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received: 04 April 2016 accepted: 14 June 2016 Published: 06 July 2016
Microbial biotransformation of DON: molecular basis for reduced toxicity Alix Pierron1,2,3,*, Sabria Mimoun1,2,*, Leticia S. Murate1,4, Nicolas Loiseau1,2, Yannick Lippi1,2, Ana-Paula F. L. Bracarense4, Gerd Schatzmayr3, Jian Wei He5, Ting Zhou5, Wulf-Dieter Moll3 & Isabelle P. Oswald1,2 Bacteria are able to de-epoxidize or epimerize deoxynivalenol (DON), a mycotoxin, to deepoxydeoxynivalenol (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 μM 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. Mycotoxins are toxic secondary metabolites produced by various molds, such as Aspergillus, Penicillium and Fusarium which may contaminate food and feed at all stages of the food/feed chain1,2. Despite the improvement of agricultural and manufacturing practices, mycotoxin contamination cannot be avoided and still represents a permanent health risk for both humans and animals. It is thus important to develop decontamination strategies3. Among mycotoxins, deoxynivalenol (DON) produced by Fusarium species, is commonly detected in cereal crops, including wheat, barley, and maize. It is the most abundant trichothecene in food with a frequent occurrence at toxicologically relevant concentrations worldwide4,5. DON causes acute and chronic disorders in humans and animals, with the gastrointestinal tract being an organ sensitive to its adverse effects6. DON affects the intestinal histomorphology, impairs barrier function and nutrient absorption7,8. DON also disrupts the local intestinal immune response; it triggers and potentiates intestinal inflammation9,10. At the cellular and subcellular level, DON binds to the ribosome, inhibits protein and nucleic acid synthesis and triggers ribotoxic stress11–13 leading to the activation of kinases, MAPKs and their downstream signaling pathways14. Several strategies have been developed to limit DON toxicity15, among them, bacterial biotransformation which depends on the ability of microorganisms to generate DON metabolites with reduced toxicity. De-epoxidation is a reductive chemical reaction opening the 12,13-epoxy ring transforming DON into its de-epoxide metabolite de-epoxy-deoxynivalenol (deepoxy-DON or DOM-1) (Supplementary Figure S1)16. Several microbial strains are capable of DON de-epoxidation15,17. Several in vitro studies demonstrated the reduced toxicity of deepoxy-DON. In vivo trials on farm animals receiving feed contaminated with DON have 1
Toxalim, Research Center in Food Toxicology, Université de Toulouse, INRA, UMR 1331, ENVT, INP-Purpan Toulouse, France. 2Université de Toulouse, INP, UMR 1331, Toxalim, Toulouse, France. 3BIOMIN Research Center Technopark 1, 3430 Tulln, Austria. 4Universidade Estadual de Londrina, Lab. Patologia Animal, CP 6001, Londrina, Paraná, Brazil. 5 Guelph Food Research Center Agriculture & Agri-Food Canada, Guelph, Ontario N1G 5C, Canada. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to I.P.O. (email:
[email protected]) Scientific Reports | 6:29105 | DOI: 10.1038/srep29105
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Figure 1. Effects of deepoxy-DON or 3-epi-DON on human intestinal epithelial cells . Activation of cytotoxicity (Panel A), TEER (Panel B) and OCR (Panel C). (Panel A): Proliferative Caco-2 cells were incubated with increasing concentrations of diluent (♦), DON (◾), deepoxy-DON (▴) or 3-epi-DON (●) for 48 hours. Cell viability evaluated by measurement of ATP, is expressed as % of control cells. (Panel B): Caco-2 cells, differentiated on inserts, were treated with 10 μM of diluent (♦), DON (◾), deepoxy-DON (▴) or 3-epiDON (●) and TEER was measured. (Panel C): After establishment of baseline oxygen consumption rate in proliferated Caco-2 cells seeded to 1.5 × 104 cells/well, diluent (♦), DON (◾), deepoxy-DON (▴) or 3-epi-DON (●), was injected at final concentration of 10 μM as indicated by the arrow. The rate of oxygen consumption was then measured for the indicated time. For visual clarity, statistical indicators were omitted from the graph. The OCR values are shown as the percent of baseline for each group. Results are expressed as mean ± SEM of 3–4 independent experiments, ***p
