisum), the latter being known to feed on legumes which are no host for F. graminearum. Using a non-targeted high resolution mass spectrometric approach, we detected DON-diglucosides in aphids probably as a result of sequential glucosylation reactions. Data are discussed in the light of an eventual co-evolutionary adaptation of S. avenae to DON.
Project description:Trichothecene mycotoxins, such as deoxynivalenol (DON) produced by the fungal pathogen, Fusarium graminearum, are not only important for plant infection but are also harmful to human and animal health. Trichothecene targets the ribosomal protein Rpl3 that is conserved in eukaryotes. Hence, a self-defense mechanism must exist in DON-producing fungi. It is reported that TRI (trichothecene biosynthesis) 101 and TRI12 are two genes responsible for self-defense against trichothecene toxins in Fusarium. In this study, however, we found that simultaneous disruption of TRI101 and TRI12 has no obvious influence on DON resistance upon exogenous DON treatment in F. graminearum, suggesting that other mechanisms may be involved in self-defense. By using RNA-seq, we identified 253 genes specifically induced in DON-treated cultures compared with samples from cultures treated or untreated with cycloheximide, a commonly used inhibitor of eukaryotic protein synthesis. We found that transporter genes are significantly enriched in this group of DON-induced genes. Of those genes, 15 encode major facilitator superfamily transporters likely involved in mycotoxin efflux. Significantly, we found that genes involved in the metabolism of gamma-aminobutyric acid (GABA), a known inducer of DON production in F. graminearum, are significantly enriched among the DON-induced genes. The GABA biosynthesis gene PROLINE UTILIZATION 2-2 (PUT2-2) is downregulated, while GABA degradation genes are upregulated at least twofold upon treatment with DON, resulting in decreased levels of GABA. Taken together, our results suggest that transporters influencing DON efflux are important for self-defense and that GABA mediates the balance of DON production and self-defense in F. graminearum.
Project description:Deoxynivalenol (DON) is a secondary fungal metabolite and contaminant mycotoxin that is widely detected in wheat and corn products cultivated around the world. Bio-remediation methods have been extensively studied in the past two decades and promising ways to reduce DON-associated toxicities have been reported. Bacterial epimerization of DON at the C3 carbon was recently reported to induce a significant loss in the bio-toxicity of the resulting stereoisomer (3-epi-DON) in comparison to the parental compound, DON. In an earlier study, we confirmed the diminished bio-potency of 3-epi-DON using different mammalian cell lines and mouse models and mechanistically attributed it to the reduced binding of 3-epi-DON within the ribosomal peptidyl transferase center (PTC). In the current study and by inspecting the chromatographic behavior of 3-epi-DON and its molecular interactions with a well-characterized enzyme, Fusarium graminearum Tri101 acetyltransferase, we provide the evidence that the C3 carbon epimerization of DON influences its molecular interactions beyond the abrogated PTC binding.
Project description:During an exposure, humans and animals are most often exposed to a mixture rather than individual mycotoxins. In this study, a Human Embryonic Kidney 293 cell (HEK-293) fluorescence sensor was developed to detect and evaluate mycotoxins, deoxynivalenol (DON) and zearalenone (ZEN) compounds, produced by Fusarium culmorum that are common food contaminants. TRE-copGFP (green fluorescent protein) and ERE-TagRFP (red fluorescent protein) plasmids were constructed and cotransfected into HEK-293 cells through a highly efficient, lipid-mediated, DNA-transfection procedure. Results show that fluorescence intensity was proportional to DON and ZEN concentrations, ranging from 2 to 40?ng/mL and 10 to 100?ng/mL respectively, with a detection limit of 0.75?ng/mL and 3.2?ng/mL respectively. The EC50 of DON and ZEN are 30.13?ng/mL and 76.63?ng/mL respectively. Additionally, ZEN may have a synergistic effect on enhancing AP-1 activity of the toxicity pathway of DON. These data indicate the high sensitivity and effectiveness of our biosensor system in the evaluation of the combined toxicity of ZEN, DON and their derivatives. In addition, this approach is suitable for an early warning method for the detection of ZEN and DON family mycotoxins contamination without higher-priced, conventional analytical chemistry methods.
Project description:This study aimed to investigate the potential accumulation of mycotoxins in the lesser mealworm (Alphitobius diaperinus, LMW) and black soldier fly (Hermetia illucens, BSF) larvae. Feed was spiked with aflatoxin B?, deoxynivalenol (DON), ochratoxin A or zearalenone, and as a mixture of mycotoxins, to concentrations of 1, 10, and 25 times the maximum limits set by the European Commission for complete feed. This maximum limit is 0.02 mg/kg for aflatoxin B?, 5 mg/kg for DON, 0.5 mg/kg for zearalenone and 0.1 mg/kg for ochratoxin A. The mycotoxins and some of their metabolites were analysed in the larvae and residual material using a validated and accredited LC-MS/MS-based method. Metabolites considered were aflatoxicol, aflatoxin P?, aflatoxin Q?, and aflatoxin M?, 3-acetyl-DON, 15-acetyl-DON and DON-3-glycoside, and ?- and ?-zearalenol. No differences were observed between larvae reared on mycotoxins individually or as a mixture with regards to both larvae development and mycotoxin accumulation/excretion. None of the mycotoxins accumulated in the larvae and were only detected in BSF larvae several orders of magnitude lower than the concentration in feed. Mass balance calculations showed that BSF and LMW larvae metabolized the four mycotoxins to different extents. Metabolites accounted for minimal amounts of the mass balance, except for zearalenone metabolites in the BSF treatments, which accounted for an average maximum of 86% of the overall mass balance. Both insect species showed to excrete or metabolize the four mycotoxins present in their feed. Hence, safe limits for these mycotoxins in substrates to be used for these two insect species possibly could be higher than for production animals. However, additional analytical and toxicological research to fully understand the safe limits of mycotoxins in insect feed, and thus the safety of the insects, is required.