RESUMEN
The incidence of inflammatory bowel diseases (IBD) is increasing in both Western and developing countries. IBD are multifactorial disorders involving complex interactions between genetic, immune, and environmental factors such as exposure to food contaminants. Deoxynivalenol (DON) is the most prevalent mycotoxin that contaminates staple food and induces intestinal breakdown and inflammatory response. To delineate the role of DON oral exposure in IBD, we used a Dextran sulfate sodium (DSS) colitis model in rats fed with a DON-contaminated diet or a control diet for 4 weeks. Colitis was induced in the 4th week by increasing concentrations of DSS in the drinking water (0, 2, 3 or 5%). DON exacerbated body weight loss and accelerated the appearance of symptoms in animals treated with DSS. DON increased morphological damage, pro-inflammatory markers (myeloperoxidase, CXCL-1 and IL-1ß) and immune cell responses. In lamina propria of the rat with colitis, DON increased adaptive and innate immune responses after anti-CD3/28 or LPS stimulation, respectively. In the spleen, DON increased IFNγ secretion and reduced Treg populations. Interestingly, De-epoxy-DON (DOM-1) a detoxified form of DON did not have any consequences on colitis. These results suggest that DON is a risk factor in the onset of IBD.
Asunto(s)
Contaminación de Alimentos , Enfermedades Inflamatorias del Intestino/inducido químicamente , Micotoxinas/toxicidad , Linfocitos T Reguladores/efectos de los fármacos , Tricotecenos/toxicidad , Animales , Colitis , Sulfato de Dextran , Dieta , Modelos Animales de Enfermedad , Intestinos , Masculino , RatasRESUMEN
Fumonisin B1 (FB1), a congener of fumonisins produced by Fusarium species, is the most abundant and most toxicologically active fumonisin. FB1 causes severe mycotoxicosis in animals, including nephrotoxicity, hepatotoxicity, and disruption of the intestinal barrier. However, mechanisms associated with FB1 toxicity are still unclear. Preliminary studies have highlighted the role of liver X receptors (LXRs) during FB1 exposure. LXRs belong to the nuclear receptor family and control the expression of genes involved in cholesterol and lipid homeostasis. In this context, the toxicity of FB1 was compared in female wild-type (LXR+/+) and LXRα,ß double knockout (LXR-/-) mice in the absence or presence of FB1 (10 mg/kg body weight/day) for 28 days. Exposure to FB1 supplemented in the mice's drinking water resulted in more pronounced hepatotoxicity in LXR-/- mice compared to LXR+/+ mice, as indicated by hepatic transaminase levels (ALT, AST) and hepatic inflammatory and fibrotic lesions. Next, the effect of FB1 exposure on the liver transcriptome was investigated. FB1 exposure led to a specific transcriptional response in LXR-/- mice that included altered cholesterol and bile acid homeostasis. ELISA showed that these effects were associated with an elevated FB1 concentration in the plasma of LXR-/- mice, suggesting that LXRs participate in intestinal absorption and/or clearance of the toxin. In summary, this study demonstrates an important role of LXRs in protecting the liver against FB1-induced toxicity, suggesting an alternative mechanism not related to the inhibition of sphingolipid synthesis for mycotoxin toxicity.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Fumonisinas/toxicidad , Receptores X del Hígado/metabolismo , Alanina Transaminasa/metabolismo , Animales , Aspartato Aminotransferasas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Femenino , Fumonisinas/sangre , Regulación de la Expresión Génica/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/fisiología , Receptores X del Hígado/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Esfingolípidos/metabolismoRESUMEN
Probiotics have been explored to stimulate gut health in weaned pigs, when they started to consume solid diet where mycotoxins could be present. The aim of this study was to evaluate the effect of Lactobacillus rhamnosus RC007 on the intestinal toxicity of deoxynivalenol (DON) in an ex vivo model. Jejunal explants, obtained from 5-week-old crossbred castrated male piglets, were kept as control, exposed for 3 h to 10 µM DON, incubated for 4 h with 109 CFU/mL L. rhamnosus, or pre-incubated 1 h with 109 L. rhamnosus and exposed to DON. Histological lesions were observed, para- and transcellular intestinal permeability was measured in Ussing chambers. The expression levels of mRNA encoding six inflammatory cytokines (CCL20, IL-10, IL-1ß, TNFα, IL-8 and IL-22) were determined by RT-PCR. The expressions of the phosphorylated MAP kinases p42/p44 and p38 were assessed by immunoblotting. Exposure to DON induced histological changes, significantly increased the expression of CCL20, IL-1ß, TNFα, IL-8, IL-22 and IL-10, increased the intestinal paracellular permeability and activated MAP kinases. Incubation with L. rhamnosus alone did not have any significant effect. By contrast, the pre-incubation with L. rhamnosus reduced all the effects of DON: the histological alterations, the pro-inflammatory response, the paracellular permeability and the phosphorylation of MAP kinases. Of note, L. rhamnosus did not adsorb DON and only slightly degrade the toxin. In conclusion, L. rhamnosus RC007 is a promising probiotic which, included as feed additive, can decrease the intestinal toxicity of DON.
Asunto(s)
Yeyuno/efectos de los fármacos , Yeyuno/microbiología , Lacticaseibacillus rhamnosus , Probióticos , Tricotecenos/toxicidad , Alimentación Animal , Animales , Citocinas/metabolismo , Técnicas In Vitro , Masculino , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Permeabilidad , Fosforilación , PorcinosRESUMEN
Deoxynivalenol (DON) is the most abundant trichothecene in food and feed. It causes both acute and chronic disorders of the human and animal intestine, liver and the immune system. The structural basis for the toxicity of DON has not been fully elucidated. Using the pig as a target and a model species for human, the toxicity of DON and its deepoxy-metabolite (DOM-1) was compared. Animals were exposed by gavage to 1 and 0.5 nmol toxin/kg b.w./day for 2 and 3 weeks respectively. Whatever the dose/duration, DOM-1 was less toxic than DON in terms of weight gain and emesis. In the 3-week experiment, animals were vaccinated with ovalbumin, and their immune response was analyzed in addition to tissue morphology, biochemistry and hematology. DON impaired the morphology of the jejunum and the ileum, reduced villi height, decreased E-cadherin expression and modified the intestinal expression of cytokines. Similarly, DON induced hepatotoxicity as indicated by the lesion score and the blood biochemistry. By contrast, DOM-1 only induced minimal intestinal toxicity and did not trigger hepatotoxicity. As far as the immune response was concerned, the effects of ingesting DOM-1 were similar to those caused by DON, as measured by histopathology of lymphoid organs, PCNA expression and the specific antibody response. Taken together, these data demonstrated that DOM-1, a microbial detoxification product of DON, was not toxic in the sensitive pig model but retained some immune-modulatory properties of DON, especially its ability to stimulate a specific antibody response during a vaccination protocol.
Asunto(s)
Sistema Inmunológico/efectos de los fármacos , Tricotecenos/toxicidad , Animales , Intestino Delgado/efectos de los fármacos , Intestino Delgado/inmunología , Hígado/efectos de los fármacos , Masculino , Porcinos , Tricotecenos/farmacología , Aumento de Peso/efectos de los fármacosRESUMEN
INTRODUCTION: The food contamination by mycotoxins is of increasing public health concerns. Deoxynivalenol (DON), a mycotoxin contaminating cereals, has been associated with the exacerbation of inflammatory bowel diseases (IBD), thereby raising the question of its role in the development of IBD. Moreover, the effect of DON on the colon is poorly described. METHODS AND RESULTS: Wistar rats exposed (1-4 weeks) to low doses of DON (2 or 9 mg kg-1 feed) show microscopic alterations of colonic tissue (dilated lymphatic vessels, luminal debris, and cubic and flattened enterocytes). Ingestion of DON also alters colonic functions by increasing paracellular permeability while reducing the expression of the tight junction proteins and increased apoptosis in colonic tissue. Pro-apoptotic factors Bax/Bak, cytochrome C, and caspase 9 are upregulated, whereas expression of anti-apoptotic protein Bcl2 tends to decrease for the mitochondrial pathway. An increased expression of FasR and caspase-8 is observed for the extrinsic pathway. An increase in the pro-inflammatory markers TNFα, IL-17, and myeloperoxidase is also observed. CONCLUSION: These results indicate that the dietary exposure to low levels of DON in food targets the colon inducing a health-threatening breakdown of the colonic barrier, highlighting oral exposure to DON as a potential risk factor in triggering IBD.
Asunto(s)
Exposición Dietética , Mucosa Intestinal , Animales , Colon , Contaminación de Alimentos , Mucosa Intestinal/metabolismo , Ratas , Ratas Wistar , Receptores de Muerte Celular , TricotecenosRESUMEN
Deoxynivalenol (DON) is a common mycotoxin that contaminates cereals and their by-products. The gastrointestinal tract is the first physical barrier against ingested food contaminants. DON contributes to the loss of barrier function of the intestine through the decreased expression of claudin-4 protein, a tight junction protein. The mechanism by which DON alters the intestinal barrier function remains poorly characterized. Therefore, we investigated the involvement of mitogen-activated protein kinases (MAPK) in the DON-induced loss of barrier function. We first verified that 30 µmol/L of DON activated MAPK in a highly sensitive porcine intestinal epithelial cell line (IPEC-1). Inhibition of p44/42 extracellular signal-regulated kinase (ERK) phosphorylation, with 0.5 µmol/L of the specific MAPK pharmacological inhibitor U0126 for 2 h, restored the barrier function of the differentiated intestinal epithelial cell monolayers. The restoration of barrier function was evaluated by trans-epithelial electrical resistance measurements and tracer flux paracellular permeability experiments. The U0126 also restored the intestinal expression of claudin-4 protein, thereby demonstrating that MAPK activation is involved in claudin-4 protein expression and claudin-4 is involved in the maintenance of the intestinal epithelial cell barrier function. Further experiments indicated that p44/42 ERK is not involved in the transcriptional regulation of claudin-4. In conclusion, we demonstrated that DON-induced activation of the p44/42 ERK signaling pathway inhibits the expression of claudin-4 protein, which leads to impaired intestinal barrier function. Given the high levels of DON in cereal grains, these observations of impaired barrier function have implications for human and animal health.
Asunto(s)
Regulación hacia Abajo/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Mucosa Intestinal/efectos de los fármacos , Proteínas de la Membrana/fisiología , Transducción de Señal/efectos de los fármacos , Uniones Estrechas/efectos de los fármacos , Tricotecenos/farmacología , Animales , Línea Celular , Permeabilidad de la Membrana Celular/efectos de los fármacos , Claudina-4 , Impedancia Eléctrica , Enterocitos/efectos de los fármacos , Enterocitos/metabolismo , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Enfermedades Transmitidas por los Alimentos/fisiopatología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Proteínas de la Membrana/genética , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación/efectos de los fármacos , Venenos/farmacología , ARN Mensajero/metabolismo , Porcinos , Uniones Estrechas/metabolismoRESUMEN
Aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus flavus or A. parasiticus, is a frequent contaminant of food and feed. This toxin is hepatotoxic and immunotoxic. The present study analyzed in pigs the influence of AFB1 on humoral and cellular responses, and investigated whether the immunomodulation observed is produced through interference with cytokine expression. For 28 days, pigs were fed a control diet or a diet contaminated with 385, 867 or 1807 microg pure AFB1/kg feed. At days 4 and 15, pigs were vaccinated with ovalbumin. AFB1 exposure, confirmed by an observed dose-response in blood aflatoxin-albumin adduct, had no major effect on humoral immunity as measured by plasma concentrations of total IgA, IgG and IgM and of anti-ovalbumin IgG. Toxin exposure did not impair the mitogenic response of lymphocytes but delayed and decreased their specific proliferation in response to the vaccine antigen, suggesting impaired lymphocyte activation in pigs exposed to AFB1. The expression level of pro-inflammatory (TNF-alpha, IL-1beta, IL-6, IFN-gamma) and regulatory (IL-10) cytokines was assessed by real-time PCR in spleen. A significant up-regulation of all 5 cytokines was observed in spleen from pigs exposed to the highest dose of AFB1. In pigs exposed to the medium dose, IL-6 expression was increased and a trend towards increased IFN-gamma and IL-10 was observed. In addition we demonstrate that IL-6 impaired in vitro the antigenic- but not the mitogenic-induced proliferation of lymphocytes from control pigs vaccinated with ovalbumin. These results indicate that AFB1 dietary exposure decreases cell-mediated immunity while inducing an inflammatory response. These impairments in the immune response could participate in failure of vaccination protocols and increased susceptibility to infections described in pigs exposed to AFB1.
Asunto(s)
Aflatoxina B1/toxicidad , Formación de Anticuerpos/efectos de los fármacos , Citocinas/efectos de los fármacos , Inmunidad Celular/efectos de los fármacos , Inmunotoxinas/toxicidad , Aflatoxina B1/administración & dosificación , Animales , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Isotipos de Inmunoglobulinas/efectos de los fármacos , Isotipos de Inmunoglobulinas/metabolismo , Inmunotoxinas/administración & dosificación , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Masculino , Mitógenos/metabolismo , Ovalbúmina/inmunología , Reacción en Cadena de la Polimerasa , Bazo/metabolismo , Porcinos , Vacunas/inmunologíaRESUMEN
Pigs are highly affected by dietary mycotoxin contamination and particularly by fumonisin. The effects of fumonisin on pig intestinal health are well documented, but little is known regarding its impact on gut microbiota. We investigate the effects of the fumonisin (FB1, 12 mg/kg feed) on the fecal microbiota of piglets (n = 6) after 0, 8, 15, 22, and 29 days of exposure. A control group of six piglets received a diet free of FB1. Bacterial community diversity, structure and taxonomic composition were carried out by V3â»V4 16S rRNA gene sequencing. Exposure to FB1 decreases the diversity index, and shifts and constrains the structure and the composition of the bacterial community. This takes place as early as after 15 days of exposure and is at a maximum after 22 days of exposure. Compared to control, FB1 alters the ecological succession of fecal microbiota species toward higher levels of Lactobacillus and lower levels of the Lachnospiraceae and Veillonellaceae families, and particularly OTUs (Operational Taxonomic Units) of the genera Mitsuokella, Faecalibacterium and Roseburia. In conclusion, FB1 shifts and constrains age-related evolution of microbiota. The direct or indirect contribution of FB1 microbiota alteration in the global host response to FB1 toxicity remains to be investigated.
Asunto(s)
Heces/microbiología , Fumonisinas/toxicidad , Microbioma Gastrointestinal , Envejecimiento , Alimentación Animal , Animales , Dieta , Masculino , Porcinos , DesteteRESUMEN
Gliotoxin is a mycotoxin having a considerable number of immuno-suppressive actions and is produced by several moulds such as Aspergillus fumigatus. In this study, we investigated its toxic effects on human neutrophils at concentrations corresponding to those found in the blood of patients with invasive aspergillosis. Incubation of the cells for 10min with 30-100ng/ml of gliotoxin inhibited phagocytosis of either zymosan or serum-opsonized zymosan without affecting superoxide production or the exocytosis of specific and azurophil granules. Gliotoxin also induced a significant re-organization of the actin cytoskeleton which collapsed around the nucleus leading to cell shrinkage and the disappearance of filopodia. This gliotoxin-induced actin phenotype was reversed by the cAMP antagonist Rp-cAMP and mimicked by pCPT-cAMP indicating that it probably resulted from the deregulation of intracellular cAMP homeostasis as previously described for gliotoxin-induced apoptosis. By contrast, gliotoxin-induced inhibition of phagocytosis was not reversed by Rp-cAMP but by arachidonic acid, another member of a known signalling pathway affected by the toxin. This suggests that gliotoxin can affect circulating neutrophils and favour the dissemination of A. fumigatus by inhibiting phagocytosis and the consequent killing of conidia.
Asunto(s)
Actinas/metabolismo , Aspergillus fumigatus/metabolismo , Citoesqueleto/efectos de los fármacos , Gliotoxina/toxicidad , Neutrófilos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Actinas/fisiología , Ácido Araquidónico/metabolismo , Aspergilosis/inmunología , Aspergilosis/metabolismo , Aspergilosis/microbiología , Aspergillus fumigatus/química , Aspergillus fumigatus/inmunología , AMP Cíclico/metabolismo , Citoesqueleto/metabolismo , Gliotoxina/inmunología , Gliotoxina/aislamiento & purificación , Gliotoxina/metabolismo , Humanos , NADPH Oxidasas/metabolismo , Neutrófilos/enzimología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Fagocitosis/inmunología , Transducción de Señal/efectos de los fármacosRESUMEN
Consequences of subchronic exposure to aflatoxin B1 (AFB1) on liver monooxygenase and transferase enzymes were compared in control pigs and pigs given 385, 867 or 1,807 microg AFB1/kg of feed for 4 weeks. Animals exposed to the highest dose of toxin developed clinical signs of aflatoxicosis, like liver fibrosis, hepatic dysfunction and decreased weight gain. This group had significantly lower levels of liver cytochrome P450, ethoxyresorufin O-deethylase (EROD) activity, testosterone metabolism, P450 1A and P450 3A protein expression. By comparison, mild degenerative hepatic changes, no hepatic dysfunction but a similar pattern of liver P450 enzymes activity without changes in P450 3A expression were observed in pigs exposed to 867 microg AFB1/kg of feed. Benzphetamine and aminopyrine N-demethylase activities were increased in pigs exposed to 867 or 1,807microg AFB1/kg of feed. Pigs exposed to 385 microg AFB1/kg of feed had low levels of EROD activity and all other biotransformation and clinical parameters remained at control levels. Aniline hydroxylase activity, P450 2C protein expression, UDP-glucuronosyl and glutathione S-transferase activities were unaffected at all doses of AFB1. In conclusion, P450 1A and P450 3A appear to be specific targets of AFB1 even if pig did not display clinical sign of liver toxicosis.
Asunto(s)
Aflatoxina B1/toxicidad , Hígado/enzimología , Oxigenasas de Función Mixta/antagonistas & inhibidores , Porcinos , Transferasas/antagonistas & inhibidores , Alimentación Animal , Animales , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Contaminación de Alimentos , Regulación Enzimológica de la Expresión Génica , Hígado/efectos de los fármacos , Masculino , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Transferasas/genética , Transferasas/metabolismoRESUMEN
An increasing number of human beings from developed countries are colonized by Escherichia coli strains producing colibactin, a genotoxin suspected to be associated with the development of colorectal cancers. Deoxynivalenol (DON) is the most prevalent mycotoxin that contaminates staple food-especially cereal products-in Europe and North America. This study investigates the effect of the food contaminant DON on the genotoxicity of the E. coli strains producing colibactin. In vitro, intestinal epithelial cells were coexposed to DON and E. coli producing colibactin. In vivo, newborn rats colonized at birth with E. coli producing colibactin were fed a DON-contaminated diet. Intestinal DNA damage was estimated by the phosphorylation of histone H2AX. DON exacerbates the genotoxicity of the E. coli producing colibactin in a time- and dose-dependent manner in vitro Although DON had no effect on the composition of the gut microbiota, and especially on the number of E. coli, a significant increase in DNA damage was observed in intestinal epithelial cells of animals colonized by E. coli strains producing colibactin and coexposed to DON compared to animals colonized with E. coli strains unable to produce colibactin or animals exposed only to DON. In conclusion, our data demonstrate that the genotoxicity of E. coli strains producing colibactin, increasingly present in the microbiota of asymptomatic human beings, is modulated by the presence of DON in the diet. This raises questions about the synergism between food contaminants and gut microbiota with regard to intestinal carcinogenesis.IMPORTANCE An increasing number of human beings from developed countries are colonized by Escherichia coli strains producing colibactin, a genotoxin suspected to be associated with the development of colorectal cancers. Deoxynivalenol (DON) is the most prevalent mycotoxin that contaminates staple food-especially cereal products-in Europe and North America. Our in vitro and in vivo results demonstrate that the intestinal DNA damage induced by colibactin-producing E. coli strains was exacerbated by the presence of DON in the diet. This raises questions about the synergism between food contaminants and gut microbiota with regard to intestinal carcinogenesis.
Asunto(s)
Daño del ADN/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Tracto Gastrointestinal/microbiología , Mutágenos/toxicidad , Péptidos/toxicidad , Policétidos/toxicidad , Tricotecenos/metabolismo , Animales , Técnicas de Cocultivo , Células Epiteliales/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Histonas/análisis , RatasRESUMEN
The few data available on fusarenon-X (FX) do not support the derivation of health-based guidance values, although preliminary results suggest higher toxicity than other regulated trichothecenes. Using histo-morphological analysis and whole transcriptome profiling, this study was designed to obtain a global view of the intestinal alterations induced by FX. Deoxynivalenol (DON) served as a benchmark. FX induced more severe histological alterations than DON. Inflammation was the hallmark of the molecular toxicity of both mycotoxins. The benchmark doses for the up-regulation of key inflammatory genes by FX were 4- to 45-fold higher than the previously reported values for DON. The transcriptome analysis revealed that both mycotoxins down-regulated the peroxisome proliferator-activated receptor (PPAR) and liver X receptor - retinoid X receptor (LXR-RXR) signaling pathways that control lipid metabolism. Interestingly, several pathways, including VDR/RXR activation, ephrin receptor signaling, and GNRH signaling, were specific to FX and thus discriminated the transcriptomic fingerprints of the two mycotoxins. These results demonstrate that FX induces more potent intestinal inflammation than DON. Moreover, although the mechanisms of toxicity of both mycotoxins are similar in many ways, this study emphasize specific pathways targeted by each mycotoxin, highlighting the need for specific mechanism-based risk assessments of Fusarium mycotoxins.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Yeyuno/efectos de los fármacos , Micotoxinas/toxicidad , Transducción de Señal/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Tricotecenos/toxicidad , Animales , Castración , Línea Celular , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Fusarium/química , Fusarium/patogenicidad , Perfilación de la Expresión Génica , Yeyuno/citología , Yeyuno/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Receptores X del Hígado/genética , Receptores X del Hígado/metabolismo , Masculino , Análisis por Micromatrices , Micotoxinas/aislamiento & purificación , Receptores Activados del Proliferador del Peroxisoma/genética , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Receptores X Retinoide/genética , Receptores X Retinoide/metabolismo , Transducción de Señal/genética , Porcinos , Técnicas de Cultivo de Tejidos , Tricotecenos/aislamiento & purificaciónRESUMEN
Pattern recognition receptors (PRRs) play a critical role in the detection of microorganisms and the induction of inflammatory and immune responses. Using PCR and Western-blot analysis, this study investigated the differential expression in the intestine of 14 PRRs and nine associated cytokines. Thirty-two pigs were used to determine the expression of these markers (1) along the proximal/distal axis of the small intestine (duodenum, jejunum, and ileum) and (2) between the intestinal segments and their respective lymphoid organs (Peyer's patches [PP] and mesenteric lymph nodes [MLN]). Six additional animals were used to quantify the expression of these genes along the crypt/villus axis of jejunum, using microdissected samples. Most genes showed increased expression (1) in the distal than in the proximal parts of the small intestine (TLR3, 5, RIG-I, IL-1ß, IL-8, and IFN-γ); (2) in lymphoid organs (TLR1, 2, 6, 9, 10, IL-10, TNF-α), especially the MLN (TLR4, 7, 8, NOD1, NOD2, NALP3, IFN-α, IL-6, IL-12, and TGF-ß), than in intestinal segments. The analysis along the crypt/villus identified: (1) genes with higher expression in lamina propria (TLR1, 2, 4, 9, NOD1, NOD2, IL-1ß, IL-10, TGF-ß, TNF-α) and (2) genes with higher expression in the villus (TLR3, 5, 6, RIG-I, IL-6). These results highlight the differential expression of PRRs and cytokines along the proximal/distal and the crypt/villus axis of the intestine, contributing to a fine analysis of the complex functional architecture of the small intestine and should be related to the gut microbiota.
RESUMEN
Annexin 1 is secreted by mammalian cells but lacks a leader signal sequence necessary to lead it to the classical secretory pathway via the endoplasmic reticulum. The mechanisms involved in the secretion of leaderless proteins remain uncertain. It has been suggested to involve membrane translocation via an ABC-transporter (ATP binding cassette). Using cultured inflamed mucosa from rectocolitis induced in rats, we studied if annexin 1 secretion followed the two main characteristics of ABC-transporter substrates: dependency on ATP hydrolysis and competitive inhibition by several other ABC-transporter substrates. Annexin 1 secretion is inhibited in a dose-dependent manner by two ATPase inhibitors. The inhibition reached 63.2+/-3.2%, 66.1+/-3.73% and 88.6+/-1.4% in the presence of 2mM vanadate, 0.5 and 1mM pervanadate, respectively. The efflux of calcein, a known ABC-transporter substrate, is similarly inhibited by 69.4+/-2.8% in the presence of 1mM pervanadate. Probenecid, an inhibitor of several ABC-transporters of the subfamilly ABCC or MRP (multidrug resistant associated protein), also inhibited annexin 1 secretion in a dose-dependent manner. As compared to control, 10mM probenecid reduced annexin 1 secretion by 72+/-20% and 20mM by 95.0+/-9%. By contrast, annexin 1 secretion is not blocked by other inhibitors of MRP1 (indomethacin, MK571), MRP2 (ochratoxin A1 or MK571), MRP5 (trequinsin or sulfinpyrazone) or by verapamil, cyclosporin A or glyburide. Taken together, our results show that annexin 1 secretion appears to share the efflux properties of ABC-transporter substrates.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Anexina A1/metabolismo , Membrana Mucosa/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Transporte Biológico , Fluoresceínas/metabolismo , Inflamación/inducido químicamente , Inflamación/patología , Ratones , Probenecid/farmacología , Ratas , Ácido TrinitrobencenosulfónicoRESUMEN
Patulin is a mycotoxin produced by fungal genera such as Aspergillus, Penicillium and Byssochlamys. It induces neurological, gastrointestinal and immunological effects, which is why patulin belongs to a short list of mycotoxins whose level in food is regulated in many countries around the world. Recently, a cluster gathering 15 genes involved in the biosynthesis of patulin has been identified in Aspergillus clavatus, but so far, only 4 genes encoding 6-methylsalicylic acid synthase, m-cresol hydroxylase, m-hydroxybenzyl alcohol hydroxylase and isoepoxydon dehydrogenase have been characterized. Previous studies have shown the involvement of a decarboxylase in the transformation of 6-methylsalicylic acid, the first stable patulin precursor, into m-cresol. In this study a putative decarboxylase gene, PatG, was identified in the genome sequence of A. clavatus. This gene is located near two P450 cytochrome genes PatH and PatI responsible respectively for the hydroxylation of m-cresol and m-hydroxybenzyl alcohol. This decarboxylase encoded by PatG (ACLA_093620) consists of 325 amino acids. The search for putative conserved domain revealed that the gene product belongs to the AminoCarboxyMuconate Semialdehyde Decarboxylase (ACMSD) related protein family. This family includes decarboxylases such as the γ-resorcylate decarboxylase or o-pyrocatechuate decarboxylase. The substrates of these enzymes display strong structural similarities with 6-methylsalicylic acid. PatG was strongly expressed during patulin production whereas it was very weakly expressed in non-patulin permissive conditions. The coding sequence was used to enable heterologous expression of functional enzymes in Saccharomyces cerevisiae. The presence of decarboxylase was confirmed by Western blot. The bioconversion assays showed that PATG catalyzed the decarboxylation of 6-methylsalicylic acid into m-cresol. These results confirm for the first time that 6-methylsalicylic acid is the substrate for PATG, the 6-methylsalicylic acid decarboxylase. With this study, the four genes involved in the four first steps of patulin biosynthesis pathway (acetateâgentisyl alcohol) are now identified.
Asunto(s)
Aspergillus/enzimología , Aspergillus/genética , Carboxiliasas/genética , Carboxiliasas/metabolismo , Patulina/biosíntesis , Aspergillus/química , Aspergillus/clasificación , Carboxiliasas/química , Sistema Enzimático del Citocromo P-450/metabolismo , Microbiología de Alimentos , Regulación Fúngica de la Expresión Génica , Datos de Secuencia Molecular , Patulina/análisisRESUMEN
SCOPE: Deoxynivalenol (DON), a mycotoxin produced by Fusarium spp., is toxic to many animal species, with pigs being the most sensitive species to the toxin. The aim of the present study was to determine the effects of DON on pig polymorphonuclear cells (PMNs), the first line of defense against infection. METHODS AND RESULTS: PMNs isolated from pig blood samples were stimulated with LPS to mimic infection. DON (0.5-10 µM) altered three main functions of pig PMNs: LPS-induced secretion of IL-8, chemotaxis, and phagocytosis capability. This alteration of PMN properties was due to apoptotis induced by DON exposure. Using Western blot and flow cytometry, we demonstrated that this process included the permeabilization of the mitochondrial outer membrane and the activation of caspase-3. The effect of DON was mediated by the phosphorylation of the p38 mitogen-activated protein kinase within the first 30 min of exposure. CONCLUSION: This study provides evidence that low concentrations of DON can alter the immune functions of porcine PMNs and suggests the involvement of p38 mitogen-activated protein kinase in the signal transduction pathway. These immunosuppressive effects of DON may have implications for humans and/or animals when eating contaminated food/feed.
Asunto(s)
Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Tricotecenos/toxicidad , Animales , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Quimiotaxis/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Escherichia coli/patogenicidad , Peróxido de Hidrógeno/metabolismo , Interleucina-8/metabolismo , Lipopolisacáridos/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Micotoxinas/toxicidad , Neutrófilos/microbiología , Fagocitosis/efectos de los fármacos , Porcinos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Trichothecenes induce changes in the intestinal barrier function through decreased expression of cell junction proteins and apoptosis of enterocytes. The mitogen activated protein kinases (MAPK) play an important role in the signaling pathways of cell turnover and differentiation. Using ex vivo and in vivo approaches, the purpose of this study was to investigate the ability of low doses of DON to induce histological changes in the intestine and to activate the MAPK ERK 1/2, p38 and JNK. Twelve weaning piglets received during four weeks a control diet or a DON-contaminated diet (2.3 mg DON/kg feed). Six weaning piglets were used to prepare jejunal explants (ex vivo model). Explants were exposed during 4 h to vehicle, 5 or 10 µM DON. Intestinal changes were graded using a histological score. Pigs fed a DON-diet and explants exposed to DON showed a significant decrease in the jejunal score. In both models, the toxin significantly enhanced phosphorylation of ERK 1/2 and p38, whereas the increased phosphorylation of JNK was non significant. Taken together these results indicate that in vivo or ex vivo exposure of intestinal tissue to DON lead to similar intestinal lesions and activation of MAPK. These effects could impair the homeostasis of intestinal tissue in the aspects of barrier function and immune protection. The similarity of the in vivo and ex vivo results provides also strong evidence that the jejunal explant model is a good alternative for toxicological studies in intestinal tissue.
Asunto(s)
Alternativas a las Pruebas en Animales , Contaminación de Alimentos , Yeyuno/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Tricotecenos/toxicidad , Alimentación Animal , Animales , Relación Dosis-Respuesta a Droga , Yeyuno/enzimología , MAP Quinasa Quinasa 4/metabolismo , Masculino , Orquiectomía , Fosforilación , Porcinos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The intestinal epithelium is the first barrier against food contaminants and is highly sensitive to mycotoxins, especially de oxynivalenol (DON). Consumption of DON-contaminated food is associated with outbreaks of gastroenteritis. In cereals and their byproducts, DON is present together with two acetylated derivatives, 3-ADON and 15-ADON. The aim of this study was to compare the intestinal toxicity of DON and A-DONs, using noncytotoxic doses. The toxicity was assessed using in vitro (intestinal epithelial cell line), ex vivo (intestinal explants), and in vivo (animals exposed to mycotoxin-contaminated diets) models. The effects were studied on cell proliferation, barrier function, and intestinal structure. The mechanism of toxicity was investigated by measuring the expression of the tight junction proteins and of phosphorylated ERK1/2, p38, and JNK, which are effectors of signaling pathway involved in cellular programs including embryogenesis, proliferation, differentiation, and apoptosis. On proliferating cells, 3-ADON was less toxic than DON, which was less toxic than 15-ADON. On differentiated cells, 15-ADON impaired the barrier function, whereas DON and 3-ADON did not have a significant effect. Similarly, ex vivo and in vivo, 15-ADON caused more histological lesions than DON or 3-ADON. At the molecular level, the 15-ADON activated the mitogen-activated protein kinases (MAPK) ERK1/2, p38, and JNK in the intestinal cell line, explants, and the jejunum from exposed animals at lower dose than DON and 3-ADON. Our results show that the higher toxicity of 15-DON is due to its ability to activate the MAPK. Given that cereal-based foods are contaminated with DON and acetylated-DON, the higher toxicity of 15-ADON should be taken into account.
Asunto(s)
Intestino Delgado/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Uniones Estrechas/efectos de los fármacos , Tricotecenos/toxicidad , Animales , Animales Recién Nacidos , Línea Celular , Proliferación Celular/efectos de los fármacos , Fenómenos Electrofisiológicos , Contaminación de Alimentos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Intestino Delgado/metabolismo , Intestino Delgado/patología , Técnicas de Placa-Clamp , Porcinos , Proteínas de Uniones Estrechas/metabolismo , Uniones Estrechas/fisiologíaRESUMEN
BACKGROUND: Increasing robustness via improvement of resistance to pathogens is a major selection objective in livestock breeding. As resistance traits are difficult or impossible to measure directly, potential indirect criteria are measures of immune traits (ITs). Our underlying hypothesis is that levels of ITs with no focus on specific pathogens define an individual's immunocompetence and thus predict response to pathogens in general. Since variation in ITs depends on genetic, environmental and probably epigenetic factors, our aim was to estimate the relative importance of genetics. In this report, we present a large genetic survey of innate and adaptive ITs in pig families bred in the same environment. METHODOLOGY/PRINCIPAL FINDINGS: Fifty four ITs were studied on 443 Large White pigs vaccinated against Mycoplasma hyopneumoniae and analyzed by combining a principal component analysis (PCA) and genetic parameter estimation. ITs include specific and non specific antibodies, seric inflammatory proteins, cell subsets by hemogram and flow cytometry, ex vivo production of cytokines (IFNα, TNFα, IL6, IL8, IL12, IFNγ, IL2, IL4, IL10), phagocytosis and lymphocyte proliferation. While six ITs had heritabilities that were weak or not significantly different from zero, 18 and 30 ITs had moderate (0.1
0.4) heritability values, respectively. Phenotypic and genetic correlations between ITs were weak except for a few traits that mostly include cell subsets. PCA revealed no cluster of innate or adaptive ITs. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that variation in many innate and adaptive ITs is genetically controlled in swine, as already reported for a smaller number of traits by other laboratories. A limited redundancy of the traits was also observed confirming the high degree of complementarity between innate and adaptive ITs. Our data provide a genetic framework for choosing ITs to be included as selection criteria in multitrait selection programmes that aim to improve both production and health traits.
Asunto(s)
Inmunidad Adaptativa/genética
, Marcadores Genéticos
, Variación Genética
, Inmunidad Innata/genética
, Enfermedades de los Porcinos/genética
, Enfermedades de los Porcinos/inmunología
, Animales
, Cruzamiento
, Citocinas/metabolismo
, Femenino
, Citometría de Flujo
, Masculino
, Mycoplasma hyopneumoniae/inmunología
, Fenotipo
, Neumonía Porcina por Mycoplasma/genética
, Neumonía Porcina por Mycoplasma/inmunología
, Neumonía Porcina por Mycoplasma/prevención & control
, Análisis de Componente Principal
, Selección Genética
, Porcinos
, Vacunación
RESUMEN
Patulin is an acetate-derived tetraketide mycotoxin produced by several fungal species, especially Aspergillus, Penicillium and Byssochlamys species. The health risks due to patulin consumption by humans have led many countries to regulate it in human food. Previous studies have shown the involvement of cytochrome P450 monooxygenases in the hydroxylation of two precursors of patulin, m-cresol and m-hydroxybenzylalcohol. In the present study, two cytochrome P450 genes were identified in the genome sequence of Aspergillus clavatus, a patulin-producing species. Both mRNAs were strongly co-expressed during patulin production. CYP619C2, encoded by the first gene, consists of 529 aa, while the second cytochrome, CYP619C3, consists of 524 aa. The coding sequences were used to perform the heterologous expression of functional enzymes in Saccharomyces cerevisiae. The bioconversion assays showed that CYP619C3 catalysed the hydroxylation of m-cresol to yield m-hydroxybenzyl alcohol. CYP619C2 catalysed the hydroxylation of m-hydroxybenzyl alcohol and m-cresol to gentisyl alcohol and 2,5-dihydroxytoluene (toluquinol), respectively. Except for the last compound, all enzyme products are known precursors of patulin. Taken together, these data strongly suggest the involvement of CYP619C2 and CYP619C3 in the biosynthesis of patulin. CYP619C2 and CYP619C3 are located near to two other genes involved in patulin biosynthesis, namely the 6-methylsalicylic acid synthase (6msas) and isoepoxydon dehydrogenase (idh) genes. The current data associated with an analysis of the sequence of A. clavatus suggest the presence of a cluster of 15 genes involved in patulin biosynthesis.