Development of a pig jejunal explant culture for studying the gastrointestinal toxicity of the mycotoxin deoxynivalenol: histopathological analysis

The digestive tract is a target for the mycotoxin deoxynivalenol (DON), a major cereals grain contaminant of public health concern in Europe and North America. Pig, the most sensitive species to DON toxicity, can be regarded as the most relevant animal model for studying the intestinal effects of DON. A pig jejunal explants culture was developed to assess short-term effects of DON. In a first step, jejunal explants from 9-13 week-old and from 4-5 week-old pigs were cultured in vitro for up to 8 hours. Explants from younger animals were better preserved after 8 hours, as assessed by morphological scores and by villi lengths. In a second step, dose-related alterations of the jejunal tissue were observed, including shortened and coalescent villi, lysis of enterocytes, oedema. After 4h of DON exposure of explants from 4-5 week-old pigs, a no-effect concentration level of 1 µM was estimated (corresponding to diet contaminated with 0.3 mg DON/kg) based on morphological scores, and of 0.2 µM based on villi lengths. In conclusion, our data indicate that pig intestinal explants represent a relevant and sensitive model to investigate the effects of food contaminants

Effects of Mycotoxins on the Intestine

Mycotoxins are secondary metabolites produced by several fungal species. They can contaminate human food and animal feed, and have been a threat for thousands of years. The gastrointestinal tract is the first target when ingesting mycotoxin-contaminated food or feed. As unlikely as it sounds, the investigations concerning the effects of mycotoxins on the intestine are still in their early stages. This book gathers the most recent advances related to the characterization of the intestinal toxicity of mycotoxins. Substantial data assembled on the damage caused to a number of histological structures and functions of the intestine remove any remaining doubt about this organ being a primary target for the toxicity of mycotoxins. An interesting overview of the detrimental effects of mycotoxins on the gut-hosted microbiota—now regarded as a fully-fledged organ associated with the gut—is also given. Finally, outstanding contributions in this book address questions relating to the suitability of current regulations to protect against alterations of the intestine, and to the efficacy assessment of new detoxification strategies using the intestinal toxicity of mycotoxins as a relevant endpoint

The low intestinal and hepatic toxicity of hydrolyzed fumonisin B1 correlates with its inability to alter the metabolism of sphingolipids

Fumonisins are mycotoxins frequently found as natural contaminants in maize, where they are produced by the plant pathogen Fusarium verticillioides. They are toxic to animals and exert their effects through mechanisms involving disruption of sphingolipid metabolism.Fumonisin B1 (FB1) is the predominant fumonisins in this family. FB1 is converted to its hydrolyzed analogs HFB1, by alkaline cooking (nixtamalization) or through enzymatic degradation. The toxicity of HFB1 is poorly documented especially at the intestinal level. The objectives of this study were to compare the toxicity of HFB1 and FB1 and to assess the ability of these toxins to disrupt sphingolipids biosynthesis. HFB1 was obtained by a deesterification of FB1, with a carboxylesterase. Piglets, animals highly sensitive to FB1, were exposed by gavage for 2 weeks to 2.8 µmol FB1 or HFB1/kg body weight/day. FB1 induced hepatotoxicity as indicated by the lesion score, the level of several biochemical analytes and the expression of inflammatory cytokines. Similarly, FB1 impaired the morphology of the different section of the small intestine, reduced villi height and modified intestinal cytokine expression. By contrast, HFB1 did not trigger hepatotoxicity, did not impair intestinal morphology and slightly modified the intestinal immune response. This low toxicity of HFB1 correlates with a weak alteration of the sphinganine/sphingosine ratio in the liver and in the plasma. Taken together, these data demonstrate that HFB1 does not cause intestinal or hepatic toxicity in the sensitive pig model, and slightly disrupts sphingolipids metabolism. This finding suggests that conversion to HFB1 could be a good strategy to reduce FB1 exposure

Deciphering the Anti-Aflatoxinogenic Properties of Eugenol Using a Large-Scale q-PCR Approach

Produced by several species of Aspergillus, Aflatoxin B1 (AFB1) is a carcinogenic mycotoxin contaminating many crops worldwide. The utilization of fungicides is currently one of the most common methods; nevertheless, their use is not environmentally or economically sound. Thus, the use of natural compounds able to block aflatoxinogenesis could represent an alternative strategy to limit food and feed contamination. For instance, eugenol, a 4-allyl-2-methoxyphenol present in many essential oils, has been identified as an anti-aflatoxin molecule. However, its precise mechanism of action has yet to be clarified. The production of AFB1 is associated with the expression of a 70 kB cluster, and not less than 21 enzymatic reactions are necessary for its production. Based on former empirical data, a molecular tool composed of 60 genes targeting 27 genes of aflatoxin B1 cluster and 33 genes encoding the main regulatory factors potentially involved in its production, was developed. We showed that AFB1 inhibition in Aspergillus flavus following eugenol addition at 0.5 mM in a Malt Extract Agar (MEA) medium resulted in a complete inhibition of the expression of all but one gene of the AFB1 biosynthesis cluster. This transcriptomic effect followed a down-regulation of the complex composed by the two internal regulatory factors, AflR and AflS. This phenomenon was also influenced by an over-expression of veA and mtfA, two genes that are directly linked to AFB1 cluster regulation

The food contaminant deoxynivalenol, decreases intestinal barrier permeability and reduces claudin expression

The gastrointestinal tract represents the first barrier against food contaminants as well as the first target for these toxicants. Deoxynivalenol (DON) is a mycotoxin that commonly contaminates cereals and causes various toxicological effects. Through consumption of contaminated cereals and cereal products, human and pigs are exposed to this mycotoxin. Using in vitro, ex vivo, and in vivo approaches, we investigated the effects of DON on the intestinal epithelium. We demonstrated that, in intestinal epithelial cell lines from porcine (IPEC-1) or human (Caco-2) origin, DON decreases trans-epithelial electric resistance (TEER) and increases in a time and dose-dependent manner the paracellular permeability to 4 kDa dextran and to pathogenic Escherichia Coli across intestinal cell monolayers. In pig explants treated with DON, we also observed an increased permeability of intestinal tissue. These alterations of barrier function were associated with a specific reduction in the expression of claudins, which was also seen in vivo in the jejunum of piglets exposed to DON-contaminated feed. In conclusion, DON alters claudin expression and decreases the barrier function of the intestinal epithelium. Considering that high levels of DON may be present in food or feed, consumption of DON-contaminated food/feed may induce intestinal damage and has consequences for human and animal health

Pathophysiological changes occurring during Escherichia coli endotoxin and Pasteurella multocida challenge in piglets: relationship with cough and temperature and predicitive value for intensity of lesions.

The aims of this study were (1) to correlate cough and body temperature (BT) with the severity of bronchopneumonia in pigs, (2) to determine whether these clinical signs can be used to early diagnose bronchopneumonia and (3) to assess the predictive values of cough and BT regarding lung lesions. Bronchopneumonia was induced by administering E. coli endotoxin (LPS) combined with Pasteurella multocida type A (PmA) in the trachea of 13 piglets. Saline-instilled negative controls (n = 8), PmA inoculated (n = 6) and LPS instilled (n = 5) groups were also constituted. Cough and BT were recorded daily while the bronchopneumonia severity was assessed using bronchoalveolar lavage fluid (BALF) cytology, cytokines and measurement of lung lesion volume. Changes in expiratory breathing pattern were also measured (Penh). The combination of LPS and PmA induced a subacute bronchopneumonia characterised by macrophage, neutrophil, and lymphocyte infiltration, changes in Penh and an increase in the mRNA level of IFN-gamma while IL8, IL-18 and TNF-alpha mRNA levels remained unchanged. The daily body weight gain of infected animals was significantly reduced. Cough and BT changes were proportional to the intensity of the lung inflammatory process, functional respiratory changes and to the extent of macroscopic lesions. When comparing the individual values of cough and BT to thresholds defined for both parameters, an early diagnosis of pneumonia was possible. Considering the pooled data of each group, it was possible to define thresholds allowing an early segregation between the groups of diseased and healthy piglets. The daily values of cough and BT were predictive for the volume of lung lesions recorded at the end of the trial. In conclusion, cough and BT appear as potential indicators for the intensity and the evolution of the respiratory disease. They also seem to be good predictors for the magnitude of lung lesions and weight gain recorded at the study endpoint

Effect of fumonisins and Salmonella on digestive flora profiles assessed using a molecular tool (CE-SSCP).

Fumonisins (FB) are mycotoxins frequently found in vegetal feedstuffs, especially in maize used for pig feeding. Among fumonisins, FB1 was the better described toxin. It caused pulmonary and hepatic damages as well as immune response disorders in pigs that were recognised as especially sensitive to FB Intoxication. The FB1 immunosuppressor induced a higher susceptibility of pigs to gut pathogens such as E coli. Effects on Salmonella have poorly been studied despite the frequent asymptomatic carnage in pigs and the presumptive role of nora equilibrium on prevention of Salmonella excretion or re-excretion. To determine the influence of Salmonella carriage, fumonisins or both on digestive flora equilibrium, the use of a molecular technique CE-SSCP (Capillary-Electrophoresis Single Strand Conformation Polymorphism) appeared a good complement to the conventional bacteriological techniques. The objective was to assess the perturbation of nora associated with co-exposition in experimental conditions in absence of clinical sign

The food contaminant fumonisin B1 reduces the maturation of porcine CD11R1+ intestinal antigen presenting cells and antigen-specific immune responses, leading to a prolonged intestinal ETEC infection

Consumption of food or feed contaminated with fumonisin B1 (FB1), a mycotoxin produced by Fusarium verticillioides, can lead to disease in humans and animals. The present study was conducted to examine the effect of FB1 intake on the intestinal immune system. Piglets were used as a target and as a model species for humans since their gastro-intestinal tract is very similar. The animals were orally exposed to a low dose of FB1 (1 mg/kg body weight FB1) for 10 days which did not result in clinical signs. However, when compared to non-exposed animals, FB1-exposed animals showed a longer shedding of F4+ enterotoxigenic Escherichia coli (ETEC) following infection and a lower induction of the antigen-specific immune response following oral immunization. Further analyses to elucidate the mechanisms behind these observations revealed a reduced intestinal expression of IL-12p40, an impaired function of intestinal antigen presenting cells (APC), with decreased upregulation of Major Histocompatibility Complex Class II molecule (MHC-II) and reduced T cell stimulatory capacity upon stimulation. Taken together, these results indicate an FB1-mediated reduction of in vivo APC maturation

Porcine small and large intestinal microbiota rapidly hydrolyze the masked mycotoxin deoxynivalenol-3-glucoside and release deoxynivalenol in spiked batch cultures in vitro

This study was supported by the Scottish Government Rural and Environment Science and Analytical Services division (RESAS) and by the French Agence Nationale de la Recherche (project ANR-13-CESA-0003-03). We thank Anne-Marie Cossalter for her excellent technical assistance with pigs.Peer reviewedPostprin

MOLEKULARNE INTERAKCIJE MYCOTOXINA SA JETRENIM ENZIMIMA KOJI SUDJELUJU U METABOLIZMU MEDIKAMENATA U GLODAVCA I DOMAĆIH ŽIVOTINJA

Mycotoxins are well known for underging liver biotransformation in humans and animal species. Metabolites correspond to either oxydative derivatives such as hydroxymetabolites of aflatoxin B1 or ochratoxin A or hydrolytic derivatives in case of trichothecenes. In some cases, highly reactive epoxides represent the first step in the formation of carcinogenic intermediates like exo-epoxides of aflatoxins. Hepatic phase II enzymes including transferases and hydrolases are involved in the conjugation of such oxidative metabolites. In this respect, they are generally considered as detoxifying enzymes: glucuronidation of deacetylated trichothecenes or hydroxy-aflatoxins, or glutathione conjugation of epoxides. The major metabolism of zearalenone consists of reduction leading to estrogenic zearalenols which is characterized by large interspecies differences. Concerning fumonisin B1, this toxin would be poorly absorbed from the gastrointestinal tract and metabolised into hydrolytic products with lower toxic effect as apoptotic compounds. Interactions between mycotoxins and liver drug metabolizing are crucial in terms of detoxication or bioactivation of these toxins in the organism of the human or animal consumers. Most of these interactions are consequences of the metabolic processes occurring in the liver. They result generally from the activity of cytochromes P450 and transferases. In relation to their hepatotoxicity, several studies demonstrate the inhibitory effects of mycotoxins on certain hepatic biotransformation enzymes, as recently demonstrated in pigs exposed to low doses of aflatoxin B1 or T-2 toxin. In other cases, specific cytochromes P450 or glutathione transferases are significantly increased in terms of both activity and protein expression, namely by aflatoxins, deoxynivalenol or fumonisins. Such results have been obtained in rodents and in farm animals like pigs, rabbits or poultry. The data strengthen the hypothesis that the normal metabolism of endobiotes or xenobiotics by the liver could be altered during chronic exposure to mycotoxins, particularly in farm animals or in humans exposed to aflatoxin B1, ochratoxin A, T-2 toxin, deoxynivalenol or fumonisin B1.Ulaskom u organizam ljudi i životinja mnogi mikotoksini podliježu biotransformaciji u jetri. Njihovi su metaboliti najčešće oksidativni derivati (hidroksimetaboliti aflatoksina B1 i okratoksina A) ili hidrolitički derivati trihotecena. U nekim slučajevima visoko reaktivni epoksidi su prvi korak u stvaranju karcinogenih intermedijera kao što su ekso-epoksidi aflatoksina. Hepatički enzimi II faze (transferaze i hidrolaze) sudjeluju u konjugaciji tih oksidativnih metabolita tj. njihovoj detoksifikaciji: glukoronidaciji deacetiliranih trihotecena i hidroksi-aflatoksina ili konjugaciji epoksida s glutationom. Zearalenon se reducira u više različitih estrogenih zearalenola, što ovisi o vrsti organizma koji ga metabolizira. Fumonizin B1 se slabo apsorbira iz probavnog sustava te se metabolizira u hidrolitičke produkte koji su manje toksični. Interakcije mikotoksina i hepatičkog metabolizma ključni su koraci u njihovoj detoksifikaciji ili bioaktivaciji u organizmu ljudi i životinja. Večina tih interakcija posljedica su upravo metaboličkog procesa u jetri, posebice aktivnosti citokroma P450 i transferaza. Nedavno je dokazan inhibitorni učinak aflatoksina B1 i trihotecena na određene hepatičke biotransformacijske enzime u svinja. S druge strane, aktivnost i ekspresija proteina specifičnih citokroma P450 i glutation transferaze značajno su povećani nakon djelovanja aflatoksina, deoksinivalenola i fumonizina, što je zabilježeno u glodavaca te domaćih životinja (svinje, kunići i perad). Ova istraživanja pokazuju da normalni hepatički metabolizam endobiotika i ksenobiotika može biti narušen tijekom kroničnog unosa mikotoksina, posebice u domaćih životinja i ljudi izloženih aflatoksinu B1, okratoksinu A, T-2 toksinu, deoksinivalenolu i fumonizinu B1