Toxicity of three biological derivatives of deoxynivalenol: deepoxy-deoxynivalenol, 3-epi-deoxynivalenol and deoxynivalenol-3-glucoside on pigs

Les mycotoxines sont des métabolites secondaires de moisissures contaminant de façon naturelle de nombreuses denrées alimentaires, notamment les céréales. Le déoxynivalénol (DON), produit par Fusarium sp., est la mycotoxine la plus répandue dans le monde. Du fait de sa grande stabilité chimique, le DON est difficile à éliminer, et se retrouve dans les céréales et les produits finis ou il induit des effets toxiques pour l'homme et l'animal. De nouvelles stratégies de lutte sont mises en places, telle la transformation biologique utilisant des bactéries ou des plantes. En effet certaines bactéries possèdent des enzymes capables de transformer le DON en de nouveaux composés, le déepoxy-déoxynivalénol (DOM-1) et le 3-épi-déoxynivalénol (3-epi-DON). De plus, certaines plantes sont naturellement capables de transformer le DON dans le but de l'éliminer et de le détoxifier, formant ainsi le deoxynivalénol-3-ß-D-glucoside (D3G). L'objectif de cette thèse était d'évaluer la toxicité de ces dérivés du DON au niveau de l'intestin et du système immunitaire par le biais d'analyses in silico, in vitro, ex vivo et in vivo. Les tests de toxicité in vitro sur la lignée humaine intestinale cellulaire Caco-2 montrent que le DOM-1, le 3-epi-DON et le D3G n'étaient pas cytotoxiques, ils ne modifiaient ni la viabilité, ni la fonction de barrière des cellules, mesurée par la résistance électrique transépithéliale. Les tests de toxicité ex vivo sur des explants jéjunum porcin ont montré que le DOM-1, le 3-epi-DON ou le D3G n'induisaient pas de modifications histomorphologiques. En revanche, les explants exposés au DON montraient des lésions morphologiques et une régulation positive de l'expression des cytokines pro-inflammatoires. L'impact de ces trois dérivés a été également analysé sur l'expression de l'ensemble des gènes du tissu, avec une analyse microarray. Ceci a montré que ces dérivés du DON n'induisaient aucun changement dans l'expression des gènes par rapport au groupe contrôle. Le DON quand a lui exprimait différentiellement 747 sondes, correspondantes à 333 gènes impliqués dans l'immunité, la réponse inflammatoire, le stress oxydatif, la mort cellulaire, le transport moléculaire et la fonction mitochondriale. L'analyse in silico a montré que le D3G, contrairement au DON était incapable de se lier au site-A du ribosome, principale cible de la toxicité pour le DON. Les deux dérivés microbiens eux, étaient capables de se fixer au site-A au sein du ribosome, mais contrairement au DON ils ne formaient que deux liaisons hydrogènes au lieu de trois. De plus, ces trois dérivés n'induisaient pas de stress ribotoxique, d'activation des MAPKs (mitogen-activated protein kinases), et de réponse pro-inflammatoire. Une étude complémentaire a été menée in vivo pour évaluer la toxicité du DOM-1 chez le porc (gavage pendant 21 jours avec .0.14mg / kg de poids vif). Les résultats ont montré que le DOM-1, contrairement au DON n'induisait pas les effets toxiques du DON au niveau des paramètres zootechniques (pas de vomissements, aucune diminution de la consommation alimentaire ou de perte de poids), sur l'intestin et le foie (pas de dommages tissulaires), ou sur la réponse immunitaire (pas de réponse inflammatoire induite). En conclusion, nos résultats montrent l'efficacité de ces transformations enzymatiques. La déepoxydation et l'épimérisation bactérienne, ainsi que la glycosylation par les plantes permettent de sensiblement diminuer la toxicité du DON, passant par une absence de toxicité sur le ribosome avec une absence d'activation des MAPKs et de réponses inflammatoires. Dans ce contexte de contamination par les mycotoxines, ces méthodes de luttes alternatives semblent être des approches prometteuses.The Fusarium sp. mycotoxin deoxynivalenol (DON) is one of the most frequently widespread mycotoxin worldwide. Due to its high structural stability, the elimination of DON, once present in cereals or feed materials, becomes difficult. Thereby, it is present in many cereals and final feed products, inducing several toxic effects on human and animals, and causing big economic losses. New strategies of to fight against mycotoxins were developed, as biological transformation, either by the use of bacteria or plants. Indeed, some microorganisms are able to transform DON in new products, by enzymatic reaction, forming the deepoxy-deoxynivalenol (DOM-1) and the 3-epi-deoxynivalenol (3-epi-DON). Moreover, some plants naturally own the capacity to glycosylate DON in the aim to detoxify it, forming the deoxynivalenol-3-ß-D-glucoside (D3G). The aim of this thesis was to assess the toxicity of these DON derivatives, on the intestine and immune response, using several approaches such as in silico, in vitro, ex vivo and in vivo models. On the human intestinal Caco-2 cell line, DOM-1, 3-epi-DON and D3G were not cytotoxic; they did not alter its viability and barrier function, as measured by the trans epithelial electrical resistance. The expression profile of DOM-1, 3-epi-DON and D3G-treated jejunal explants was similar to that of controls and these explants did not show any histomorphology alteration. On the other hand, the treatment of intestinal explants with DON, induced morphological lesions and upregulated the expression of proinflammatory cytokines. The impact of these three derivatives was also studied on intestinal explants with a pan-genomic transcriptomic analysis. Results show that the derivatives of DON did not induce any change on the gene expression in comparison to the control-treated explants. In contrary, DON-treated explants differentially expressed 747 probes, representing 323 genes involved in immune and inflammatory responses, oxidative stress, cell death, molecular transport and mitochondrial function. In silico analysis revealed that D3G, opposing to DON, was unable to bind to the A site of the ribosome, which is the main target for DON toxicity. Both DOM-1 and 3-epi-DON were able to fit into the pockets of the A site of the ribosome but only by forming two hydrogen bonds, while in this position, DON forms three hydrogen bonds. Moreover, the three derivatives do not elicit a ribotoxic stress, MAPKinase activation, and inflammatory response. Then, an in vivo study was carried out to assess the toxicity of DOM-1 on pig (feed forced during 21 days at 0.14 mg/Kg BW). The results showed that DOM-1 does not have as much toxic effects as DON on zootechnical parameters (no emesis induced, no decrease of food consumption or weight loss observed), on intestine and liver (no tissues damages), or on the immune response (no inflammatory response induced). Our data demonstrate that bacterial de-epoxidation or epimerization of deepoxy-DON modified its interaction with the ribosome, leading to an absence of MAPKinase activation and toxicity; and that the glycosylation of DON suppresses its ability to bind to the ribosome and decreases its intestinal toxicity. The mycotoxin deoxynivalenol (DON) remains an important challenge in many regions in the world. Thus, these biological detoxifications of DON seem to represent a new promising approach helping manage the problem of its contamination

Effect of DON and ZEN and their metabolites DOM-1 and HZEN on B cell proliferation and antibody production

IntroductionThe mycotoxins deoxynivalenol (DON) and zearalenone (ZEN), produced by Fusarium fungi, are frequently found in the cereal-rich diet of pigs and can modulate the immune system. Some enzymes or bacteria present in the digestive tract can de-epoxydize DON to deepoxy-deoxynivalenol (DOM-1) and biotransform ZEN into hydrolyzed ZEN (HZEN). The effects of these metabolites on immune cells, particularly with respect to the vaccine responses, are poorly documented. The aim of this study was to address the impact of DON and ZEN and their respective derivatives, on proliferation, and antibody production of porcine B cells in vitro.MethodsPeripheral blood mononuclear cells (PBMCs), isolated from healthy pigs, were stimulated with the Toll-like receptor (TLR) 7/8-agonist Resiquimod (R848) or the TLR/1/2-agonist Pam3Cys-SKKKK in combination with DON [0.1-1.6 µM] or DOM-1 [1.6 µM and 16 µM] and ZEN [2.5-40 µM] or HZEN [40 µM].ResultsA strong decrease in B-cell proliferation was observed at DON concentrations equal to or exceeding 0.8 µM and at ZEN concentrations equal to or exceeding 20 µM. Treatment with 1.6 µM DON or 40 µM ZEN led to almost a complete loss of live CD79α+ B cells. Moreover, CD21 expression of proliferating IgG+ and IgM+ B-cell subsets was decreased at DON concentrations equal to and exceeding 0.4 µM and at ZEN concentrations equal to or exceeding 10 µM. ELISpot assays revealed a decrease of IgG-secreting B cells at concentrations of and exceeding 0.4 µM and at ZEN concentrations equal to and exceeding 10 µM. ELISA assays showed a decrease of IgM, IgG, and IgA secretion at concentrations equal to or exceeding 0.4 µM DON. ZEN reduced IgM secretion at 20-40 µM (both R848 and Pam3Cys-SKKKK), IgG secretion at 40 µM (both R848 and Pam3Cys-SKKKK) and IgA secretion at 20-40 µM.DiscussionOur in vitro experiments show that while DON and ZEN impair immunoglobulin production and B-cell proliferation, this effect is abrogated by HZEN and DOM-1

Toxicité de trois dérivés biologiques du déoxynivalénol : déepoxy-déoxynivalénol, 3-epidéoxynivalénol et édoxynivalénol-3-glucoside chez le porc

The Fusarium sp. mycotoxin deoxynivalenol (DON) is one of the most frequently widespread mycotoxin worldwide. Due to its high structural stability, the elimination of DON, once present in cereals or feed materials, becomes difficult. Thereby, it is present in many cereals and final feed products, inducing several toxic effects on human and animals, and causing big economic losses. New strategies of to fight against mycotoxins were developed, as biological transformation, either by the use of bacteria or plants. Indeed, some microorganisms are able to transform DON in new products, by enzymatic reaction, forming the deepoxy-deoxynivalenol (DOM-1) and the 3-epi-deoxynivalenol (3-epi-DON). Moreover, some plants naturally own the capacity to glycosylate DON in the aim to detoxify it, forming the deoxynivalenol-3-ß-D-glucoside (D3G). The aim of this thesis was to assess the toxicity of these DON derivatives, on the intestine and immune response, using several approaches such as in silico, in vitro, ex vivo and in vivo models. On the human intestinal Caco-2 cell line, DOM-1, 3-epi-DON and D3G were not cytotoxic; they did not alter its viability and barrier function, as measured by the trans epithelial electrical resistance. The expression profile of DOM-1, 3-epi-DON and D3G-treated jejunal explants was similar to that of controls and these explants did not show any histomorphology alteration. On the other hand, the treatment of intestinal explants with DON, induced morphological lesions and upregulated the expression of proinflammatory cytokines. The impact of these three derivatives was also studied on intestinal explants with a pan-genomic transcriptomic analysis. Results show that the derivatives of DON did not induce any change on the gene expression in comparison to the control-treated explants. In contrary, DON-treated explants differentially expressed 747 probes, representing 323 genes involved in immune and inflammatory responses, oxidative stress, cell death, molecular transport and mitochondrial function. In silico analysis revealed that D3G, opposing to DON, was unable to bind to the A site of the ribosome, which is the main target for DON toxicity. Both DOM-1 and 3-epi-DON were able to fit into the pockets of the A site of the ribosome but only by forming two hydrogen bonds, while in this position, DON forms three hydrogen bonds. Moreover, the three derivatives do not elicit a ribotoxic stress, MAPKinase activation, and inflammatory response. Then, an in vivo study was carried out to assess the toxicity of DOM-1 on pig (feed forced during 21 days at 0.14 mg/Kg BW). The results showed that DOM-1 does not have as much toxic effects as DON on zootechnical parameters (no emesis induced, no decrease of food consumption or weight loss observed), on intestine and liver (no tissues damages), or on the immune response (no inflammatory response induced). Our data demonstrate that bacterial de-epoxidation or epimerization of deepoxy-DON modified its interaction with the ribosome, leading to an absence of MAPKinase activation and toxicity; and that the glycosylation of DON suppresses its ability to bind to the ribosome and decreases its intestinal toxicity. The mycotoxin deoxynivalenol (DON) remains an important challenge in many regions in the world. Thus, these biological detoxifications of DON seem to represent a new promising approach helping manage the problem of its contamination.Les mycotoxines sont des métabolites secondaires de moisissures contaminant de façon naturelle de nombreuses denrées alimentaires, notamment les céréales. Le déoxynivalénol (DON), produit par Fusarium sp., est la mycotoxine la plus répandue dans le monde. Du fait de sa grande stabilité chimique, le DON est difficile à éliminer, et se retrouve dans les céréales et les produits finis ou il induit des effets toxiques pour l'homme et l'animal. De nouvelles stratégies de lutte sont mises en places, telle la transformation biologique utilisant des bactéries ou des plantes. En effet certaines bactéries possèdent des enzymes capables de transformer le DON en de nouveaux composés, le déepoxy-déoxynivalénol (DOM-1) et le 3-épi-déoxynivalénol (3-epi-DON). De plus, certaines plantes sont naturellement capables de transformer le DON dans le but de l'éliminer et de le détoxifier, formant ainsi le deoxynivalénol-3-ß-D-glucoside (D3G). L'objectif de cette thèse était d'évaluer la toxicité de ces dérivés du DON au niveau de l'intestin et du système immunitaire par le biais d'analyses in silico, in vitro, ex vivo et in vivo. Les tests de toxicité in vitro sur la lignée humaine intestinale cellulaire Caco-2 montrent que le DOM-1, le 3-epi-DON et le D3G n'étaient pas cytotoxiques, ils ne modifiaient ni la viabilité, ni la fonction de barrière des cellules, mesurée par la résistance électrique transépithéliale. Les tests de toxicité ex vivo sur des explants jéjunum porcin ont montré que le DOM-1, le 3-epi-DON ou le D3G n'induisaient pas de modifications histomorphologiques. En revanche, les explants exposés au DON montraient des lésions morphologiques et une régulation positive de l'expression des cytokines pro-inflammatoires. L'impact de ces trois dérivés a été également analysé sur l'expression de l'ensemble des gènes du tissu, avec une analyse microarray. Ceci a montré que ces dérivés du DON n'induisaient aucun changement dans l'expression des gènes par rapport au groupe contrôle. Le DON quand a lui exprimait différentiellement 747 sondes, correspondantes à 333 gènes impliqués dans l'immunité, la réponse inflammatoire, le stress oxydatif, la mort cellulaire, le transport moléculaire et la fonction mitochondriale. L'analyse in silico a montré que le D3G, contrairement au DON était incapable de se lier au site-A du ribosome, principale cible de la toxicité pour le DON. Les deux dérivés microbiens eux, étaient capables de se fixer au site-A au sein du ribosome, mais contrairement au DON ils ne formaient que deux liaisons hydrogènes au lieu de trois. De plus, ces trois dérivés n'induisaient pas de stress ribotoxique, d'activation des MAPKs (mitogen-activated protein kinases), et de réponse pro-inflammatoire. Une étude complémentaire a été menée in vivo pour évaluer la toxicité du DOM-1 chez le porc (gavage pendant 21 jours avec .0.14mg / kg de poids vif). Les résultats ont montré que le DOM-1, contrairement au DON n'induisait pas les effets toxiques du DON au niveau des paramètres zootechniques (pas de vomissements, aucune diminution de la consommation alimentaire ou de perte de poids), sur l'intestin et le foie (pas de dommages tissulaires), ou sur la réponse immunitaire (pas de réponse inflammatoire induite). En conclusion, nos résultats montrent l'efficacité de ces transformations enzymatiques. La déepoxydation et l'épimérisation bactérienne, ainsi que la glycosylation par les plantes permettent de sensiblement diminuer la toxicité du DON, passant par une absence de toxicité sur le ribosome avec une absence d'activation des MAPKs et de réponses inflammatoires. Dans ce contexte de contamination par les mycotoxines, ces méthodes de luttes alternatives semblent être des approches prometteuses

Impact of mycotoxin on immune response and consequences for pig health

Mycotoxins are fungal secondary metabolites detected in many agricultural commodities, especially cereals. Due to their high consumption of cereals, pigs are exposed to these toxins. In the European Union, regulations and/or recommendations exist in pig feed for aflatoxins, ochratoxin A, fumonisins, zearalenone, and trichothecenes, deoxynivalenol and T-2 toxin. These mycotoxins have different toxic effects, but they all target the immune system. They have immunostimulatory or immunosuppressive effects depending on the toxin, the concentration and the parameter investigated. The immune system is primarily responsible for defense against invading organisms. The consequences of the ingestion of mycotoxin-contaminated feed are an increased susceptibility to infectious diseases, a reactivation of chronic infection and a decreased vaccine efficacy. In this review we summarized the data available on the effect of mycotoxins on the immune system and the consequences for pig health

Deoxynivalenol affects proliferation and expression of activation-related molecules in major porcine T-cell subsets

The Fusarium mycotoxin deoxynivalenol (DON) contaminates animal feed worldwide. In vivo, DON modifies the cellular protein synthesis, thereby also affecting the immune system. However, the functional consequences of this are still ill-defined. In this study, peripheral blood mononuclear cells from healthy pigs were incubated with different DON concentrations in the presence of Concanavalin A (ConA), a plant-derived polyclonal T-cell stimulant. T-cell subsets were investigated for proliferation and expression of CD8α, CD27, and CD28, which are involved in activation and costimulation of porcine T cells. A clear decrease in proliferation of all ConA-stimulated major T-cell subsets (CD4+, CD8+, and γδ T cells) was observed in DON concentrations higher than 0.4 µM. This applied in particular to naïve CD4+ and CD8+ T cells. From 0.8 μM onwards, DON induced a reduction of CD8α (CD4+) and CD27 expression (CD4+ and CD8+ T cells). CD28 expression was diminished in CD4+ and CD8+ T cells at a concentration of 1.6 µM DON. None of these effects were observed with the DON-derivative deepoxy-deoxynivalenol (DOM-1) at 16 µM. These results indicate that DON reduces T-cell proliferation and the expression of molecules involved in T-cell activation, providing a molecular basis for some of the described immunosuppressive effects of DON

Zymosan-Induced Murine Peritonitis Is Associated with an Increased Sphingolipid Synthesis without Changing the Long to Very Long Chain Ceramide Ratio

Sphingolipids are key molecules in inflammation and defense against pathogens. Their role in dectin-1/TLR2-mediated responses is, however, poorly understood. This study investigated the sphingolipidome in the peritoneal fluid, peritoneal cells, plasma, and spleens of mice after intraperitoneal injection of 0.1 mg zymosan/mouse or PBS as a control. Samples were collected at 2, 4, 8, and 16 h post-injection, using a total of 36 mice. Flow cytometry analysis of peritoneal cells and measurement of IL-6, IL-1β, and TNF-α levels in the peritoneal lavages confirmed zymosan-induced peritonitis. The concentrations of sphingoid bases, dihydroceramides, ceramides, dihydrosphingomyelins, sphingomyelins, monohexosylceramides, and lactosylceramides were increased after zymosan administration, and the effects varied with the time and the matrix measured. The greatest changes occurred in peritoneal cells, followed by peritoneal fluid, at 8 h and 4 h post-injection, respectively. Analysis of the sphingolipidome suggests that zymosan increased the de novo synthesis of sphingolipids without change in the C14–C18:C20–C26 ceramide ratio. At 16 h post-injection, glycosylceramides remained higher in treated than in control mice. A minor effect of zymosan was observed in plasma, whereas sphinganine, dihydrosphingomyelins, and monohexosylceramides were significantly increased in the spleen 16 h post-injection. The consequences of the observed changes in the sphingolipidome remain to be established

Toxicology of deoxynivalenol and its acetylated and modified forms

Mycotoxins are the most frequently occurring natural contaminants in human and animal diet. Among them, deoxynivalenol (DON), produced by Fusarium, is one of the most prevalent and thus represents an important health risk. Recent detection methods revealed new mycotoxins and new molecules derivated from the "native" mycotoxins. The main derivates of DON are the acetylated forms produced by the fungi (3- and 15-acetyl-DON), the biologically "modified" forms produced by the plant (deoxynivalenol-3-beta-d-glucopyranoside), or after bacteria transformation (de-epoxy DON, 3-epi-DON and 3-keto-DON) as well as the chemically "modified" forms (norDON A-C and DON-sulfonates). High proportions of acetylated and modified forms of DON co-occur with DON, increasing the exposure and the health risk. DON and its acetylated and modified forms are rapidly absorbed following ingestion. At the molecular level, DON binds to the ribosome, induces a ribotoxic stress leading to the activation of MAP kinases, cellular cell-cycle arrest and apoptosis. The toxic effects of DON include emesis and anorexia, alteration of intestinal and immune functions, reduced absorption of the nutrients as well as increased susceptibility to infection and chronic diseases. In contrast to DON, very little information exists concerning the acetylated and modified forms; some can be converted back to DON, their ability to bind to the ribosome and to induce cellular effects varies according to the toxin. Except for the acetylated forms, their toxicity and impact on human and animal health are poorly documented

Bovine Peripheral Blood Mononuclear Cells Are More Sensitive to Deoxynivalenol Than Those Derived from Poultry and Swine

Deoxynivalenol (DON) is one of the most prevalent mycotoxins, contaminating cereals and cereal-derived products. Its derivative deepoxy-deoxynivalenol (DOM-1) is produced by certain bacteria, which either occur naturally or are supplemented in feed additive. DON-induced impairments in protein synthesis are particularly problematic for highly proliferating immune cells. This study provides the first comparison of the effects of DON and DOM-1 on the concanavalin A-induced proliferation of porcine, chicken, and bovine peripheral blood mononuclear cells (PBMCs). Therefore, isolated PBMCs were treated with DON (0.01–3.37 µM) and DOM-1 (1.39–357 µM) separately, and proliferation was measured using a bromodeoxyuridine (BrdU) assay. Although pigs are considered highly sensitive to DON, the present study revealed a substantially higher sensitivity of bovine (IC50 = 0.314 µM) PBMCs compared to chicken (IC50 = 0.691 µM) and porcine (IC50 = 0.693 µM) PBMCs. Analyses on the proliferation of bovine T-cell subsets showed that all major subsets, namely, CD4+, CD8β+, and γδ T cells, were affected to a similar extent. In contrast, DOM-1 did not affect bovine PBMCs, but reduced the proliferation of chicken and porcine PBMCs at the highest tested concentration (357 µM). Results confirm the necessity of feed additives containing DON-to-DOM-1-transforming bacteria and highlights species-specific differences in the DON sensitivity of immune cells

COVEVOL: Natural Evolution at 6 Months of COVID-19

Many studies have investigated post-COVID symptoms, but the predictors of symptom persistence remain unknown. The objective was to describe the natural course of the disease at 6 months and to identify possible factors favoring the resurgence or persistence of these symptoms. COVEVOL is a retrospective observational descriptive study of 74 patients. All patients with positive SARS-CoV-2 PCR from March 2020 were included. We compared a group with symptom persistence (PS group) with another group without symptom persistence (no-PS group). Fifty-three out of seventy-four patients (71.62%) described at least one persistent symptom at 6 months of SARS-CoV-2 infection. In the PS group, 56.6% were women and the average age was 54.7 years old [21–89.2] ± 16.9. The main symptoms were asthenia (56.6%, n = 30), dyspnea (34%, n = 18), anxiety (32.1% n = 17), anosmia (24.5%, n = 13) and agueusia (15.1% n = 8). Ten patients (13.51%) presented a resurgence in symptoms. Patients in the PS group were older (p = 0.0048), had a higher BMI (p = 0.0071), and were more frequently hospitalized (p = 0.0359) compared to the no-PS group. Odynophagia and nasal obstruction were less present in the inaugural symptoms of COVID-19 in the PS group (p = 0.0202 and p = 0.0332). Persistent post-COVID syndromes are common and identification of contributing factors is necessary for understanding this phenomenon and appropriate management