Oswald I P La

Dr Isabelle P Oswald
email: isabelle.oswald@toulouse.inra.fr

Alix Pierron

Sabria Mimoun

Leticia S Murate

Nicolas Loiseau

Yannick Lippi

Ana-Paula F L Bracarense

Gerd Schatzmayr

Jianwei He

Ting Zhou

Wulf- Dieter Moll

Microbial biotransformation of DON: molecular basis for reduced toxicity

Keywords: Pig, mycotoxins, feed contamination, intestine, barrier function, immune response trichothecenes, modified mycotoxins, gut, Fusarium, wheat

Mycotoxins are secondary metabolites of fungi that grow on a variety of substrates. Due to their high consumption of cereals and their sensitivity, pigs are highly impacted by the presence of mycotoxins.

Pigs can be exposed to different mycotoxins such as aflatoxins, ochratoxins, fumonisins, zearalenone, and trichothecenes especially deoxynivalenol. At the European level, regulations and recommendations exist for these mycotoxins in pig feed. The intestine is the first barrier to food contaminants and can be exposed to high concentrations of mycotoxins upon ingestion of contaminated feed. Mycotoxins target this organ, they alter the intestinal barrier, impair the immune response, reduce feed intake and weight gain. Among them, deoxynivalenol and fumonisin have been studied especially for their toxicity in the intestine. Their presence in feed increases the translocation of bacteria; mycotoxins can also impair the immune response and enhance the susceptibility to infectious diseases. In conclusion, because of their effect on the intestine, mycotoxins are a major threat to pig health, welfare and performance.

THANKS

The present work was possible by a fellowship from CIFRE (2012/0572, jointly financed by the BIOMIN Holding GmbH, Association Nationale de la Recherche Technique (ANRT) and the laboratory of INRA-ToxAlim, UMR 1331.

 I would like to thank all researchers who have accepted to be part of my jury:  Prof.Chiara Dall'Asta, Dr. Sven Dänicke and Prof. Alexis Valentin, who accepted to evaluate my work.

 Dr. Wulf-Dieter Moll, for having accepted to be my co-director during this PhD, for his support and all his advice and for trusting me during these 4 years.

 Dr. Isabelle Oswald :

Isabelle, merci à toi de m'avoir donné ma chance en stage d'ingénieur, puis d'avoir continué à me faire confiance pour une thèse. Merci de m'avoir permis de faire de nombreuses expérimentations, supplémentaires, non prévues… Je te remercie de m'avoir permis de participer à de nombreux congrès et réunions à l'étranger qui m'ont fait énormément progresser et évoluer! Enfin, merci de m'avoir suivie, conseillée, à toute heure, jour et nuit, 7 jours sur 7, pour rendre sans cesse mon travail meilleur. Ta capacité de travail et de gestion de nombreux projets simultanément est impressionnante. En un mot j'ai adoré ma thèse sous ta direction.

 BIOMIN :

I would like to thank the company for funding my thesis, allowing me to make many varied experiments in my research during my PhD. I thank BIOMIN France and BIOMIN Austria, as well as Gerd Schatzmayr, Chirstian Tenier, Wulf-Dieter Moll to make possible this collaboration. They gave me the opportunity to visit them several times in Austria, go to several congresses where I met many researchers. All of this was very rewarding. Dieter, Gerd, it was a great pleasure to work with you during this period.



Merci à tous les collaborateurs qui ont participé directement ou indirectement à la réalisation de ces travaux. Merci à Ting Zhou et à Franz Berthiller pour nous avoir procuré les différentes molécules nécessaires à mes analyses.

Un grand merci au Professeur Ana-Paula Loureiro Bracarensede l'école vétérinaire de Londrina au Brésil, pour avoir réalisé toutes les coupes et analyses histologiques des expérimentations in vivo. J'ai été enchantée de te rencontrer et de bénéficier de tes conseils.

 Je tiens aussi à remercier toutes les personnes qui m'ont accompagnée pendant ces années de travail au sein de ToxAlim, de près ou de loin, et que j'ai eu le plaisir de rencontrer ; qui m'ont aidé, conseillé, soutenu, encouragé et avec qui j'ai eu le plaisir de papotter!  A ma chère équipe, tu vas me manquer! Ces 4 années et 4 mois tout compris, que j'ai passé au sein de cette équipe ont été super, riches en enseignement et en rencontre. Merci à tous les membres qui la composent, qui font fonctionner la machine et qui font en sorte que notre séjour, plus ou moins long, soit toujours agréable ! Ce sera difficile de trouver une équipe aussi chouette, avec des personnes aussi travailleuses, dynamiques, enjouées et compétentes que celles qui la compose.

Joëlle, telle une abeille butineuse tu t'affaires sans relâche pour que toute la ruche fonctionne correctement et ait toujours assez pour se nourrir et fonctionner! Merci Joëlle.

Philippe, tel le sage de la montagne, tu sais tout ! Toujours patient, tu écoutes et promulgue soit un bon conseil, une astuce ou encore des précautions! Je suis contente d'avoir pu travailler avec toi.

Anne, que dire…, peut-être que de passer la meilleure des journées c'est de la passer avec toi et les cochons! Anne je te souhaite tout le meilleur pour la suite et encore plus.

Je remercie aussi tous les autres membres de l'équipe pour leur disponibilité, conseils et bonne humeur : Olivier, Sophie, Soraya, Sylviane, Jean-Denis, Arlette ; cela a été un plaisir de travailler à vos côtés.

Une pensée à tous les stagiaires, thésards, post-docs que j'ai eu le plaisir de rencontrer et de côtoyer de plus ou moins près pendant ma thèse, et pour tous les bons moments passés en Une pensée pour tous mes cochons, décédés mais non moins aimés. A mon Marshmallow trop vite parti…, ainsi que Baloo et tous les autres.



Un grand merci à mon club de Kung Fu, mon équipe de volley et à mes compagnons de sorties roller et danse…, qui m'ont permis de bien me défouler et de me vider la tête au besoin.



Merci à tout mon entourage, de plus ou moins loin, qui m'ont soutenu, réconforté et aussi permis de me changer les idées, merci d'être là ! Je citerai en particulier Sarah (ma BF), Florent (mon BF), Christelle pour votre soutien actif !  Un mot à ma famille (plus ou moins éloignée), inébranlable, parfaite, qui m'a toujours soutenue, encouragée, portée, cette thèse est aussi pour vous. Je vous aime : Jacky, Jojo, Maman.

Introduction Materials and Method Results Discussion

List of Abbreviations

I. Context of the study

Plants and cereals are subject to numerous fungal contaminations occurring either in fields or during storage processes. Many species of fungi exist and are able to grow on various types of cereals (maize, wheat, barley, soybean, rice, rye…) or on food commodities (seeds, peanuts, fruits, spices, forages…) (Figure 1) [START_REF] Afssa | Evaluation des risques liés à la présence de mycotoxines dans les chaînes alimentaires humaine et animale[END_REF]. These fungi are able to produce several toxic molecules, called mycotoxins (from Greek μύκης (mykes, mukos) 'fungus' and Latin (toxicum) 'poison') [START_REF] Aiko | Occurrence, detection and detoxification of mycotoxins[END_REF].

Mycotoxins are secondary metabolites; and unlike primary metabolites, they are not essential to the development and survival of the fungus but could constitute an advantage during the colonization of ecological niche when in competition with other microorganism. These molecules also discourage predators from eating the fungus (Keller et al. 2005).

Mycotoxins can be divided into polyketoacids, terpenes, cyclopepetides and nitrogen metabolites, depending on their origins and their structures [START_REF] Afssa | Evaluation des risques liés à la présence de mycotoxines dans les chaînes alimentaires humaine et animale[END_REF]. They can also be classified according to their toxic effects. Mycotoxins considered important in terms of food safety are aflatoxins (AF), ochratoxins (OT) (in particular ochratoxin A (OTA)), patulin (PAT), fumonisins (FB), zearalenone (ZEA) and trichothecenes (TCT), especially deoxynivalenol (DON) (Figure 2) (Bennet and Klich 2003). Several factors control fungal growth and mycotoxin production, such as weather conditions, agricultural practices or storage conditions [START_REF] Hesseltine | Mycotoxin research in india[END_REF]. The toxic effects of moulds and fungi were already known in ancient times.

Historically, many illnesses linked to mycotoxicoses have been reported [START_REF] Afssa | Evaluation des risques liés à la présence de mycotoxines dans les chaînes alimentaires humaine et animale[END_REF]. The most famous case, which occurred in the Middle Ages, is known under the name of ignis sacer (sacred fire) or St Anthony's fire. It was caused by toxins of Claviceps purpurea, the ergot alkaloids of rye (Figure 3). Ergotism reached epidemic proportions, mutilating and killing thousands of people in Europe. Victims of ergotism suffered from delirium, prostration, acute pain, abscess and gangrene of the extremities, leading to serious and incurable infirmity. Epidemics occurred from the 8 th to the 15 th century due to the bad quality of food and contamination with fungal sclerotia. Similarly, fusariotoxins (toxin T2 and ZEA), seem to have been involved in the decline of the Etruscan civilization 5 centuries B.-C [START_REF] Richard | Mycotoxins and human disease; in clinical mycology (eds) ej anaissie, mr mcginnis and ma pfaller[END_REF]. "Yellow rice disease" or shoshin-kakke disease in Japan was also a mycotoxicosis caused by unhygienic conditions and practices, which is induced by the Citreoviridin, a metabolite produced by Penicillium citreonigrum. This fungus used to grow readily on rice during its storage (after harvest), especially in the colder regions of Japan. New hygiene measures applied, more rigorous than before, made it disappear [START_REF] Udagawa | safety of rice grains and mycotoxins -a historical review of yellow rice mycotoxicoses[END_REF]. In 1960, the turkey X disease has been an important episode of mycotoxicosis on animals. It killed thousands of turkey, ducklings and other domestic animals in England.

This allowed the discovery of aflatoxins, main mycotoxin produced by Aspergillus flavus, and present in a high quantity in groundnut flour designed for poultry's food [START_REF] Blount | Turkey "X" Disease[END_REF]. Most mycotoxins have an acute toxicity, but nowadays it's exceptional to be exposed to such high doses in Europe [START_REF] Européenne | Scoop task 3.2.10. Collection of occurrence data of fusarium toxins in food and assessment of dietary intake by the population of eu members states, substask i : Trichothecenes[END_REF]. In this part of the world chronic contamination is the most threatening, due to the persistence of these mycotoxins in food and the repeated ingestion by animals for example. In 2004, a worldwide survey showed that 72% of more than 19000 samples analyzed contained detectable amounts of AF, FB, DON, ZEA or OTA (Figure 4). Among them, 38% represented a co-contamination by 2 or more mycotoxins (Schatzmayr and Streit 2013). Several toxic effects can be induced, depending of the mycotoxin and the organ targeting. At high doses, mycotoxins exposure can leads to general cytotoxicity, biochemical lesions and impact on early cellular functions in the cascade of events (Bryden 2012;Maresca and Fantini 2010). At low doses, various functions of tissues and organs can be impaired. And some mycotoxins are also genotoxic, carcinogenic and teratogenic (Maresca and Fantini 2010).

II. Problem of DON contamination

A. Occurrence

DON is produced by Fusarium fungi, one of the most common mycotoxin in the world. It and can be found in many cereals and raw materials, like wheat, barley, oat, rye, maize and sometimes on rice, sorghum and triticale. A worldwide survey to assess the contamination by mycotoxins in feed and feed raw materials, done on 19,000 samples, shows that DON was present in 56% of the tested samples (Schatzmayr and Streit 2013).

Fungal infection and DON production are difficult to predict and regulate; because largely dependent on the weather, high humidity and low temperature, and so vary greatly from year to year and between areas (Rotter et al. 1996). In developed countries, where storage conditions are well managed and controlled, DON contamination is especially a pre-harvest problem. While, in developing countries, DON can also be produced during the storage stage.

So, DON can be commonly detected at low levels (< 1 ppm) and sporadically at higher levels (5 to 20 ppm) on cereals intended to be given to animals or humans (Abouzied et al. 1991). It can also be present in end products, such as the cereal-based food for adults and infants or even at low levels in beer (Lombaert et al. 2003;Scott 1996).

Economic losses due to DON contamination are difficult to evaluate. Nevertheless a computer simulations evaluated that the annual costs for DON in the USA were $637 million in crop losses (mainly wheat and corn), $18 million in feed losses and $2 million in livestock losses (EFSA 2013).

B. Toxicity

The toxicity of DON is well known and numerous studies bring information on its toxic effects at high and chronic doses (Maresca 2013;Pestka 2010;Wang et al. 2014). A high concentration of DON causes effects and symptoms that are similar to those observed during an exposure to ionizing radiation, such as abdominal distress, salivation, discomfort, diarrhea, vomiting, leukocytosis and gastrointestinal bleeding. It also has high emetic and anorexic effects, that are equal or even higher to those of observed with the most toxic trichothecene B (Pestka and Smolinski 2005). Actually, the first name of DON was "vomitoxin" due to its emetic effects seen in pigs (Vesonder et al. 1973).

A chronic exposure can impact growth (by anorexia and disregulation of nutrients efficacy), immunity (increased or decreased) and reproduction in animals. At acute doses it can induces emesis, abdominal distress, malaise, diarrhea and increases the salivation (Pestka 2010). At low dose it impairs the growth and the immune function in human and interferes with nutritional efficiency on pigs (Rotter et al. 1996). At higher doses it causes diarrhea, emesis, leukocytosis, hemorrage, endotexemia and ultimately shock-like death (Ueno 1983).

C. Detoxification methods for DON

The effectiveness of detoxification methods of mycotoxins depends on several parameters, the nature of the food/feed, the environmental conditions such as moisture content, temperature, as well as the type of mycotoxin, its concentration and the extent of binding between mycotoxin and constituents (Grenier and Oswald 2011).

DON resist to most of the industrial processes; it is stable at high temperature, due to its high chemical stability and can be found in numerous final processed products (Hazel and Patel 2004). Actually DON is completely stable at 120°C, quite stable at 180°C and partially stable at 210°C (OMS 2001). At concentrations below 1mg/kg, DON is mainly found on the seed surface but at higher concentrations, it can be found in the entire grain (Charmley and Prelusky 1994). To reduce the occurrence and the impact of mycotoxins and especially DON, several detoxifying strategies were established in the feed chain, including the prevention of fungal growth and the production of mycotoxin, strategies to reduce or eliminate mycotoxins from contaminated raw or finished materials or even in diverting contaminated product to low risk uses including animal feeds (Bryden 2012). However, the amount of information related to mycotoxins detoxifying methods is still limited. From the described detoxifying strategies there are three principal categories used: the physical, the chemical and the biological methods.

Physical methods

Some processes used to detoxify mycotoxins (such as milling, irradiation, ethanol fermentation or extrusion) were initially developed for other purposes, and some were specifically developed for the detoxification itself (such as sorting, cleaning or washing).

These practices, are linked to the FAO guidelines, namely fulfilled: cheap and simple, no production of toxic metabolites, and no change in the nutritional value or properties of raw materials. However, all these approaches present some inconvenient. The standard processes, like milling and baking, do not allow the elimination of DON with efficacy (Abbas et al. 1985;Hart and Braselton 1983). Dry milling, permit an elimination that is up to 40% of DON present in the flour; sieving or cleaning can reduce the concentration in DON by over 60% (Pestka and Smolinski 2005). The problem of the milling and grain separation process, commonly used for human's food, is that it concentrates all the mycotoxins in bran and Germ, fractions will be used later for animal feed. However, in the sieving and cleaning procedures, an important loss of grains is reported. In their study, Trenholm et al. (1991) did observe a 73% reduction of DON, but they have also observed that up to 69% of the total weights of the corns was removed as well. And after flotation and washing, the cost of drying grains is high.

2.

Chemical methods

Several chemical processes, using molecules like ammonia, calcium hydroxide, chlorine, hydrochloric acid, ozone, sodium bisulphate and sodium hydroxide are able to degrade DON. In fact ammoniation has been proved to reduce the aflatoxin levels but this process is not accepted in all countries and is quite expensive (Norred et al. 1991;Park and Price 2001). With alkalization, DON can be transformed into different products, with various toxicity (Bretz et al. 2005;Bretz et al. 2006;Young et al. 1986).

In majority, the chemical methods can reduce mycotoxins' levels, but they can also severely damage the nutrient quality of the grains and can be health hazards on their own. Not only that, they can result in the formation of degraded products that might be constituted of new and unknown biologically active mycotoxins (Humpf and Voss 2004).

Biological methods

Two strategies are possible to manage DON, once present in plants and cereals. The first strategy consist on preventing the production of DON in infected crop by controlling the plant pathogens (Fusarium spp.). Equipping crops with DON detoxification activities can reduce the concentration of mycotoxin in grain and also increase the resistance against infection (Karlovsky 2011). It was shown that DON plays a role in the infection; host plants inoculated with fungal strain not able to produce TCT can't be able to infect the plant (Maier et al. 2006;Proctor et al. 1995). Another study shows that a major QTL responsible for the resistance of wheat to FHB co-segregated with the ability to detoxify DON by glycosylation (Lemmens et al. 2005); It has been proved that by selecting a plant naturally resistant to Fusarium, its capacity to glycosylate DON into D3G can be increased by 2.7 times more (Sasanya et al. 2008).

Some companies also tried to build transgenic plants, by transferring the 3-Oacetyltransferase gene issued from F. sporotrichioides to the plant in order to reduce the pathogenicity of Fusarium (Karlovsky 2011).

The second strategy consists in detoxifying DON that has been produced, by physical and chemical methods as we saw but more innovative by biological methods. The biodetoxification of mycotoxins, by isolating microorganisms and/or enzymes that will degrade or metabolize the mycotoxins, is currently an innovative and promising strategy aiming to control mycotoxicoses in animals (Schatzmayr et al. 2006). (Cheng et al. 2010) obtained two Bacillus strains able to detoxify DON in wheat and maize. In another study, Bacillus sp.

LS100, which transforms deoxynivalenol (DON) to a less toxic chemical de-epoxy DON (DOM-1) has been assessed. This intestinal bacteria, Genus novus species novus of family Coriobacteriaceae BBSH 797, isolated from digestive tracts, is able to de-epoxydize DON to DOM-1 (Fuchs et al. 2002). There is also the bacterial strain Devosia mutans 17-2-E-8, isolated from an alfalfa soil enriched with F. graminearum-infested corn that is able to highly reduce DON level, in producing an epimer, the 3-epi-DON (He et al. 2015b;Zhou andHe 2009, 2010).

Bringing the enzymatic kit to animals, by the use of bacteria, will allow them detoxify mycotoxins, and easily and effectively protect them against the toxic effects of mycotoxins.

Definitely, since decontaminated or detoxified crops are cheaper (since they are considered as products of lower quality), they are mainly used for feed production and animal feeding (Grenier and Oswald 2011), in which explains why animals are very exposed. The need of feed additives preventing the absorption of mycotoxins and by that occurrence of their toxic effects in farm animals has increased significantly. Indeed, the adsorption is not a viable option regarding trichothecenes, zearalenone and ochratoxins, that's why the mycotoxin inactivation by biotransformation is a very promising strategy to detoxify these mycotoxins.

However, all the additives and bacterial products have to be tested before coming into the market to assure their efficiency and safety. In vitro and in vivo tests are mostly important to check and follow their scientific development and improvement. Sensitive parameters such as biochemistry, gross pathology, histopathology, immune parameters and animal performances have to be measured to evaluate their toxicity. This is why the aim of this thesis was to evaluate the toxicity of the products issued from biological detoxification and to assess the efficiency of this process in order to protect animals or humans from the toxic effects of DON.

Zhou T, He J. 2010. Bacterial isolate, methods of isolating bacterial isolates and mathods for detoxification of trichothecene mycotoxins. Patent Cooperation Treaty Application No61/249,023 Filed October 6, 2010 (International).

III. Literature review

The literature review consists on three reviews covering different aspects studied during this thesis. The first two reviews deal with the different effects caused by mycotoxins and the intestine or the immunity of pigs. The third review focuses on new forms of mycotoxins derived from these mycotoxins, the "masked" and "modified" mycotoxins.

To date, contamination by mycotoxins cannot be avoided. Mycotoxins can be present in several types of cereals (maize, wheat, barley, oats, rice ...) and end up in high concentrations due to cultural practices or storage. These mycotoxins can be found in cocontamination in pig feed. All these mycotoxins have toxic effects on pig that is particularly sensitive because of its simple digestive system and its high cereal rich diet. The first two reviews have a look on the two most affected parameters after a contamination by one or more mycotoxin, on the gut and the immune system of the pig. Mycotoxins contaminations, mainly by ingestion, cause many toxic effects on the digestive system and small intestine.

The first review presents the different mycotoxins that are found in pig's feed and their effects and consequences on the intestine and the general health of the pig. Mycotoxins have been also described as responsible of modifying important functions of the intestine (barrier function, mucus production, nutrient absorption…).

The second review reports the effect of mycotoxins on the immune system of pigs. Certainly, many mycotoxins have an immune-modulatory effect on the immune response and may affect the vaccine response as well as induce an increased susceptibility to infections or chronic infectious diseases.

Finally, the last review presents advances in terms of new analytical methods allowing the identification of new forms of mycotoxins, the mycotoxins called maskedand modified. It is important to study these new forms of mycotoxins to evaluate their impact on pig health and to assess whether they can represent an additional threat that will have to be taken into account in the overall management of the risk of mycotoxins.

A. Feed mycotoxins: impact on pig intestinal health

Nowadays, many mycotoxins can contaminate cereals and feeding stuffs designated to the pig consumption. These mycotoxins have several toxic effects on pigs, which are greatly impacted, due to their high sensibility and their cereals rich diet.

Due to the way of exposure, by ingestion, intestine is the major organ targeted by mycotoxins. This review summarizes the major effects induced by these mycotoxins on the intestine, on its integrity, its biological function and on its immune response. It also highlights the consequences of this contamination, which increases the translocation of bacteria and enhances the susceptibility to other diseases and thus impairs the global health of pigs.

This review is currently submitted to Porcine Health Management.

Mycotoxins in Feed: impact on pig intestinal health

Alix PIERRON 1,2,3 , Imourane ALASSANE-KPEMBI 1,2 and Isabelle P. OSWALD 1,2*

Introduction

Food safety is a major issue throughout the world. In this respect, much attention needs to be paid to the possible contamination of food and feed by fungi and the risk of mycotoxin production. Mycotoxins are secondary metabolites produced by filamentous fungi, mainly by species from the genus Aspergillus, Fusarium and Penicillium. They are produced on a wide variety of substrates before, during and after harvest. Mycotoxins are very resistant to technological treatments and difficult to eliminate, and therefore they can be present in human food and animal feed. The ingestion of mycotoxin-contaminated feed can induce acute diseases, and the ingestion of low doses of fungal toxins also causes damage in case of repeated exposure.

Monogastric livestock, pig and poultry, are particularly vulnerable to mycotoxins because of the high percentage of cereals in their diet and because they lack a rumen with a microbiota able to degrade mycotoxins before their intestinal absorption. From a pig health perspective, the most notorious mycotoxins (Fig. 1) are aflatoxins (AF), ochratoxin A (OTA), fumonisins B (FB), zearalenone (ZEN), and trichothecenes, especially deoxynivalenol (DON) [START_REF] Cast | Council for Agricultural Science and Technology-Potential economic costs of mycotoxins in United States Task Force Rep[END_REF].

This review will summarize the effect of mycotoxins on the intestine and analyze the consequences in terms of pig health.

I-Toxicity of the main mycotoxins in pig feed

The toxicity of mycotoxins varies according to several parameters such as the dose, the duration of exposure, the age and the sex of the animal, as well as nutritional factors (Andretta et al. 2012;Bryden 2007;Wild 2007). For example, the effects of AF, FB or DON on performance are greater in males and young pigs (Andretta et al. 2012;Marin et al. 2006). In the European Union, only AFs are regulated in animal feed; recommendations exist for OTA, DON, T2 and HT-2 toxins, FB1, FB2 and ZEN (Tab. 1). The AFs are rapidly absorbed and metabolized in the liver (Haschek et al. 2002); they are hepatotoxic, and have some impacts on growth and on the immune response of the pig (Meissonnier et al. 2006). OTA is nephrotoxic and hepatotoxic and its oxydative metabolites are genotoxic (Aish et al. 2004;Pfohl-Leszkowicz and Manderville 2007). Among mycotoxins, pigs are very sensitive to DON, the most common mycotoxin of the type B trichothecene. Short exposure to high doses of DON induces vomiting and lower doses cause feed refusal (Haschek et al. 2002); chronic exposure is associated with weight loss, anorexia, immunomodulation and alteration of intestinal barrier functions (Haschek et al. 2002;Pestka 2010;Pinton and Oswald 2014). Type A trichothecene T2 and HT2 toxins have similar but more pronounced effects than DON. They also induce irritation of the intestine and the skin and increase the sensibility of pigs to diseases (Bryden 2012).

Complete and complimentary feeding stuffs:

-for piglets and gilts FB1 is the most prevalent toxin of the fumonisin family. It has a carcinogenic effect in humans and induces multiple toxic effects in different animal species. In pigs, this toxin induces pulmonary oedema (Haschek et al. 2002) and alters the immune response with a dysregulation of the T helper lymphocytes TH1/TH2 balance (Marin et al. 2006;Taranu et al. 2005). The last mycotoxin with a recommendation for pig feed is ZEN. This toxin has an impact on pig fertility and reproduction. ZEN and its principal derivatives, α-zearalenol (α-ZEL) and β-zearalenol (β-ZEL) (more toxic than the other two), are non-steroidal oestrogens inducing an oestrogenic response in animals (Fink-Gremmels and Malekinejad 2007). In pigs, especially young sows, ZEN induces red patching and tumefaction of the vulva, a prolapse of the vulva and sometimes of the rectum (Gaumy et al. 2001).

In terms of intestinal toxicity, the effects of DON and FB have been studied in detail in pigs; by contrast only few papers are concerned with the effect of OTA or AF on this organ.

II-Effects of mycotoxins on the pig intestine

The intestinal tract is the first target for mycotoxins following ingestion of contaminated feed. The intestinal epithelium is a single layer of cells lining the gut lumen that acts as a selective filter, allowing the translocation of dietary nutrients, essential electrolytes, and water from the intestinal lumen into the blood circulation. It also constitutes the largest and most important barrier to prevent the passage of harmful intraluminal substances from the external environment into the organism, including foreign antigens, microorganisms, and their toxins. Following the ingestion of mycotoxin-contaminated feed, intestinal epithelial cells may be exposed to high concentrations of toxins, potentially affecting intestinal functions (Alassane-Kpembi and Oswald 2015; Ghareeb et al. 2015;Grenier and Applegate 2013).

A. Effect on intestinal histomorphology

Consumption of mycotoxin-contaminated feed induces histological damage on intestinal tissue. Epithelial lesions in the intestine of pigs fed with a diet naturally contaminated with DON were observed (Bracarense et al. 2012;Eriksen and Pettersson 2004). Jejunal lesions, including shortened and coalesced villi, lysis of enterocytes, and edema, were also observed in an ex-vivo model of intestinal tissues after exposure to DON (Lucioli et al. 2013;Pinton and Oswald 2014). Exposure to FB also induces changes in intestinal villi morphology such as reduced villi height and villi fusion and atrophy (Bracarense et al. 2012).

A study on pigs showed that low doses of ZEN do not impair the morphology and ultrastructure of the small intestine (Obremski et al. 2005), in contrast to what has been observed in rats (Liu et al. 2014).

As far as AFB1 is concerned, no data on the effect of this toxin on the histomorphology of the pig intestine are available. Nevertheless, exposure of broiler chicken to AFB1 induced a decreased jejunal villus height, villus height/crypt ratio, and shedding of epithelial cells on the tip of jejunal villi (Zhang et al. 2014).

B. Effect on intestinal digestion and nutrient absorption

The regressive intestinal lesions observed upon exposure to mycotoxins may explain, at least in part, the reduced absorption of nutrients and the impaired digestion observed after ingestion of mycotoxins. Pigs consuming corn culture extracts containing FB showed a markedly lowered activity of aminopeptidase N (Lessard et al. 2009). Likewise, exposure to 1.5mg/kg b.w. FB1 has been shown to induce sphingolipid depletion in pig intestinal epithelium, which can result in a deficiency of folate uptake (Grenier and Applegate 2013;Loiseau et al. 2007). The sodium-glucose dependent transporter (SGLT-1) activity is particularly sensitive to DON inhibition. SGLT-1 is the main apical transporter for active glucose uptake in the small intestine. Inhibition of SGLT-1 has nutritional consequences and could explain diarrhea associated with DON ingestion, since this transporter is responsible for daily absorption of water in the gut (Maresca 2013). Conversely, sodium-dependent glucose absorption might be up-regulated in pigs after acute or long term exposure to the mycotoxin FB1 (Lessard et al. 2009).

C. Effect on barrier function

Several mycotoxins are able to alter intestinal barrier functions (Ghareeb et al. 2015;Grenier and Applegate 2013). They affect the intestinal epithelium permeability through modulation of the tight junction complexes. A defective expression of occludin and Ecadherin has been observed in the ileum of piglets fed low doses of FB1 (Lucioli et al. 2013).

The FB-induced alteration of the sphingolipid biosynthesis pathway and the associated lipid rafts could also contribute to impairing the establishment and maintenance of tight junctions.

Likewise, the activation of MAP kinases by DON affects the expression and cellular localization of proteins forming or being associated with tight junctions such as claudins and ZO-1, which results in increased intestinal paracellular permeability (Pinton and Oswald 2014). Similarly to DON, T2-toxin, FB 1 and ZEN have been shown, in vitro and in vivo, to impair the pig intestinal barrier function and to promote oral absorption of antibiotics such as doxycycline, chlortetracycline and paromomycin (Goossens et al. 2012;Goossens et al. 2013).

D. Intestinal immune system

Some mycotoxins impact the systemic and/or the local immune response. At the intestinal level, they decrease the immunity leading to enhanced intestinal infections. They also have a direct or indirect proinflammatory effect (Cano et al. 2013;Maresca 2013).

Indeed, the intestine is a major source of cytokines and chemokines, molecules involved in the regulation of the immune system. Among cytokines, IL-8, which is a chemoattractant cytokine, is of particular interest because it is involved in the recruitment of polymorphonuclear neutrophils at the site of infection, mediating a non-specific acute inflammatory response.

Ingestion of FB 1 specifically decreases expression of IL-8 mRNA in the ileum of exposed piglets while expression of other inflammatory cytokines is not affected. This decrease of IL-8 caused by FB 1 may lead to reduced recruitment of inflammatory cells in the intestine during infection, and may contribute to the observed increased susceptibility of FB 1treated piglets to intestinal infections (Bouhet and Oswald 2007).

DON modulates intestinal innate immunity both directly (through activation of signal pathways) and indirectly (through crossing of luminal bacterial antigens, which was observed together with bacterial translocation following mucus layer alteration and tight junction opening) (Maresca et al. 2008). DON affects expression of proteins involved in epithelial innate immunity, including inflammatory cytokines, COX-2 and β-defensins (Cano et al. 2013;Lessard et al. 2015). Numerous studies have demonstrated that DON stimulates expression and secretion of IL-8 and thus potentially participates indirectly in the central effects of DON in terms of feed refusal and emesis. As described for immune cells (Pestka 2010), DON has a biphasic effect on the secretion of IL-8 by intestinal epithelial cells: Low doses of toxin cause a massive increase in secretion of IL-8, whereas higher doses inhibit it.

Such a biphasic effect explains why DON acts: (i) as a proinflammatory toxin leading to intestinal inflammation at low doses; and (ii) as an inhibitor of intestinal immunity leading to higher susceptibility of animals to intestinal infections at higher doses (Maresca 2013).

The ability of ZEN to interact with the pig immune system has been poorly investigated.

However, it is known that exposure to high concentrations of ZEN (5-250mg/Kg feed or 200-1000 µg/Kg b.w./day) induces chronic inflammation of the genital tract in females pigs (EFSA 2011;JECFA 2011). In vitro analyses also show that ZEN and its metabolites have differential effects on synthesis of the inflammatory cytokines IL-8 and IL-10 in swine intestinal epithelial cells (Marin et al. 2015).

There is no report of OTA-induced impairment of local immunity. However, this mycotoxin decreases the level of inflammatory cytokines (TNF-alpha and IL-10) in the plasma of exposed pig (Bernardini et al. 2014).

E. Intestinal microbiota

The gut hosts an important microflora. Surprisingly, the impact of mycotoxins on the intestinal microflora has been poorly investigated. As far as pigs are concerned, only two studies have investigated the impact of mycotoxins on the intestinal microflora (Burel et al. 2013;Wache et al. 2009). The first study indicates that consumption of feed naturally contaminated with DON (2.8 mg/kg) for four weeks had a moderate effect on cultivable bacteria in the pig intestine, but changed the microflora (Wache et al. 2009). In the second study, pigs received feed contaminated with 12 mg FB/kg feed for 63 days. This diet transiently affected the balance of the digestive microbiota during the first four weeks of exposure; a co-infection with Salmonella typhimurium amplified this phenomenon (Burel et al. 2013).

Two recent studies performed on rats have also demonstrated an effect of OTA and AF on the intestinal microbiota (Guo et al. 2014;Wang et al. 2016). The effects of mycotoxins on the intestinal microbiota are not surprising; indeed other secondary metabolites produced by the same fungi, antibiotics, are well known for their effect on the gut flora.

Recent advances in next-generation sequencing technologies and metagenomics should give us a comprehensive analysis of the effect of mycotoxins on the structure and function of gut microbial ecosystem in the near future.

III-Consequences of intestinal toxicity of mycotoxins for pig health

A. Impairment of zootechnical performance

All damage induced by mycotoxins on the intestine level and on the different functions lead to different symptoms expressed by the pig. Such symptoms are either directly associated with local toxicity in the intestine, or indirectly with a systemic effect, and with visible impact on the overall health of the pig.

The colloquial name of DON, vomitoxin, refers to its emetic effect observed both in field reports and in experimental intoxications where high doses of the toxin were given orally or intravenously to pigs. Complete feed refusal was observed at levels of 12 and vomiting at 20 mg DON/kg feed. Pig feeding trials with naturally or artificially contaminated diets have shown decreased feed consumption and weight gain at doses from 0.6 to 3mg DON/kg feed (Bracarense et al. 2012). A meta-analysis showed that deoxynivalenol reduced feed intake and weight gain by 26%; the same analysis also demonstrated a 16% reduction of feed intake in response to AFB1 (Andretta et al. 2012).

Consumption of pure FB1 or FB1-contaminated feed also induces a slight reduction of body weight in piglets. Although FB are poorly absorbed and metabolized in the intestine, they induce intestinal disturbances (abdominal pain or diarrhea) and cause extra-intestinal organ pathologies (pulmonary edema in pigs, leukoencephalomalacia in horses, or neural tube defects in rodents).

Ingestion of ZEN and OTA doesn't alter zootechnical performance (Bernardini et al. 2014;Schoevers et al. 2012). However ZEN can induce a decrease in reproductive performance with a reduction of healthy follicles leading to premature oocyte depletion in adulthood and so leading to abortion (Schoevers et al. 2012).

B. Bacterial translocation

The intestinal disturbance induced by mycotoxins may lead to increased bacterial translocation across the intestine and increased susceptibility to enteric infections. The loss of tight junction integrity and resulting increased paracellular permeability can lead to entry of bacteria that are normally restricted to the gut lumen. Such an increase in bacterial passage through intestinal epithelial cells after mycotoxin exposure has major implications for pig health in terms of sepsis, inflammation and enteric infection.

Porcine ileal loops were used to reproduce Salmonella typhimurium induced intestinal inflammation. Co-exposure to bacteria and DON dramatically enhances the inflammatory response to S. typhimurium in the ileal loops, with a clear potentiation of expression of IL-1β, IL-8 or IL-6 (Vandenbroucke et al. 2011). It has been suggested that this potentiation coincided with significantly enhanced Salmonella invasion in and translocation over intestinal epithelial cells.

A higher susceptibility of the gastrointestinal tract to other bacteria was reported in pigs exposed to FB1. Two separate studies analyzed the effect of low to moderate doses of FB1 on intestinal colonization and mucosal response to pathogenic strains of Escherichia coli (Devriendt et al. 2009;Oswald et al. 2003). Besides, translocation of bacteria to the mesenteric lymph nodes and dissemination to the lungs, and to a lesser extent to liver and spleen, were observed in FB1-treated pigs in comparison to untreated animals (Oswald et al. 2003).

A study on human enterocytes exposed to low doses of DON or OTA showed an increase of translocation of commensal bacteria across the epithelium even without alteration of the intestinal permeability (Maresca et al. 2008). The mechanism involved in this increase is not elucidated, but this phenomenon could be due to an energetic modification of the cell status with a reduction of ATP levels (Grenier and Applegate 2013).

Conclusion

The intestine is a target for mycotoxins and as illustrated in this paper this may have some consequences in terms of pig health (fig. 2). Regulations and recommendations exist for six mycotoxins (AF, FB, OTA, ZEN, T2/HT2 and DON) present in pig feed. Among them, DON and FB have been studied especially for their toxicity in the intestine. They are not only locally toxic for this organ, but also dysregulate many intestinal functions and impair the immune response. This results in systemic toxicity leading to many symptoms and impairment of zootechnical parameters. Feed contamination with mycotoxins also increases translocation of bacteria across the intestine and thus intestinal and systemic infections, and so aggravates pigs' condition. For AF, ZEN and OTA, little is known about their intestinal toxicity on pigs.

The increased performance of analytical methods reveals new toxins, especially emerging ones, as well as "masked" or "modified" forms. Occurrence and toxicity of these new mycotoxins are poorly documented (Broekaert et al. 2015;Pierron et al. 2015), and thus it still needs to be determined if they represent a new risk in pig production. Global surveys indicate that animals are generally exposed to more than one mycotoxin (Streit et al. 2012). Indeed fungi are able to produce several mycotoxins simultaneously; and it is common practice to use multiple grains in animal diets.

Unfortunately, the toxicity of mycotoxin mixtures cannot be predicted based on their individual toxicities. Interactions between concomitantly occurring mycotoxins can be antagonistic, additive, or synergistic (Alassane-Kpembi et al. 2015). The data on combined toxicity of mycotoxins are limited and therefore, the health risk from exposure to a combination of mycotoxins is incompletely understood (Alassane-Kpembi et al. 2016;Grenier and Oswald 2011) and deserves further investigation.

B. Impact of mycotoxin on immune response and consequences for pig health

Many mycotoxins, alone or in co-exposure, can contaminate cereals and feeding stuffs for pig consumption. These mycotoxins induce different toxic effects on pigs and especially on the immune system. They are able to modulate or dysregulate the immune response. This review summarizes the major effects induced by these mycotoxins on the immune response. It also highlights the consequences of these contaminations, which increase the susceptibility to infectious diseases or to chronic infection and can also decrease the vaccine efficacy. Moreover these mycotoxins can be found in co-contamination, with, as a consequence, a potential increase of the effects observed and an impairment of the global health of the pig.

This review was published this year in Animal Nutrition.

C.

Masked mycotoxins: a risk in pig production?

Currently, many mycotoxins induce toxic effects on pigs and are regulated in pig feed.

New analytical methods of detection allow highlighting new types of molecules that derivate from these mycotoxins. There are also present in cereals and feed and their toxicity is not very well-known.

This review summarizes the knowledge on mycotoxins, their occurrence, their effects on pigs and their regulation in pig feed at the European level. It presents new forms of mycotoxins, the "masked" and the "modified" mycotoxins, which are derivated from these mycotoxins and which are recovered in co-occurrence with them in pig feed. It makes a statement on the way of production, the occurrence, the toxicity and the metabolization of these molecules in the pig. It summarizes the knowledge on these "masked" and "modified" mycotoxins, and talks about the necessity to take into account these molecules in the regulation of mycotoxins in pig feed.

This review was published in the book of the 48 th days of the Journées de la Recherche Porcine (JRP), and as been presented as an oral synthesis at these same days of JRP in February 2016.

Translated Version (English)

« Masked » mycotoxins : new risk in porcine production ?

Alix PIERRON [START_REF] Akbari | Deoxynivalenol: A trigger for intestinal integrity breakdown[END_REF][START_REF] Alassane-Kpembi | New insights into mycotoxin mixtures: The toxicity of low doses of type b trichothecenes on intestinal epithelial cells is synergistic[END_REF][START_REF] Awad | Decontamination and detoxification strategies for the fusarium mycotoxin deoxynivalenol in animal feed and the effectiveness of microbial biodegradation[END_REF], Imourana ALASSANE-KPEMBI [START_REF] Akbari | Deoxynivalenol: A trigger for intestinal integrity breakdown[END_REF][START_REF] Alassane-Kpembi | New insights into mycotoxin mixtures: The toxicity of low doses of type b trichothecenes on intestinal epithelial cells is synergistic[END_REF],Delphine PAYROS [START_REF] Akbari | Deoxynivalenol: A trigger for intestinal integrity breakdown[END_REF][START_REF] Alassane-Kpembi | New insights into mycotoxin mixtures: The toxicity of low doses of type b trichothecenes on intestinal epithelial cells is synergistic[END_REF], Philippe PINTON [START_REF] Akbari | Deoxynivalenol: A trigger for intestinal integrity breakdown[END_REF][START_REF] Alassane-Kpembi | New insights into mycotoxin mixtures: The toxicity of low doses of type b trichothecenes on intestinal epithelial cells is synergistic[END_REF], Isabelle P. OSWALD [START_REF] Akbari | Deoxynivalenol: A trigger for intestinal integrity breakdown[END_REF][START_REF] Alassane-Kpembi | New insights into mycotoxin mixtures: The toxicity of low doses of type b trichothecenes on intestinal epithelial cells is synergistic[END_REF] ( Les mycotoxines sont des métabolites secondaires de moisissures qui peuvent contaminer différentes céréales et par conséquent l'alimentation du porc. Au niveau européen, des réglementations et des recommandations pour l'alimentation animale ont été édictées pour six mycotoxines dont la toxicité est documentée. Les avancées dans les techniques de détection ont permis de mettre en évidence des dérivés de ces mycotoxines "natives", appelés mycotoxines "modifiées" ou plus spécifiquement mycotoxines "masquées" lorsqu'elles sont issues d'une métabolisation par la plante.

Du fait de leur caractérisation récente, peu d'informations sont disponibles sur leur occurrence dans l'alimentation du porc et leur toxicité pour cette espèce. Les données préliminaires indiquent que ces toxines peuvent être présentes à de fortes concentrations dans les aliments. Le porc pourrait être une espèce cible également pour ces "nouvelles" mycotoxines, du fait de sa grande sensibilité à la présence de mycotoxines conventionnelles, et à son régime alimentaire composé en grande partie de céréales. Ces mycotoxines "modifiées" peuvent augmenter la somme de mycotoxines auquel le porc est exposé, si elles sont hydrolysées dans l'organisme de l'animal.

INTRODUCTION

Nowadays, contamination by fungi cannot be avoided. Ecological conditions allowing the development of fungi (bad weather, humidity, high heat, plant sensitivity…), makes the management of the contamination of raw materials difficult. During a fungi contamination, mycotoxins, which are secondary metabolites, are produced and found into the seeds. These toxins are present in numerous cereals and by-products. A survey realized on 1100 different samples of animals' feed show that about 70% of the samples are contaminated (Streit et al. 2013).

The presence of these mycotoxins is considered as a serious threat to the health (Bryden, 2007;Wild and Gong 2010). The syndromes caused by the ingestion of high or medium doses of mycotoxins are well characterized and can go from acute mortality to reduced growth or problems in reproduction (Bryden, 2012). Consuming smaller amounts of toxins can lead to an alteration in the immune response and decrease the resistance to infectious diseases (Oswald, 2007). Some mycotoxins have an acute toxicity (a single exposure at a high dose) that is very strong, but it is exceptional in Europe being exposed to toxic doses in a single ingestion of contaminated food. Chronic effects (repeated exposure to low or very low doses) are the most feared due to the repetitive diet of the animals and because of the persistence of these toxins that are often resistant to high temperatures and technological processes used in the animal feed industry.

Recent advances in analytical methods have revealed new forms of mycotoxins and "masked" mycotoxins, not detected by conventional analytical methods. Currently, only "native" mycotoxins are regulated and taken into account in the calculation of the total exposure in raw or processed food. Actually, only few data are available on these new molecules where the risk to underestimate the toxicity induced by these molecules is not included in the regulation. Therefore, it becomes important to better know the risk that these "masked" mycotoxins can pose to humans and animals.

After the presentation of the conventional mycotoxins usually detected, this review provides a summary of current knowledge on "masked" mycotoxins, their identity, their occurrence, their metabolism and toxicity. It concludes on the potential danger that these "masked" toxins can represent on the health of the pig.

THE MYCOTOXINS REGULATED IN PIG'S FEED

In animal feed, only aflatoxins (AF) are regulated in Europe. There are some recommendations (Table 1) for five other toxins, which occur regularly and which are known to be toxic to swine. There are the ochratoxin A (OTA), the deoxynivalenol (DON), the toxins T2 and HT2, the fumonisins (FB1, FB2) and the zearalenone (ZEN) (Bennett et Klich, 2003). These molecules belongs to different families of mycotoxins, with various chemical structures, and so various toxic effects on pig. The dose, time of exposure, the specie, the age and the status of the animal (Bryden, 2007;Wild, 2007).

The Table 2 lists the major known effects of these mycotoxins on pig health.

AF are quickly absorbed and metabolized in the liver by the microsomal system which actives or modifies the metabolites (Riley, 1998;Haschek et al., 2002). AF alters the global immune response (innate and cellular) in pigs (Meissonnier et al., 2006).

OTA is mainly toxic to the liver and kidneys and causes kidney diseases in pigs. OTA affects the renal proximal tubule (Krogh, 1987;Marquardt and Frohlich, 1992). Moreover OTA acquires a genotoxic effect after its metabolization in the body (Ash et al., 2004;Pfohl-Leszkowicz and Manderville, 2007;Steyn et al., 2009). DON is the most common trichothecenes B. Pig is very sensitive to this mycotoxin, which can induce at low concentration feed refusal, and in higher concentrations vomiting (Haschek et al., 2002).

Chronicle doses of DON (low concentrations on the long term), induce in pigs weight loss, anorexia, immunomodulation and a modification of the intestinal barrier function (Trenholm et al, 1984;. And Rotter al., 1996;Haschek et al., 2002;Pinton, Oswald, 2014). Toxins T2 and HT2 that belong to the family of trichothecenes A have similar effects but more pronounced than Trichothecenes B. They induce irritation to the gastrointestinal tract and skin, and they increase the sensitivity of the animal disease (Bryden, 2012).

Fumonisins are constituted of 12 compounds including fumonisin B1 (FB1), which is the most toxic and most studied. Fumonisins induce multiple toxic effects on animals with a known carcinogenic effect. In pigs, the FB1 affects the specific and the humoral responses by altering the balance of helper T cells, TH1 / TH2 (Taranu et al., 2005;Marin et al., 2006) FB1 induced pulmonary edema in pigs (Haschek et al., 2002). Zearalenone (ZEN) has a significant effect on reproduction and fertility especially in swine. The α-zearalenol (α-ZEL) and β-zearalenol (β-ZEL), from the reduction of ketones by ZEN-reductase of the host, are non-steroidal estrogens that induce estrogenic activity in the animal (Fink-Gremmels and Malekinejad, 2007). ZEN and its derivatives cause redness and swelling of the vulva, vaginal prolapse and sometimes rectal prolapse in sows. In young sows, they can induce a significant swelling of the vulva (Gaumy et al., 2001).

« MODIFIED» MYCOTOXINS IN PIG FEED

Presentation

New analytical methods allowed putting in evidence new secondary metabolites and some molecules derivate from these mycotoxins. The term of "masked" mycotoxins" was introduced in 1990 by Gareis to describe a glucoside zearalenone not detected during routine analysis, but hydrolyzed during digestion (Gareis et al., 1990). Indeed, different changes can occur in the structure of mycotoxin, which make them undetectable by conventional analytical techniques (Table 3). There are biological changes (did by a plant, fungus or animal body) or chemical ones such as the ones caused during thermal food processing methods.

The name of "masked mycotoxin" has often been an ambiguous use, and recently some authors have proposed a more precise terminology for the various forms of mycotoxins [START_REF] Berthiller | Masked mycotoxins: A review[END_REF]. Rychlik et al, 2014).

These authors have redefined the terminology of "masked mycotoxin" strictly and introduced the concept of "modified mycotoxins." Figure 1 shows for example, all the forms described for DON.

Mycotoxins called "native or free" correspond to the basic structures of mycotoxins formed by molds. Most likely to be found in the pig supply is DON, ZEN, fumonisin, aflatoxin and OTA.

Matrix-associated mycotoxins correspond to the "native" mycotoxins bound to a matrix, i.e.

physically dissolved and / or trapped and / or forming a covalent bond with the matrix. Thus, Fumonisins are able to bind to polysaccharides or proteins by their two tricarballyliques acids chains, thus forming the hidden fumonisins (hidden F) or linked with starch (F related to starch) (Seefelder et al., 2003).

Excepted these binding phenomena in a matrix, "native" mycotoxins can undergo biological or purely chemical transformations. The term "modified mycotoxin" was proposed to describe any biological or chemical modification of the chemical structure of a "native" mycotoxin (Rychlik et al., 2014).

"Biologically modified" mycotoxins indicate compounds derived from biotransformation in an animal body, plant or a mold. Biotransformation are divided into two main types: Phase I reactions (oxidation, reduction or hydrolysis) and phase II reactions (conjugation).

Generally, biotransformation allows detoxification of toxics, for example in facilitating their excretion. However in some cases, it can lead to a more toxic molecule than the parent compound. This is for example the case of aflatoxin B1-epoxy which is derived from the oxidation of AFB1 by cytochromes P450 during the biotransformation reactions of stage I in animals. Glucuronide forms (DON3-GlcA, ZEN14-GlcA, T2-GlcA, HT2-3 / 4-GlcA) come from the Phase II biotransformation of the "native" mycotoxins corresponding by the animal,

and represent examples of mycotoxins called "biologically modified -conjugated". They correspond to the excretion of the native mycotoxins in animal body.

DON-3-β-D-glucopyranoside (D3G) and zearalenone-14-β-D-glucopyranoside (ZEN14G) are issued from DON or ZEN respectively after the phase II biotransformation of metabolization by plant. By convention, the terminology of "masked mycotoxin" was reserved only for the "biologically modified" mycotoxins from the conjugation reaction in a plant [START_REF] Berthiller | Masked mycotoxins: A review[END_REF].

At present, the four major "hidden" mycotoxins in the strict sense are the ZEN14G, the D3G, T2 toxin-glucoside (T2-Glc) and HT2 toxin-glucoside (HT2-Glc) (Lattanzio et al. 2012). It is interesting to note the case of the acetylated derivative of 3ADON, an acetylated derivative of DON. This compound can be produced both by the fungus, in this case it is a "native" mycotoxin, and by transgenic varieties of rice, wheat and barley expressing the gene of the 3-O-acetyltransferase, and therefore considered as a "hidden"mycotoxin. Gene transfer of the 3-O-acetyltransferase to plants is a promising strategy to reduce the pathogenicity of Fusarium that affect some plant species. Indeed, it's established that the conversion of DON into 3ADON by the plant can limit the aggressiveness of Fusarium (Karlovsky, 2011). Other mycotoxins may be "biologically modified" by the action of a microorganism, and are grouped under the term of "mycotoxins differently modified". The Deepoxy-DON (DOM-1) and 3-epi-DON, resulting from the transformation of DON by bacteria extracted from human microbiota or animal, belong to this group (Eriksen et al., 2002;Karlovsky, 2011;Gratz et al., 2013).

"Chemically modified" mycotoxins are the last group. The chemical modifications may or may not depend from the heat. "Chemically modified-thermally formed" mycotoxins appear during food processes such as baking, roasting, freezing or extrusion. These thermodependent changes are known for many mycotoxins, in particular fumonisins capable of entering into a Maillard reaction, due to the reduction of sugars with the production for example of fumonisin B1 N-(1-deoxy D-Fructos-1-yl) and fumonisin N-(carboxymethyl) (Hmph and Voss, 2004).

We can also mention the derivatives of DON (Nordon A-F and 9-hydroxymethyl DON lactone) as thermal degradation products; some of which can be found in commercial food samples (Bretz et al., 2005). "chemically modifiednon-thermally formed" mycotoxins are formed by different processes, including hydrolysis carried out with fumonisins (HFBx), sulfation of DON leading to DON-sulfonate or the degradation products of ochratoxins by UV rays (Beyer et al, 2010;. Heydt-Schmidt et al, 2012.).

2.2. Occurrence of "natives" and "modified" mycotoxins Some "modified" mycotoxins, particularly the "hidden" forms but also the "matrixassociated" forms and some chemically modified forms can be found in pig feed. Table 4 shows data of occurrence of major mycotoxins and their "modified" forms in cereal samples (wheat, barley, corn, oats and rice) over the period from 2010 till 2014.

The "native" mycotoxins represent the major part in food contamination. However, other forms are also concomitantly found in foods. It is currently possible to detect many "modified" mycotoxins, but few quantitative data are available, particularly because of a lack of analytical standards and reference materials.

Table 5 provides more information on the proportion of certain "modified» mycotoxins for which few data are available, compared to their "native" form. For some mycotoxins, such as T2-HT2-Glc and Glc, the occurrence data are from only one study. Their presence was reported for the first time in 2012 in wheat and oats naturally contaminated (Lattanzio et al, 2012).

For the D3G, discovered earlier, more data are available on its occurrence and its ratio to DON. The proportion of this "masked" mycotoxin is stable in food and corresponds, to almost, 20% of the DON present (Berthiller et al, 2009). However, the ratios vary depending on the cereal, genotype concerned, the country and the year of harvest and can increase up to 46%. (Berthiller et al, 2009;De Boevre et al, 2012). Also the increasing use of Fusarium resistant plants, able to glucosylate DON in D3G, could increase the ratio D3G / DON. Some studies of these resistant plants have even found up to 2.7 times more D3G present in the plant than DON (Sasanya et al, 2008). 1 Abbreviations: Nivalenol (NIV), see also Tables 1 and3. The "native" mycotoxins are in italics and "modified" mycotoxins in bold. 2 Country of different grains analyzed: Austria, Belgium, China, Denmark, Finland, Italy, Nigeria, Norway, Czech Republic and Sweden.

In terms of matrix-associated fumonisins (physically entrapped), their proportions compared to the ones of free fumonisins are more variable. Their presence has been shown after a hydrolysis step of raw materials (Dall'Asta et al., 2009). The proportion of physically trapped forms change according to the genotype of corn and according to the culture conditions (Dall'Asta et al., 2012).

In conclusion, more occurring data on different crops and in different countries are needed to properly assess the risk associated with the presence of these new mycotoxins.

Metabolization and toxicity of "modified" mycotoxins on pig

The occurrence of "modified" mycotoxins in feed and animal exposure to these new toxins raise a number of questions and the need to investigate the metabolism and toxicity of these compounds (EFSA, 2014). It is important to study the intrinsic toxicity of these toxins, but also to know their metabolism and in particular to determine if "modified" mycotoxins are converted into their "native" forms.

Some recent studies were interested in the effects of these "modified" mycotoxins on the pig, on in vitro or in vivo models. Most of these studies focus on the metabolism of these molecules and few about their toxicity.

Inherent toxicity of "modified" mycotoxins for pigs

Studies on the toxicity of "modified" mycotoxins mainly concern the "modified" forms of DON and ZEN. The majority of these studies were conducted in vitro on human cells and only a few are interested in vivo toxicity in animals, including mice and pigs.

The toxicity of DON were compared to those of its acetylated derivatives (3ADON and 15ADON) taking into account the cell proliferation, activation of MAPKs (mitogen-activated protein kinase) and expression of tight junction proteins as well that the expression of cytokines in pigs [START_REF] Pinton | Toxicity of deoxynivalenol and its acetylated derivatives on the intestine: Differential effects on morphology, barrier function, tight junction proteins, and mitogen-activated protein kinases[END_REF]. Percentages of cell viability of the pig intestinal cells (IPEC-1) incubated for 24 hours in the presence of DON, 3ADON and 15ADON were decreased to 60%, 13% and 69%. The expression of the junction proteins by these porcine intestinal cells is decreased by 40% in the presence of 15ADON, and is equivalent to the DON and 3ADON. The 15ADON also showed more toxicity than DON and 3ADON on the activation of MAPKs, in vitro on IPEC-1 cells, ex vivo on explant cultures of porcine jejunum or in vivo in the tissue jejunal piglets [START_REF] Pinton | Toxicity of deoxynivalenol and its acetylated derivatives on the intestine: Differential effects on morphology, barrier function, tight junction proteins, and mitogen-activated protein kinases[END_REF].

One of the known derivatives of DON is the DOM-1 coming from a bacterial transformation reducing the 12,13-epoxy group, which is essential in the toxicity of DON and trichothecenes in general (Schatzmayr et al., 2006;Zhou et al. 2008). Thus the DOM-1 is considered as a non-toxic metabolite of DON. An in vitro study showed that the DOM-1 was 54 times less toxic than DON level of DNA synthesis in mouse fibroblasts (Eriksen and Pettersson, 2004).

The toxicity of DOM-1 on production parameters was evaluated in vivo in pigs. Animals receiving the contaminated food with DON 5mg / kg feed and the bacterial strain capable of deepoxydise DON, showed no decrease in food intake or weight gain (He et al., 1993;Li et al., 2011). However the in vivo evaluation of the pure DOM-1 toxicity on intestine and on the pig organism has not been evaluated.

Compared to DON, the D3G was nontoxic on pig jejunum ex vivo study with an inability to induce a D3G ribotoxic stress and to activate the MAPK pathway central, in the implementation of the observed pro-inflammatory response with DON [START_REF] Pierron | Intestinal toxicity of the masked mycotoxin deoxynivalenol-3-beta-d-glucoside[END_REF].

This inability of D3G to induce a pro-inflammatory response, on the contrary to DON, is confirmed in the rat with an absence of an over-expression of cytokines and chemokines (Wu et al., 2014A). Moreover, the power of the emetic D3G seems much lower than that of DON (Wu et al., 2014b).

Several studies have compared estrogenic powers of the ZEN and its two derivatives α-ZEL and β-ZEL. Estrogenicity of these molecules thus ranks as follows: β-ZEL <ZEN <α-ZEL (Mukherjee et al, 2014.). At the cellular level, higher cytotoxicity and genotoxicity of β-ZEN compared to the α-ZEN were shown on endometrial pig cells (Tiemann et al., 2003;Othmen et al, 2008), while the α-ZEL was more toxic than the β-ZEL on pig oocytes (Alm et al., 2002). In summary, the hierarchy in the toxicity of these two "modified" mycotoxins, α-β-ZEL and ZEL, is not clearly established and it depends on the cell type considered. Overall, their toxicity is lower than ZEN.

Generally, glycosylated and sulfated forms of ZEN, as ZEN14G and ZEN14S, seem unable to bind to estrogen receptors and induce in vitro toxicity (Poppenberger et al., 2006;Berthiller et al., 2009). However, Plasencia and Mishra (1991) found in our study that ZEN4S was able to induce the same estrogenic activity than ZEN in the rat uterus enlargement bioassay.

Overall, the compounds from the detoxification pathways of plants, "masked" forms sensu stricto are less toxic or inactivated with respect to the "native" molecule. The metabolic pathways of the plant which are similar to those of animals (e.g. conjugation reactions to different molecules sulfate, glutathione or glucuronic acid), increase the polarity of these molecules, thereby facilitating their excretion and decreasing their toxicity (Yiannikouris and Jouany 2002;Homolya et al, 2003).

Metabolization of "modified" mycotoxins in pig

Questions about the conversion of "modified" mycotoxins into "native" mycotoxins are as old as the discovery of the first "modified" mycotoxin.

Very early, it has been shown that exposing orally a piglet for 2 weeks to the ZEN14G, it was possible to find in his urine and feces varying amounts of ZEN and its estrogenic metabolite α-ZEL (Gareis and al., 1990). This study highlights the fact that a significant part of the pig exposure to mycotoxin could come from the conversion of "modified" mycotoxins. Such conversion may be due to the activity of digestive enzymes and metabolism of the animal. It may also result from the enzymatic activity of the digestive flora followed by reabsorption of the "native" mycotoxin and / or its metabolites. Table 6 summarizes the features of some "modified" mycotoxin along the digestive tract and their hydrolyzed to "native" form.

Pig is physiologically very close to man, particularly in terms of gastrointestinal tract. In an in vitro system mimicking human in successive stages, the action of salivary juice, gastric juice, intestinal juice and bile, D3G, ZEN14G and ZEN14S are retained respectively to 99.5%, 97.3% and 98.6% (De Nijs et al, 2012;. Dall'Erta et al, 2013.). However, D3G that is not converted by the enzymes present in saliva and mammalian stomach, may be hydrolyzed by lactic acid produced by certain bacterial species such as Enterococcus mundtii and Lactobacillus plantarum present in the gastrointestinal tract [START_REF] Berthiller | Hydrolytic fate of deoxynivalenol-3-glucoside during digestion[END_REF]. and "stable" if the molecule is not transformed in its"native" form in the compartment. 3 Based on in vitro and in vivo experiments 4

Based on in vivo experiments

As for DON, "modified" forms of ZEN can be deconjugated after fermentation in presence of human feces, and so the "native" forms can be released (Dall'Erta et al, 2013). An increase of 30-50% of detectable fumonisins is observed, after food matrix digestion (Dall'Asta et al, 2010). This suggests that the gastrointestinal enzymes are capable of destroying the interactions between the matrix and fumonisin, salting-out forms of "native" mycotoxins. For the DOM-1, from a bacterial transformation, there is no in vivo study with pure DOM-1.

The epimerization forming the 3-epi-DON, is an irreversible reaction (Karlovsky, 2011). But currently there is no data available on its toxicity. In terms of acetylated forms of DON, they are quickly deacetylated in DON within the organism of animal (Wu et al., 2010).

These in vitro results therefore show that some "modified" mycotoxins can be transformed back into their "native" mycotoxins in variable proportions. This suggest that this transformation would be mainly due to intestinal microbiota.

One study looked at the becoming of DON or D3G orally and intravenously administered to piglets [START_REF] Nagl | Metabolism of the masked mycotoxin deoxynivalenol-3-glucoside in pigs[END_REF]. DON is excreted largely in the urine, mainly in the first eight postexposure hours by mainly "native" form and to a minor form of DON-3-glucuronide (DON3-GlcA) and DON-15-glucuronide (DON15-GlcA). Urinary excretion of D3G administered orally, although a majority, appearing between the 8th and the 24th hour post-exposure. Only a tiny fraction of D3G was found unprocessed in the urine, most of which is converted into DON, and as well in DON3 GlcA-and-DON15 GlcA. In the case of D3G parenteral administered, most of the administered dose was recovered unchanged in the urine within the first eight hours. This study demonstrates that (i) the contaminant D3G pig feed can be converted to DON in the digestive tract, (ii) due to the delayed urinary excretion, this conversion takes place probably in the lower portion of the digestive tube, and (iii) the DON from the D3G of microbial hydrolysis may well be absorbed and contribute significantly to the total exposure of pig to DON.

ASSESSMENT OF THE EXPOSURE AND CHARACTERIZATION OF THE RISK FOR THE "MODIFIED" MYCOTOXINS IN PIG

In 2014, EFSA performed a work of assessing exposure of pig to certain "modified" mycotoxins and on the characterization of the risk associated to these molecules (EFSA, 2014). Mycotoxins concerned are ZEN, nivalenol (NIV) (a mycotoxin of the family of trichothecenes such as DON), T2 toxin and HT2 and fumonisins.

Exposure calculation traduces in cumulative exposure the increase of exposure of one mycotoxin due to the conversion of "modified" forms into "native" mycotoxin.

As in human, the increase was estimated at 100% for ZEN, 30% for NIV, 10% for T2 and HT2, and 60% for fumonisins.

Table 7 presents the calculation of estimated exposure to these "modified" and "natives" mycotoxins.

Based on NOEL (No Observed effect Level) of ZEN fixed at 10µg/Kg BW per day for these estrogenic effects, EFSA has estimated that the increase exposure, linked to the "modified" forms, was not enough to overcome the regulation of ZEN in pig feed.

The LOAEL (Lowest Observed Adverse Effect Level) established for NIV in pigs is 100 µg/Kg BW/day (EFSA, 2013). The calculation of estimated exposure in NIV and NIV from the "modified" forms retransformed will represent 2-3% of the LOAEL (Table 7). So, EFSA estimated that the addition of "modified" forms of NIV, was not likely to jeopardize the safety of pig feed. Starting from the most pessimistic hypothesis, the cumulative exposure in T2 and HT2, and their "modified" forms correspond to 9% of the LOAEL group established at 29 µg/kg BW/day for these trichothecenes of group A (EFSA, 2011). On this basis, the EFSA considered that the inclusion of "modified" forms of T2 and HT2 also was not likely to jeopardize the level of food safety for pigs for these mycotoxins. In the highest hypothesis, cumulative exposure to "native" fumonisin and their "modified" forms represent 25% of the LOAEL established by EFSA in 2005 at 200 µg/kg BW/day. For these mycotoxins the safety of food for pigs does not appear compromised.

To sum up, the EFSA issued an opinion on four mycotoxins or groups of mycotoxins (ZEN, NIV, T2/HT2 and fumonisins) for which no new concern seem to emerge following the consideration of exposure to "modified" forms. One advice for DON and its "modified" forms is being written. Such advices of reference are not yet available for aflatoxins and ochratoxin, which are two families of mycotoxins respectively subject to regulation and recommendation in pig feed.

CONCLUSION

New methods in toxicology and in analysis allow to increase the knowledge on the hazards present in food and to put in evidence new types of mycotoxins. However, effects of these "modified" and "masked" mycotoxins on the health is not well known. Actually, only few studies in vivo and in vitro are available on the metabolization of these molecules into the animal. More studies are necessary to better understand their impact on the health of pigs and humans.

The percentage of "modified" mycotoxins are generally lower than the one of "native" mycotoxins which means that the observed toxic effects are mainly due to "native mycotoxins". However, the feasible hydrolyze of "modified" mycotoxins in the intestine, could increase the total amount of mycotoxins to which pigs are exposed, and so minimize the real risk of exposure for the animal.

Many "modified" mycotoxins are retransformed into their "native" mycotoxins. For this reason, it's important to take into account both "modified" and "natives" mycotoxins in the risk assessment of exposure. Actually, only "natives" mycotoxins are regulated, but "modified" forms can be found in important proportion in cereals. Indeed, for example, glucosylated forms, as D3G can reach in proportion 30% of the "native" mycotoxins.

The EFSA recent works tend to consider the amount of "natives" and "modified" mycotoxins in order to calculate the risk linked to the exposure to mycotoxins.

Aims of the thesis

The Fusarium mycotoxin deoxynivalenol (DON) is one of the most frequently widespread mycotoxin worldwide, in cereals and feed raw materials. DON is known for its toxic effects on animals, and causing big economic losses. Due to its high structural stability of the DON makes its elimination difficult, once present in cereals or feed materials. Several Indeed, one of the major questions associated with these metabolites of DON is related to their toxicity: are they toxic by themselves? Indeed, little is known about the toxicity, of these masked and modified forms of DON, compared to the extensively investigated DON.

As part of this thesis, I assessed the toxicity of these DON derivatives with a special emphasis on DOM-1 with an in vivo experiment.

Intestine is the first exposed organs to xenobiotics or mycotoxins, present in food, and so constitutes the first barrier upon exposure of toxins and could be exposed to high level of mycotoxins (Rotter et al. 1996). Intestinal viability cells could be directly damage or it is the barrier function that can be impaired, and so promote the entry of contaminant in the blood and in all the organism. Thus, analysis focused on intestinal tissue.

Moreover, DON is well known for its immunomodulatory effects and for its great impact on the intestine, the first organ targeted following the ingestion of mycotoxins. DOM-1, 3-epi-DON and D3G derivatives of DON may also have an impact on these two functions.

Therefore, it becomes important to know whether these transformations of DON will lead to less toxic molecules for animal or whether they will induce other effect on the animal.

The specific aims of this research were: To study the toxicity of DON and its derivatives on intestinal epithelial cells, the choice was to use the human intestinal cell line Caco-2. It has advantages to express characteristics markers of adults' intestinal cells and to be a reference line in toxicology studies, and to be largely used to study intestinal absorption (Sambruy et al. 2001). The explant model was also chose to enable directly testing the toxicity of molecules on intestinal living tissue keeping its polarity and extracellular integrity (Gonzalez-Vallina et al. 1996). Moreover, in the context of 3R, it limits the use of animals, and to test many conditions with limited number of animals.


In vivo pig model allowed studying more deeply the comparative toxicity of DON and DOM-1, on the intestine and on the immune response. Therefore in the context of risk assessment, in vivo experiment allows to investigate the metabolism of modified mycotoxin into the animal.

Experimental animal phases were conducted on pigs at the ToxAlim laboratory. From an agronomic point of view, breeding a monogastric animal such a pig is particularly vulnerable to mycotoxins due to the importance of share of cereals in its diet. In addition, pig is very sensitive to mycotoxins, due to the absence of ruminal tank, known to contain microorganisms capable of degrading toxins before their intestinal absorption. Finally, considering the similarity in the immune and digestive systems of pigs and humans, the use of pig model permit also to extrapolate data to man. For the choice of doses and time of exposure, this work fits in the current dynamic of a toxicological study, for chronical exposure going from low doses to moderate ones, not inducing major clinical manifestations.

Finally, performing microarray analysis on all the genome of the pig allowed identifying Penicillium and Fusarium which may contaminate food and feed at all stages of the food/feed chain (Bennett and Klich 2003;[START_REF] Frisvad | Recommendations concerning the chronic problem of misidentification of mycotoxigenic fungi associated with foods and feeds[END_REF]. 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 strategies (Awad et al, 2010). 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 worldwide (EFSA 2013[START_REF] Cast | Council for Agricultural Science and Technology-Potential economic costs of mycotoxins in United States Task Force Rep[END_REF].

DON causes acute and chronic disorders in humans and animals, with the gastrointestinal tract being an organ sensitive to its adverse effects (Pestka 2010a). DON affects the intestinal histomorphology, impairs barrier function and nutrient absorption (Maresca 2013 ;Pinton and Oswald 2014). DON also disrupts the local intestinal immune response; it triggers and potentiates intestinal inflammation (Cano et al. 2013;Vandenbroucke et al. 2011). At the cellular and subcellular level, DON binds to the ribosome, inhibits protein and nucleic acid synthesis and triggers ribotoxic stress [START_REF] Shifrin | Trichothecene mycotoxins trigger a ribotoxic stress response that activates c-jun n-terminal kinase and p38 mitogen-activated protein kinase and induces apoptosis[END_REF][START_REF] Pestka | Cellular and molecular mechanisms for immune modulation by deoxynivalenol and other trichothecenes: Unraveling a paradox[END_REF]Garreau de Loubresse et al. 2014) leading to the activation of kinases, MAPKs and their downstream signaling pathways (Pestka 2010b).

Several strategies have been developed to limit DON toxicity (Zhou et al. 2008), 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-epoxydeoxynivalenol (deepoxy-DON or DOM-1) (Fig. 1) (Sundstol Eriksen et al. 2004). Several microbial strains are capable of DON de-epoxidation (Schatzmayr et al. 2006;Zhou et al. 2008). Several in vitro studies demonstrated the reduced toxicity of deepoxy-DON. In vivo trials on farm animals receiving feed contaminated with DON have also shown a beneficial effect of the bacteria able to de-epoxidize DON, according to zootechnical parameters and immune response (Grenier et al. 2013). The hydroxyl on carbon 3 also seems to be significant of non-essential amino acids (Sigma-Aldrich). Cells were maintained at 37° C in an atmosphere of 5% CO 2 and 90% relative humidity. The medium was changed every 2 days.

Cells were passaged once a week. The partially confluent cell monolayers were trypsinized with Trypsin-EDTA (Eurobio).

Cell viability assay

Cell viability assay was performed with the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, USA) according to manufacturer's instruction. This test measures the quantity of ATP, proportional to the quantity of cells. Cells were seeded at the density of 1.56x10 5 cells/cm 2 in 96-well microtiter plates. Cells were grown for 24 hours and exposed to DON, deepoxy-DON or 3-epi-DON, or corresponding concentrations of DMSO, for 48 hours.

Luminescence was measured with a spectrophotometer (TECAN Infinite M200, Männedorf, Switzerland).

Trans-epithelial electrical resistant measurements

To assess the integrity of individual monolayers, trans-epithelial electrical resistance (TEER)

was measured as already described [START_REF] Pinton | Toxicity of deoxynivalenol and its acetylated derivatives on the intestine: Differential effects on morphology, barrier function, tight junction proteins, and mitogen-activated protein kinases[END_REF]. Cells (1.34 x 10 5 cells/cm 2 ) were grown until differentiation on polyethylene terephthalate membrane inserts (surface area 0.3 cm 2 , pore size 0.4 µm) in 24-well format (Becton Dickinson, Pont de Claix, France). The medium was changed every two days. Differentiated cells were exposed to 10µM of diluent or toxins, DON, deepoxy-DON or 3-epi-DON. The culture medium in the apical side of differentiated cells in each well was replaced every two days with medium containing toxin.

The TEER was measured for each well daily for 11 days using a Millicell-ERS Voltohmmeter (Millipore, Saint-Quentin-en-Yvelines, France). TEER values were expressed as % of initial values.

Oxygen consumption measurements

The acute effect of toxins on oxygen consumption of Caco-2 cells was assessed using an XF24 extracellular flux analyzer (Seahorse Bioscience, North Billerica, USA). The procedure was performed according to the manufacturer's instructions. Briefly, cells were cultured in XF24 cell culture microplates at 1.5x10 4 cells per well and maintained as described above.

Oxygen consumption rates (OCR) were measured in both differentiated and undifferentiated proliferated Caco-2 cells in non-buffered DMEM (Seahorse Bioscience) supplemented with 10 mM glucose, 2 mM sodium pyruvate (Sigma-Aldrich) and 2mM glutamine (Eurobio) and adjusted to pH 7.4. OCR was monitored every 20 minutes before (basal level) and after injection of diluent or toxins (10µM). OCR values from each well were normalized against viable cell counts (calculated with a Malassez cell) and expressed as a percentage of the baseline value.

Intestinal jejunal explants

Jejunal explants were obtained from 5 week old crossbred castrated male piglets as described previously (Lucioli et al. 2013). The experiment was conducted under the guidelines of the French Ministry of Agriculture for animal research. Two authors (I.P.O. and A.P.) have an official agreement with the French Veterinary Services permitting animal experimentation.

Explants were treated for 4 hours at 39°C with 10µM of toxins (DON, deepoxy-DON or 3epi-DON) or diluent (DMSO) in complete medium. After incubation, treated explants were fixed in 10% formalin (Sigma-Aldrich) for histological analysis or stored at -80°C for RNA extraction.

Histological assessment

Explants fixed with 10% formalin for 24 hours were dehydrated and embedded in paraffin wax (Labonord, Templemars, France) according to standard histological procedures. Sections of 5 µm were stained with haematoxylin and eosin (Sigma-Aldrich) for histopathological assessment. Histological findings were scored based on histological changes and the severity of lesions as previously described (Lucioli et al. 2013).

Gene expression analysis of explants by RT-qPCR

For the gene expression analysis, total RNAs were extracted in lysing matrix D tubes (MP Biomedicals, Illkirch, France) containing guanidine thiocyanate-acid phenol (Eurobio). The quality of these RNA was assessed (Agilent RNA 6000 Nano Kit Quick, Agilent Bioanalyzer 2100); the mean RNA Integrity Number (RIN) of these mRNA preparations was 6.32 ± 0.83.

Reverse transcription and RT-qPCR steps were performed as already described (Gourbeyre et al. 2015;Halloy et al. 2005) with previously published primers (Pierron et al. 2015).

Amplification efficiency and initial fluorescence were determined by the ΔCt method.

Obtained values were normalized using two reference genes, ribosomal protein L32 (RPL32)

and cyclophilin A (CycloA). Gene expression levels of treated explants were expressed relative to the mean of the control explants.

Gene expression analysis by microarray

The microarray GPL16524 (Agilent technology, 8 x 60K) used in this experiment consisted of 43,603 spots derived from the 44K (V2:026440 design) Agilent porcine specific microarray.

This was enhanced with 9,532 genes from adipose tissue, 3,776 genes from the immune system and 3,768 genes from skeletal muscle (Pierron et al. 2015). For each sample, Cyanine-

Immunoblotting

Expression of the phosphorylated MAPK p38 and JNK (Jun amino-terminal kinases) was assessed on differentiated Caco-2 cells and jejunal explants by immunoblotting as previously described [START_REF] Pinton | Toxicity of deoxynivalenol and its acetylated derivatives on the intestine: Differential effects on morphology, barrier function, tight junction proteins, and mitogen-activated protein kinases[END_REF]. Cells differentiated on 24-well inserts or explants were treated with 10µM of diluent (DMSO) or toxins, DON, deepoxy-DON or 3-epi-DON for 1 hour.

Proteins were extracted, quantified and a total of 15 µg of protein was separated by SDS-PAGE. The membranes were probed with rabbit antibodies (Cell Signaling Technology, Danvers, USA) specific for: phospho-SPAK/JNK or phospho-p38 diluted at 1:500 or GAPDH diluted at 1:1000. After washing, the membranes were incubated with 1:10,000 CF TM 770 goat anti-rabbit IgG (Biotium, Hayward, USA) for the detection. Antibody detection was performed using an Odyssey Infrared Imaging Scanner (Li-Cor Science Tec, les Ulis, France)

with the 770nm channel. The expression of the proteins was estimated after normalization with GAPDH signal.

Molecular modeling.

All the compounds tested were modeled with NAMD under VMD1.9. The structures of deepoxy-DON and 3-epi-DON were built using molefacture from the DON structure included in PDB-file 4U53, after adding hydrogen chirality and atom verification for minimization.

Docking of deepoxy-DON and 3-epi-DON was done in VMD based on the carbon backbone of DON. To relax structures and to evaluate the interaction of H-bonds between ribosome and ligand, a semi-rigid energy minimization was calculated by NAMD using AMBER topology and parameter files with a fixed ribosome structure and a flexible structure for the ligand.

Statistical analysis

Data are expressed as a mean ± SEM of values. The results were analyzed using the Fisher test on equality of variances, one-way ANOVA and Bonferroni as a test post-hoc; p-values < 0.05 were considered significant.

Microarray data from Feature Extraction software was analyzed with R using Bioconductor packages and the limma lmFit function as previously described (Pierron et al. 2015). Probes with adjusted p-value ≤ 0.05 were considered differentially expressed between treated and control conditions. Hierarchical clustering was applied to the samples and the probes using 1-Pearson correlation coefficient as distance and Ward's criterion for agglomeration.

Results

Deepoxy-DON and 3-epi-DON do not impair proliferation of human intestinal cells

Comparative effects of deepoxy-DON, 3-epi-DON and DON were first evaluated on proliferating human Caco-2 cells. The cell viability was assessed by the quantification of ATP using the luminescent cell viability assay. As shown in figure 2, 48 hours exposure to deepoxy-DON or 3-epi-DON at concentrations up to 30µM had no significant impact on cell viability. By contrast DON markedly decreased the viability of proliferating cells in a dosedependent manner; exposure to 10µM of DON for 48 hours reduced cell viability by approximately 70%. The IC50 was calculated at 1.30µM.

Deepoxy-DON and 3-epi-DON do not impair cell viability and TEER of differentiated human intestinal cells

V.

The comparative toxicity of deepoxy-DON, 3-epi-DON and DON was also performed on differentiated Caco-2 cells through the measurement of TEER. As already described (Pierron et al. 2015), differentiated cells are more resistant to DON and at least 30µM are needed to induce a significant decrease in viability. At 10µM of DON, a significant decrease of TEER in differentiated Caco-2 cells at a non-cytotoxic dose was observed (Figure 3). The decrease was time-dependent and reached 25% after 2 days and about 90% at 10 days. By contrast, cells treated with 10µM deepoxy-DON or 3-epi-DON didn't show any decrease in TEER.

Deepoxy-DON and 3-epi-DON do not affect oxygen consumption in Caco-2 cells

The impact of deepoxy-DON, 3-epi-DON and DON on bioenergetic function in Caco-2 cells was evaluated using extracellular flux analyses. As shown in figure 4, DON linearly decreased the rate of oxygen consumption in a time-dependent manner starting at the 40 minute stage. Approximately 3 hours after DON exposure, oxygen consumption values were 32% less than the base value. By contrast, deepoxy-DON and 3-epi-DON had no effect on the cellular oxygen consumption of proliferating Caco-2 cells and displayed bioenergetics profiles comparable to that of control cells. Similar data were obtained with differentiated Caco-2 cells (data not shown).

Deepoxy-DON and 3-epi-DON do not induce histological alterations of intestinal explants

In order not to restrict the observations to an intestinal cell line, experiments were also performed on jejunal explants, a model developed to assess short-term effects of mycotoxins (Lucioli et al. 2013). The effects on intestines of deepoxy-DON, 3-epi-DON and DON were first compared with histology (Figure 5). Lymphatic vessel dilation was observed at different intensities in all groups. Control explants displayed normal villi lined with columnar enterocytes (Figure 5A). Explants exposed to deepoxy-DON (Figure 5C) and 3-epi-DON (Figure 5D) presented similar features but mild interstitial edema and cell debris on apical surface (arrow) were also observed. By contrast, multifocal to diffuse villi atrophy, multifocal villi fusion (arrows), necrosis of apical enterocytes and cellular debris (arrowhead, Figure 5B)

were observed after 4 hours of explant incubation with 10µM of DON.

Deepoxy-DON and 3-epi-DON do not induce intestinal inflammation

To complete the analysis of the intestinal toxicity of deepoxy-DON and 3-epi-DON, their effects on the expression of inflammatory genes were analyzed by RT-qPCR. As already described (Cano et al. 2013), a strong intestinal inflammatory response was observed in jejunal explants in the presence of DON and a significant increase in expression of IL-1α, TNFα, IL-1β, IL-8, IL-12p40 Il-17A and IL-22 was also observed (Table 2). By contrast, no induction in the expression of these genes was observed in deepoxy-DON and 3-epi-DON treated explants, demonstrating that microbial transformation of DON to DEEPOXY-DON or 3-epi-DON led to decreased inflammatory response in intestinal explants (Table 2).

DON but not deepoxy-DON and 3-epi-DON changes gene expression profile in intestinal explants

The effects of DON, deepoxy-DON and 3-epi-DON were investigated beyond the inflammatory response, through a genome wide transcriptomic analysis. Exposure to DON resulted in differential expression of 747 probes; 681 and 66 probes corresponding to 303 and 33 genes were up-and down-regulated respectively (Figure 6A). By contrast, no genes were differentially expressed in deepoxy-DON and 3-epi-DON treated explants when compared to control explants, indicating that microbial transformation of DON to deepoxy-DON or 3-epi-DON abolishes the toxicity of the mycotoxin.

DON differentially expressed genes were then selected to perform principal component analysis (PCA) (Figure 6B) and cluster analysis (Figure 6C). Two clusters were distinguished, indicating up-regulated and down-regulated genes (supplementary table 3). The most significantly up-regulated genes in DON-treated explants, with a change of more than 2.4 fold compared to control, were immune genes such as CCL20, CXCL2, PRDM1, AREG, CSF2, FOSL1 (Table 3). As expected, the 6 pro-inflammatory cytokines already tested in RT-qPCR analysis were also up-regulated in the DNA array analysis; a strong correlation between the two methods of analysis was observed (coefficient R²=0.96). DON also increased the expression of the ER heat shock protein HSP70 gene (HSPA2), genes of ubiquitination pathway (HSPA2, BIRC2, NEDD4L, BIRC3) and genes of metallothioneins (MT1A, MT1M and MT2B). DON decreased expression of the CHAC1 gene, genes for molecular transport including ABCC2, SLC15A1, SLC9A2, the CCL24 gene which is thought to play a role in the immune response, the MLEC gene which is connected to protein misfolding under conditions of endoplasmic reticulum (ER) stress, and other genes (table 3).

Pathway analysis of differentially expressed genes exposed to DON was performed using Ingenuity Pathway Analysis software (IPA). The top 10 scored pathways are listed in Table 4.

In silico analysis of the interaction of deepoxy-DON and 3-epi-DON with ribosomes and their inability to activate MAPKs

The above data indicate that microbial transformation of DON into deepoxy-DON or 3-epi-DON abrogates its toxicity. The last step of this study was to investigate the underlying mechanism and more specifically to determine the ability of DON, deepoxy-DON and 3-epi-DON to bind to the A site of the ribosome peptidyl transferase center and to activate the MAPKs.

As expected, after 1 hour of exposure to 10µM DON, MAPKs were activated in both differentiated monolayers Caco-2 cells and jejunal explants (Figure 7). In cells, DON significantly increased phosphorylated p38 (3.24 ± 0.75 vs. 1 ± 0.09) compared to the control (relative intensity in arbitrary unit (A.U.); p<0.05; n=3) and phosphorylated SapKjunk (7.28 ± 0.64 vs. 1 ± 0.12 for control A.U.; p<0.05; n=3). By contrast, deepoxy-DON and 3-epi-DON

were not able to activate these MAPKs in Caco-2 cells (Figure 7

Discussion

Despite good agricultural practices, contamination by mycotoxins cannot be avoided.

Several strategies have been developed to reduce mycotoxin exposure. Among them, microbial transformation is of interest but requires demonstration of the absence of toxicity of the metabolites produced. The aims of the present study were (i) to analyze the intestinal toxicity of two bacterial metabolites of DON, deepoxy-DON and 3-epi-DON and (ii) to investigate the molecular basis for their reduced toxicity.

Through the action of bacteria, DON can be epimerized on the hydroxyl group of C3 or de-epoxidized on the C12-C13 epoxide (Karlovsky 2011). Epimerization is an aerobic irreversible transformation that may require two enzymatic activities of partially overlapping substrate specificity which occur together in sequence: oxidation of DON to 3-keto-DON and then conversion to 3-epi-DON. To date only four bacterial strains have been described to epimerize DON to 3-epi-DON (Karlovsky 2011). A very recent paper shows that this bacterial metabolite is substantially less toxic than DON when tested in vitro on proliferating human Caco2 cells, as well as in vivo when given orally to mice for 14 days (He et al. 2015b).

Deepoxy-DON is obtained after de-epoxidation of DON; several bacterial species and enzymes are able to catalyze this reaction (Karlovsky 2011). The lower toxicity of deepoxy-DON, as compared to DON has been demonstrated in vitro on Swiss mouse 3T3 fibroblasts (Sundstol Eriksen 2004), lymphocytes (Schatzmayr et al. 2006) and brine shrimp (Swanson et al. 1987). In vivo supplementation of DON-contaminated feed, with bacteria and/or an enzyme able to de-epoxidize DON, induced a reduction of the toxicity as shown by measurement of zootechnical or immune parameters (Li et al. 2011;He et al. 1993;Grenier et al. 2013).

In the present study, we observed reduced intestinal toxicity of 3-epi-DON and deepoxy-DON The toxicity of DON and its bacterial metabolites on intestinal tissues was further evaluated. Because of the difficulties accessing human intestinal samples, the study was performed on porcine intestinal explants. Indeed pigs are very sensitive to DON and can be considered good models for extrapolation to humans, with a digestive physiology very similar to that of humans (Nejdfors et al. 2000;Pinton and Oswald 2014). Histological assessment showed normal villi lined with columnar enterocytes, mild interstitial edema and cell debris on the apical surface for the control, 3-epi-DON and deepoxy-DON treated explants. This is in accordance with the absence of histopathological lesions observed in mice after a 14 day oral exposure to 25 or 100 mg 3-epi-DON /Kg bw (body weight) (He et al. 2015). Effects of purified deepoxy-DON on the intestine have never been tested, however nutritional strategies including bacteria/enzyme transforming DON to deepoxy-DON have reduced the occurrence and extent of intestinal lesions (Grenier et al. 2013) and showed the same zootechnical performance as in control animals (He et al. 1993;Li et al. 2011). By contrast, as already shown, treatment with 10µM of DON induces intestinal damage indicated by villi atrophy and villi fusion (Lucioli et al. 2013). To confirm that the two microbial transformation products of DON were not toxic, a pan-genomic analysis using a DNA array containing 62,976 probes was performed on jejunal explants. It revealed that no probes were differentially expressed between control explants and the ones treated with either deepoxy-DON or 3-epi-DON. To the best of our knowledge this is the first genome wide analysis performed for deepoxy-DON and 3-epi-DON.

The global transcriptomic analysis of the effect of DON on the intestine indicated that DON does not only interfere with genes involved in the immune response. As already described for human and murine thymus cells (Van Kol et al. 2011;Katika et al. 2012;Mishra 2014), DON exposure also targets ER (endoplasmatic reticulum) stress, protein synthesis, oxidative stress, cell cycle regulation and apoptosis in intestinal tissues. The strong alteration of the gene MLEC implicated in misfolded glycoprotein quality control observed herein is likely due to the arrest of translation induced by ribotoxic stress. This leads to less protein entering the ER to temper the unfolded protein response and therefore protein synthesis (Katika et al. 2012). The increased gene expression of the ER heat shock protein HSP70 could also reduce the accumulation of unfolded protein in ER lumen. An increased expression of some genes involved in the ubiquitination pathway was observed in the presence of DON.

This result could indicate that the presence of DON may induce the increase in proteins involved in protein degradation (Shen et al. 2007;Osman et al. 2010;Katika et al. 2012). Our data also underline the decrease of the unfolded protein response pro-apoptotic gene CHAC1.

The CHAC1 protein seems to play a role in glutathione degradation (Kumar et al. 2012). ER stress could also induce leakage of calcium from the reticulum leading to activation of NFkB, NRF2-mediated oxidative stress response and apoptosis (Katika et al. 2012). The present work emphasizes the effect of DON on metallothioneins MT1A, MT1M and MT2B. A relationship between metallothionein protein levels, used as a marker of oxidative stress, and mycotoxins in the liver of rats fed on naturally contaminated wheat has been reported (Vasatkova et al. 2009). Therefore, it could be assumed that MTs are associated with pathways protecting the intestine against DON toxicity. The present study underlines the effect of DON on the genes of intestinal transporters. DON decreases the expression of the solute carrier SLC15A1 and SLC9A2 involved in proton-coupled oligopeptides transporter PepT1 and a Na+/H+ exchanger, respectively (Bookstein et al. 1997;Smith et al. 2013).

Similar effects on other mRNA expression transporters as sugars transporters were described in the jejunum and to a lesser extent in the liver of broilers exposed to DON [START_REF] Dietrich | Fusarium mycotoxincontaminated wheat containing deoxynivalenol alters the gene expression in the liver and the jejunum of broilers[END_REF]). Accordingly, it has been experimentally shown that DON decreases the intestinal uptake of various nutrients in human epithelial intestinal cell line HT-29-D4 [START_REF] Maresca | The mycotoxin deoxynivalenol affects nutrient absorption in human intestinal epithelial cells[END_REF]. This effect is likely due to a specific modulation of intestinal transporters expression, rather than a consequence of cell damage. The transcriptomic analysis demonstrates that DON down-regulates the expression of ABCC2 gene that encodes for MRP2, a protein involved in efflux of DON and other mycotoxins and also in the transport of a wide range of organic anions including bile salt flow (Videmann et al. 2007). An action of DON on mitochondrial dysfunction, attested to by the down-regulation of CPT1A mRNA was also observed in this study. CPT1A encodes for a key regulatory enzyme of β-oxidation and is required for transport of long chain fatty acids into mitochondria [START_REF] Nakamura | Regulation of energy metabolism by longchain fatty acids[END_REF]. The modulation of β-oxidation in addition to the modulation of intestinal transporters could explain the energy failure reported after DON exposure (Maresca et al.2002). It is now necessary to investigate these changes at the protein level.

The use of bacteria is a promising approach to DON decontamination. In the present study we observed that deepoxy-DON and 3-epi-DON were devoid of intestinal toxicity. The underlying mechanism was further investigated. DON is known to develop its toxic potential by interacting with the peptidyl transferase at the 60S ribosomal subunit level, blocking the protein synthesis at the elongation step, inducing a ribotoxic stress and activating MAPKinases (Maresca 2013;[START_REF] Pestka | Cellular and molecular mechanisms for immune modulation by deoxynivalenol and other trichothecenes: Unraveling a paradox[END_REF]Garreau de Loubresse et al. 2014). In accordance with literature, we observed that DON induced phosphorylation of JNK and p38

proteins [START_REF] Sergent | Deoxynivalenol transport across human intestinal caco-2 cells and its effects on cellular metabolism at realistic intestinal concentrations[END_REF]Lucioli et al 2013). By contrast deepoxy-DON and 3-epi-DON did not active these signaling pathways, suggesting an absence of ribotoxic stress. To further the analysis, a modeling of deepoxy-DON and 3-epi-DON in the ribosome peptidyl transferase center was performed. DON and its bacterial metabolites fit into the A-site pocket, however whereas DON binds to the peptidyl transferase center with three hydrogen bonds, only two hydrogen bonds were identified between deepoxy-DON or 3-epi-DON and the peptidyl transferase center. This suggests that the absence of the epoxy group or the isomeric transformation decreases the affinity of these latter metabolites for the active site pocket A of the ribosome and prevents the induction of ribotoxic stress. In silico modeling revealed that a third hydrogen bond (the one between the oxygen of the C15 group CH2OH and the hydrogen of the guanine base G2403) could be involved in the interaction of DON with the ribosome. It would be of interest to establish whether this H-bond is necessary for the toxicity of DON.

Unfortunately we were not able to identify a proper DON metabolite or another fusariotoxin metabolite to confirm the involvement of this H-bound in the structure-toxicity relationship.

In conclusion, the present study confirms that the toxicity of DON is not only linked to the epoxy group but is also influenced by the C3 group [START_REF] Sato | Comparative toxicities of trichothecenes[END_REF]Sundstol Eriksen 2004, Karlovsky 2011). It demonstrates that microbial biotransformation of DON into deepoxy-DON or 3-epi-DON decreases the intestinal toxicity of this mycotoxin. The underlying metabolism causes decreased affinity of the metabolites to the ribosome and the lack of MAPKinases activation. These data significantly increase the current knowledge of intestinal toxicity of DON, deepoxy-DON and 3-epi-DON and contribute to the evaluation of the effectiveness of the microbial biotransformation strategies in the fight against mycotoxins. The magnesium atom inside the A-site pocket has been highlighted in green.

DON forms 3 hydrogen bonds with the A-site: between the oxygen of the DON epoxy group on C12 and one hydrogen of the sugar of the uracil U2873; between the oxygen of the C15 group CH2OH and one hydrogen of the guanine basis G2403; between the hydrogen of the C3 group and one oxygen of the uracil U2869.

I. Context of the study

In the previous chapter, it was shown that DOM-1, in comparison to DON, has a reduced toxicity on intestinal cells and intestinal explants. The aim of this study was to assess the toxicity of purified DOM-1 on animal model. Actually, the toxicity of purified DOM-1 has never been assessed. Previous experiments were conducted on pigs receiving DON and the bacteria (bacillus sp. Ls100 or contents of the large intestine of chickens (CLIC)), able to deepoxidize DON into DOM-1 into the gut of the animal. In addition, in these experiments that are more focused on the zootechnical parameters such as feed intake and weight gain, they described no effect on treated animals. This prove that microbial detoxification of DON in contaminated feed can eliminate the negative effects of the mycotoxin, and the pre-feeding detoxification approach may be applied in the livestock industry (He et al. 1993;Li et al. 2011).

The aim of the present study was to evaluate the toxic effect of purified DOM-1 based on several parameters, including zootechnical parameters but also on the intestinal and immune responses. In fact, the effects of DON on the intestine and immunity are well characterized and represent a serious risk for animal health. Among animal species, pig constituted a relevant model to evaluate the toxicity of DOM-1, due to its high sensibility to mycotoxin, especially DON and its cereals rich diet.

Plasma analysis

Total concentration of the immunoglobulin subsets (IgG) was measured by ELISA as already described (Taranu et al. 2005). Briefly, plasma samples diluted 1:15.000 to detect IgG, in Tris-buffered saline and added to plates coated with immunoglobulin class specific pig antibody (Bethyl, Interchim, Montluçon, France). The different subsets were detected with the appropriate peroxidase anti-pig IgG (Bethyl) and were quantified by reference to standard curves constructed with known amounts of pig immunoglobulin subsets. Antibody titles against ovalbumin were also measured by ELISA (Meissonnier et al. 2008). Briefly, ELISA plates were coated with 2µg/mL ovalbumin diluted in 0.05 M NaHCO3 (pH 9.6). Diluted plasma, were then added to the plates and the anti-ovalbumin antibodies were detected with peroxidase-labeled anti-pig IgG (Bethyl). Absorbance was read at 450 nm using an ELISA plate reader (Spectra thermo scan, Tecan, NC, USA).

Histology

Fragments of liver, spleen, lymph node and intestine (jejunum with and without Peyer patches, ileum with and without Peyer patches) were fixed in 10% buffered formalin for 24 h, fixed in ethanol 70%, embedded in paraffin and cut into sections of 3µm. Paraffin sections of all the fragments were stained with the hematoxylin-eosin method.

Histological findings were graduated in scores. The score is based in histological changes and severity of lesions as previously reported (Bracarense et al. 2012;Grenier et al. 2011). Morphometry of intestinal villous and crypt was performed using a MOTIC Image Plus 2.0 ML® image analysis system. Thirty measurements from each fragment of intestine (duodenum, jejunum, ileum and colon) were done to determine villous height and crypts length.

mRNA expression for cytokines by real-time PCR

RNA on tissue was extracted with lysing matrix D tubes (MP Biomedicals, Illkirch, France) containing guanidine thiocyanate-acid phenol (Extract-All ; Eurobio, les Ulis, France)

for use with the FastPrep-24 (MP Biomedicals, Illkirch, France). Concentration and quality of samples were analysed using Nanodrop ND1000 (Labtech International, Paris France) and using Bioanalyser (Agilent RNA 6000 Nano Kit Quick Start Guide). Then, reverse transcription and real-time qPCR steps were performed as previously described (Meissonnier, 2008). Specificity of qPCR products was assessed at the end of the reactions by analyzing dissociation curves. Primers were purchased from Invitrogen (Invitrogen, Life Technologies Corporation, Paisley, UK). Specific sequences were specified in Table 1. Amplification efficiency and initial fluorescence were determined by the ΔCt method, then obtained values were normalized using a reference gene, the cyclophillin A (Bruel et al. 2010). Stability of these genes was demonstrated previously in intestinal tissues (Delgado-Ortega et al. 2011).

mRNA expression levels were expressed relative to the mean of the control group.

Statistical analysis

For statistical analysis, a One-way or Two-way ANOVA were realized with a Bonferroni test as Post-hoc, p<0.05.

C. Comparative effects of DOM-1 and DON on liver

Animals receiving DOM-1 were similar to the control ones. By contrasts, a significant increase on liver lesional score was observed in the DON-treated group (3 fold higher than control). In these animals the liver, showed a disorganization of hepatocytes, a vacuolization and a megalocytosis of these cells (Figure 6).

D. Comparative effect of DOM-1 and DON on immune parameters

The last part of the experiment was to evaluate the effect of DOM-1 and DON on immunity related parameters such as the histology of the spleen and lymph node, total and specific antibody response.

A significant increase in histological changes was observed in animals treated with DOM-1 in spleen (2.4 fold), and a non-significant increase in mesenteric lymph nodes in comparison to control group was also seen (1.4 fold). A similar increase in histological changes was also observed in animals treated with DON (1.5 fold and 2.5 fold for lymph node and spleen, respectively). In these organs, lymphoid depletion and apoptosis of lymphocytes were the most frequent changes observed in DOM-1

or DON treated animals. Reactive germinal center with macrophages containing tangible bodies was also observed in spleen of DON treated group (Figure 7).

The effect of DON and DOM-1 were also measured on the total and specific IgG. The plasmatic concentration of IgG was not significantly modified by the presence of toxins (Fig. 8). The immunization protocol allowed investigating the effects of mycotoxins on antigen-specific immunity.

Ingestion of either DOM-1 or DON significantly increased the production of OVA-specific IgG at day

IV. Discussion

In this present 3-week study, piglets were exposed to low doses of one major mycotoxin, the DON, and one of its derivatives, the DOM-1. Previous studies on the toxicity of DOM-1 toxicity were done with feed contaminated with DON in presence of the bacteria that is able to deepoxydise the DON and focused on zootechnical parameters (He et al. 1993;Li et al. 2011). By contrast, this study assessed the effect of purified DOM-1 on animals, and assessed major markers of toxicity, zootechnical parameters, intestine, systemic and immunologic parameters.

The use of purified toxin by gavages allowed to precisely control the dose used and to make sure that each animal received the same amount. Purified DOM-1 were given to pig at a dose of 0.14mg/Kg BW, molecular equivalent of 0.15mg/Kg BW of DON corresponding approximately to 3-4 ppm [START_REF] Sergent | Deoxynivalenol transport across human intestinal caco-2 cells and its effects on cellular metabolism at realistic intestinal concentrations[END_REF], at levels commonly found in crops (SCOOP 2003).

From an agronomic point of view, pigs are exposed to mycotoxins due to the proportion of cereals in their diet. In addition, pigs are very sensitive to mycotoxins, due to the absence of ruminal tank, known to contain microorganisms capable of degrading toxins before their intestinal absorption (Rotter et al. 2006). Finally, considering the similarity in the immune and digestive systems of pigs and humans, the use of pig model allow to extrapolate data to man (Heinritz et al. 2013;Nedjfors et al. 2000;[START_REF] Helm | Nonmurine animal models of food allergy[END_REF][START_REF] Kimber | Assessment of protein allergenicity on the basis of immune reactivity: Animal models[END_REF][START_REF] Rothkotter | The pig as a model of developmental immunology[END_REF]. For the choice of doses and time of exposure, this work fits in the current dynamic of a toxicological study, for chronic exposure going from low doses to moderate ones, not inducing major clinical manifestations [START_REF] Forsyth | Emetic and refusal activity of deoxynivalenol to swine[END_REF].

In summary, results showed that DOM-1 does not have toxic effects on zootechnical parameters, intestinal histology, intestinal and inflammatory response and liver histology. For these parameters we found toxic effects of DON as already described by others (Grenier et al. 2011;Pestka 2010;Rotter et al. 1996). The zootechnical parameters were not impacted by DOM-1 ingestion, with no decrease of weight gain and no emesis observed. Concerning the impact of DOM-1 on the intestine, which is the first organ targeted by feed contaminant [START_REF] Prelusky | Biological fate of fumonisin b1 in food-producing animals[END_REF][START_REF] Oswald | Role of intestinal epithelial cells in the innate immune defence of the pig intestine[END_REF], no tissue damage were induced. Moreover, DOM-1 didn't induce a pro-inflammatory response on intestinal tissue like it was observed with DON.

One organ of the systemic response was analyzed, the liver, to complete the information about the action of DOM-1 once absorbed in the intestine. Histomorphological analysis on liver showed no damage induced by DOM-1 in contrary to DON. Finally, concerning the global immune response, DOM-1 didn't elicit total immune response.

On secondary lymphoid organs and on total and specific immune response, DOM-1 elicited the same increased immune response as DON. Indeed, concerning the specific immune response, and after a vaccine injection DOM-1 seems to act like an adjuvant, in increasing the immune response against the vaccine. Moreover, lymphoid depletion and apoptosis of lymphocytes were observed on lymph node and spleen like in DON group, may be correlated to the important specific immune response. In a previous study (see results of In conclusion these results are in accordance with previous results found in others in vivo study. DOM-1, deepoxidated from DON by a strain BBSH 797, Gen. nov. sp. nov. of family Coriobacteriaceae, sold as a food additive, is globally not toxic for animals as DON. Taking into account that degrading product of DON (DOM-1) formed by this bacteria is much less toxic than DON, associated to the information given by previous in vivo studies (He et al. 1993;Li et al. 2011); the use of these detoxifying products seems to be a relevant strategy to protect animals, against the toxic effects of DON found in pig feed. However, this study has also shown that DOM-1 is able to act on the immune response. The underlying mechanism still need to be investigated.

I. Discussion

Once present in cereals or feed materials, the high stability of mycotoxins make them very difficult to eliminate. Several strategies have been developed to manage mycotoxins contamination, such as physical (cleaning, sieving), chemical (ammoniation) and biological (binding agents, feed additives, resistant crops) treatments. In this context, my thesis project deals with problematic of mycotoxin detoxification and was in particular focused on DON.

Indeed, among mycotoxins, DON is the most common in the world and can be present in various cereals and raw materials. As other mycotoxins, it is really hard to manage and eliminate. New strategies of biological detoxification led to the formation of metabolites, such as the deepoxydeoxynivalenol (DOM-1) and the 3-epi-deoxynivalenol (3-epi-DON). The "masked" mycotoxin, deoxynivalenol-3-glucoside (D3G) is produced in plant resistant to Fusarium and is very often present simultaneously with DON. Thereby it becomes important to assess the toxicity of these biological degradation products of detoxification of DON. Limited toxicological information on DONtransformation products are available, this may be due to the lack of suitable methods to purify sufficient amounts of the chemicals for structure identification and toxicological studies.

The main purpose of this work was to assess the toxicity of "modified" and "masked" forms of DON, to evaluate the efficacy of the detoxification. This thesis investigates the toxicity of these DON derivatives on human and animal models, mostly on immune and intestinal parameters, using several approaches. These new molecules issued from a natural way of transformation can be a promising approach to detoxify mycotoxins and protect human and animals from their hazard effects.

Problematic of modified and masked mycotoxin in context of risk assessment, effects of DON on health pig and comparative toxicological effects of DON, DOM-1, 3-epi-DON and D3G have been summarized and discussed in the previous papers and will not be repeated here. This general discussion will focus on the studied models used, the specific results obtained with in silico and IPA analysis, and to finish the perspectives on experiments that could be realized to further investigate or complete information of toxicity and mechanisms of toxicity on DON and its derivatives.

A. Discussion on the analysis performed in the thesis

In silico, in vitro, and ex vivo models, were used to compare the intestinal and immune toxicity of D3G, DOM-1 and 3-epi-DON to those of DON.

In silico analysis allowed to draw three dimensions molecules and to visualize their interaction with the ribosome, especially with the petptidyl transferase center (PTC) of the ribosome. In silico approach is widely used in medical chemistry to decipher the mechanism of binding between protein and ligands, to predict interactions between molecules or to study structure activity relationship. This approach is not so commonly used in food safety, however some studies show that this approach allow in food safety studies to discover and study, quickly and economically, new putative endocrine disruptors (Amadasi et al., 2009), like ZEN and its masked derivative [START_REF] Cozzini | In silico approach to evaluate molecular interaction between mycotoxins and the estrogen receptors ligand binding domain: A case study on zearalenone and its metabolites[END_REF].

In this thesis, modeling allow the better understanding of the molecular mechanism of DON toxicity.

In vitro model using cell lines is a powerful tool for the exploration of fundamental questions regarding drug delivery dynamics and kinetics [START_REF] Artursson | Caco-2 monolayers in experimental and theoretical predictions of drug transport[END_REF][START_REF] Boveri | Comparison of the sensitivity of different toxicological endpoints in caco-2 cells after cadmium chloride treatment[END_REF]). To study the toxicity of DON and its derivatives on intestinal epithelial cells, we chose a human intestinal cell line.

Caco-2 has the advantage to express characteristics markers of adults' intestinal cells, to be a reference line in toxicology studies, and to be largely used to study intestinal absorption (Sambruy et al. 2001).

Explant model allows a direct testing of the toxicity of molecules on intestinal living tissue keeping its polarity and extracellular integrity (Gonzalez-Vallina et al. 1996). Moreover, in the context of 3R, explants limit the use of animals, as well as the variability. Indeed many conditions are tested on the same animal, thus an animal is its own control. This model also allows to investigate in detail the impact of each derivatives on the expression of many genes on the intestinal tissue of pigs.

Using a microarray encompassing the genome, associated with a functional analysis allows to go deeper on the investigation on the pathways targeted by these derivatives.

Finally the use of in vivo model, allow assessing the toxicity on the entire organism, looking at several organs and parameters that can be targeted by mycotoxins.

1.

In silico analysis of the interaction between DON, DON derivatives and the ribosome

The in silico analysis lets us go deeper in the understanding of the mechanism of toxicity. The molecules binding of DON, DOM-1 and 3epi-DON into the peptidyl transferase center of the ribosome were modeled. The in silico analysis demonstrates that DON interacts with the two chains of the A-site of the ribosome through three hydrogen bonds (with G2403, U2873 and U2869). The deepoxydation of the epoxy group (into one hydrogen double bound) leads to a loose of the hydrogen bond U2873 and to the absence of toxicity. We can thus imagine that the three hydrogen bounds are required to maintain the molecule in the peptidyl transferase center; when only two bounds forming the stability is too weak to maintain the molecule in the peptidyl transferase center and to induce a subsequent cellular response. A similar analysis was performed with the 3-epi-DON; due to the isomeric change, the hydrogen bond with the uracile U2869 is lost. The molecule is only maintained in the ribosome pocket by two hydrogen bonds, this reduces the stability of the interaction and prevent cellular activation.

As both DOM-1 and 3-epi DON do not activate MAPKinase, the three hydrogen bonds seem required to maintain stably the molecule of DON into the A-site of the ribosome, to activate the MAPKinase and to induce the toxicity of this mycotoxin. This finding is in line with the hypothesis that the toxicity of DON is linked to the epoxy group and the C3 group (Karlovsky 2011;Sundstol Eriksen et al. 2004) (Rotter et al. 1996). The loss of one hydrogen bound decreases the toxicity of DON. Most of the A-site inhibitors molecules, such as DON, impair peptide bond formation during translation elongation. They all block protein synthesis by competing with the amino acid side chains of incoming aminoacyl-tRNAs for binding in the A-site cleft in the PTC, which is universally conserved (Garreau de Loubresse et al. 2014). In addition, these structures support the hypothesis that the species specificity exhibited by the A-site cleft inhibitors is determined by the interactions they make, or fail to make, with a single nucleotide, U2504 (Escherichia coli) [START_REF] Gurel | U2504 determines the species specificity of the a-site cleft antibiotics: The structures of tiamulin, homoharringtonine, and bruceantin bound to the ribosome[END_REF]). In our case, it confirms that just one change of interaction can change the toxicity of the molecule. However, we did not determine the role of the third hydrogen bond (between the oxygen of C15 group CH2OH and the hydrogen of the guanine basis G2403) on the binding of DON to the PTC of the ribosome and the subsequent toxicity of DON. Indeed, we couldn't identify a DON metabolite or another fusariotoxin metabolite to confirm the involvement of this H-bound in the structure-toxicity relationship. It would be interesting to test a molecule having both the epoxy and the C3 group but not the G2403 hydrogen bound. A second possibility would be to make a double mutations on the A-site of the ribosome, replacing the uracile basis U2869 by a guanine and the guanine G2403 by an adenosine, and to test the interaction of this mutated ribosome with DON.

The molecular basis to explain the lack of toxicity of D3G is easier. Due to its size, D3G can't interact with the A-site. Indeed, glucose molecule, added during the phase II of metabolization to easily eliminate the molecule, importantly increase its size.

Pan genomic analysis of the effect of DON and its derivatives

After the microarray analysis of explant treated with DON, or its derivative, a functional analysis using Ingenuity Pathways Analysis (IPA), was performed. IPA is a database gathering information on molecules (genes, proteins, metabolites, xenobiotics) and their interactions between each other or with pathologies, phenotypes, cells or cellular processes. The sources of information of IPA consist in existing databases (GenBank, Ensembl Entrez Gene, Gene Ontology, GEO ...), and knowledge extracted from the literature. This software allows to rapidly obtain information on genes expressed in the tissue and find genes who interferes (in different ways) with these genes and that are differentially expressed in our analysis. The Ingenuity Pathways Analysis demonstrates the absence of toxicity of DOM-1, 3-epi-DON and D3G, with no genes differentially expressed and thus no functions impacted. It also allows identifying key biological functions regulated by DON. This is a powerful tool to assess, on one tissue, all the functions that could be regulated by one molecule. It both confirms DON-3-glucoside, and 3-epi-deoxynivalenol (3-epi-DON) [START_REF] He | Chemical and biological transformations for detoxification of trichothecene mycotoxins in human and animal food chains: A review[END_REF][START_REF] Ikunaga | Nocardioides sp. Strain wsn05-2, isolated from a wheat field, degrades deoxynivalenol, producing the novel intermediate 3epi-deoxynivalenol[END_REF][START_REF] Sato | Thirteen novel deoxynivalenoldegrading bacteria are classified within two genera with distinct degradation mechanisms[END_REF]Zhou and He 2009). Table 1. Biological transformation of DON by microorganisms (He Jian We thesis)

Efficiency of detoxifying strategies a) Bacterial transformation

The use of feed additives is a well-established practice in the animal feed industry. Successful mycotoxin-binding agents act by preventing its intestinal absorption by the animal from the contaminated feed (Bryden 2012). However, adsorption approaches for DON are relatively ineffective and so the use of microorganisms is recommended for trichothecene (Awad et al. 2010). Moreover, due to the chemical diversity of mycotoxins, often present in food in the same time, additional approaches to detoxifying feedstuffs are required, even if adsorbing, binding or trapping agents can be very effective.

Concerning the use of detoxifying agents, the major part of my work was to assess the global in vivo toxicity of DOM-1. Indeed, one strategy to protect animal from mycotoxin, is the use of microorganisms/enzymes detoxifying mycotoxins to limit the absorption in the organism (Schatzmayr et al. 2006). These microorganisms realize some transformation not feasible by the plant or by the animal. These transformations on the basic structure will play on the original toxicity of the molecule.

The toxicity of trichothecene mycotoxins varies and is determined by their molecular structures, particularly functional groups such as epoxy, ester, and hydroxyl groups [START_REF] Betina | Structure-activity relationships among mycotoxins[END_REF][START_REF] Nagy | Hydrogen bondings in deoxynivalenol (don) conformations-a density functional study[END_REF]Zhou et al. 2008). The number and the position of hydroxyl groups influence trichothecene toxicity. Currently, information on the toxicity of these transformation products (or DON derivatives) are limited.

BIOMIN group isolated and stabilized bacterium, now called BBSH 797, Gen. nov. sp. nov. of family Coriobacteriaceae able to remove the epoxide group of trichothecenes in vivo, and to transform DON into DOM-1 [START_REF] Binder | Microbial detoxification of mycotoxins[END_REF]Fuchs et al. 2002). Since its discovery in 1997, Eubacterium specific of trichothecenes, named BBSH797 in honour of the research team who discovered it (BBSH, Binder Binder Schatzmayr Heidler), was the first additive detoxifying mycotoxins formulated from a life microorganism. This derivative is recognized to be less toxic than DON, based on in vitro and in vivo experiment with animal ingesting the bacteria in presence of DON (He et al. 1993;Li et al. 2011;Schatzmayr et al. 2006;Sundstol Eriksen et al. 2004;Swanson et al. 1987). However, actual DOM-1 cytotoxic assays are not sufficient to correctly evaluate the overall toxicological relevance (Rychlik et al. 2014). And these in vivo studies didn't evaluate the global toxicity of purified DOM-1 on the animal.

The 3-epi-DON is also issued from a bacterial transformation. A bacteria, issued from the an alfalfa soil enriched with F. graminearum-infested corn, bacterium Devosia mutans 17-2-E-8 that is capable of completely transforming DON into a major product 3-epi-DON and a minor product 3keto-DON in aerobic conditions (He et al. 2015b;Zhou andHe 2009, 2010). 3-epi-DON, is an epimer of DON, the only difference between these two chemicals is the stereochemistry at the 3-OH Group (He et al. 2015b). 3-epi-DON, has received very little attention. Nevertheless, it is known that epimers can have different physicochemical properties, biological activities and toxicities. A study shows that a epimeric form of catechin was less active than the initial form [START_REF] Mendoza-Wilson Ama | Theoretical study of the molecular properties and chemical reactivity of (+)-catechin and (âˆ')-epicatechin related to their antioxidant ability[END_REF]. A second study showed a five-fold difference in in vitro toxicity that has been demonstrated for the marine biotoxins azaspiracid-1 (AZA1) and 37-epi-AZA1 [START_REF] Kilcoyne | Epimers of azaspiracids: Isolation, structural elucidation, relative lc-ms response, and in vitro toxicity of 37-epiazaspiracid-1[END_REF]). Moreover, it seems that, once it is formed, the molecule is stable and the transformation is irreversible (Karlovsky 2011). This is a very interesting point in the context of the problem of the metabolization into the animal. 3-epi-DON could be a promising new commercial product if is not toxic and if it stay stable into the organism. Today, only one study assesses its toxicity on in vitro and in vivo models (He et al. 2015a) and there are no information on its metabolization. The above mentioned study demonstrated that 3-epi-DON is far less toxic than DON on Caco-2 cells and mouse models.

Different analysis realized in this thesis complete the information known about DOM-1 and 3-epi-DON toxicity. In conclusion, our results confirm the lack of in vitro toxicity of DOM-1 and 3-epi-DON as seen in previous studies performed on Caco-2 cells, a relevant model in toxicology studies on the intestine (He et al. 1993;Li et al. 2011;Sambruy et al. 2001;Schatzmayr et al. 2006;Sundstol Eriksen et al. 2004;Swanson et al. 1987). Our results also show that these DON metabolites did not impair the integrity of intestinal tissue and the barrier function. They also demonstrate that DOM-1 and 3-epi-DON do not alter gene expression, do not induce a pro-inflammatory response and do not induce the activation of MAPKs. Moreover, results obtained in vivo show that DOM-1 did not alter most of the parameters investigated. Finally, our results bring a mechanistic hypothesis on the lack of toxicity of DOM-1 and 3-epi-DON.

b) Plant transformation

In natural conditions, some plants are able with efficacy to manage xenobiotics and mycotoxin, as DON. Plants could accumulate toxic levels of these molecules without efficient detoxification strategy. The natural way of defence includes biosynthesis pathways, phase I transformation (hydrolysis, reduction or oxidation), phase II solubilisation (conjugaison) and phase III compartmentalisation. These different phases (I and II) aim at increasing the polarity of the molecule making it more water soluble and facilitating by that its transport mediation by ATP-dependent glutathione-conjugate transporters to the vacuole or apoplastic space outside cell (phase III) [START_REF] Coleman | Detoxification of xenobiotics by plants: Chemical modification and vacuolar compartmentation[END_REF].

Mycotoxins issued from this biosynthesis pathway, are "modified" mycotoxins and more specifically called "masked" mycotoxins by ILSI as they are issued from a conjugation transformation into the plant [START_REF] Berthiller | Masked mycotoxins: A review[END_REF]. Thereby, masked derivatives of DON can be found in cocontamination in raw cereals and processed food, such as deoxynivalenol-3-β-D-glucoside (D3G). A study showed that D3G was present in all tested wheat and maize samples (Berthiller et al. 2009).

One strategy of fight against mycotoxin is the use of plant, for instance wheat cultivars, that carry a major QTL for increased Fusarium resistance and increased ability to conjugate DON into D3G.

Indeed DON enhances the virulence on wheat [START_REF] Jansen | Infection patterns in barley and wheat spikes inoculated with wild-type and trichodiene synthase gene disrupted fusarium graminearum[END_REF]Lemmens et al. 2005). These plants resistant to fungi over express UDP-glycosyl-transferase, an enzyme able to glycosylate DON in higher proportion [START_REF] Berthiller | Masked mycotoxins: A review[END_REF]Karlovsky 2011;[START_REF] Poppenberger | Detoxification of the fusarium mycotoxin deoxynivalenol by a udp-glucosyltransferase from arabidopsis thaliana[END_REF]. So, this increased use of wheat cultivars may lead to a higher D3G/DON ratio in the future. In this case, the contribution of D3G to overall DON toxicity has to be taken into account.

Different analyses realized in this thesis bring new information about D3G toxicity, and show that it is non toxic. These results imply that the increasing use of resistant plant to Fusarium could be a promising strategy to reduce mycotoxin production and their occurrence in cereals and food process products. However, studies show that D3G can be hydrolyzed into DON by acid lactic of some bacteria inside the gut [START_REF] Berthiller | Hydrolytic fate of deoxynivalenol-3-glucoside during digestion[END_REF]. A study realized on pig shows that the major part of D3G ingested, was excreted by urine in DON form and in minority in DON-glucuronidate forms [START_REF] Nagl | Metabolism of the masked mycotoxin deoxynivalenol-3-glucoside in pigs[END_REF]. So, even if D3G is not toxic by itself, if hydrolyzed into DON by the animal, it could be reabsorbed and thus the total amount of DON to which pig is exposed would be increased.

profile to human (Maresca 2013). Plus, since it's a monogastric just like human its similar tract is organized in the same way.

Concerning the use of resistant plants to mycotoxins, the toxicity analysis and the metabolization of products transformed by these plants is important. This step is required to correctly estimate the risk of exposure to native mycotoxin. It's essential to determine if D3G or other masked mycotoxin, present in high quantity in selected or genetically modified plants, won't be all re-transformed to DON in the animal. Even if D3G is not toxic by itself (because issued from a detoxification pathway into plant), it could be transformed back to its aglycone into the organism and thus increase the amount of DON present in the organism. So, in this case to correctly assess the risk, it is necessary to take into account both "native" and "masked" mycotoxins in the calculation of exposure and the calculation of NOEL and LOAEL by regulator organism like European Food Safety Authorities (EFSA).

An alternate application of bacteria is to only use for detoxification the enzymes and/or the genes implicated in the detoxification process. The genes may be cloned and expressed in crops to develop varieties that are resistant to the production of mycotoxins or which detoxify mycotoxin and, thus prevent this mycotoxin from entering the human and animal food chains. The detoxification genes may also be cloned and expressed in microorganisms to produce recombinant microorganisms that are suitable in an industrial scale enzyme production and purification [START_REF] Altalhi | Localization of zearalenone detoxification gene(s) in pzea-1 plasmid of pseudomonas putida zea-1 and expressed in escherichia coli[END_REF]. These detoxification enzymes should have great potential to eliminate DON and other mycotoxins present in the human and animal food chains.

In conclusion, this work fits in context of animal production, where expenses linked to prophylaxis, cares and productivity loss are important points in the agricultural sector. Mycotoxins present a problem all over the world for both human and animals. Therefore, the necessity to continue developing strategies of prevention and detoxification, is encouraged by the global warming, which will have an impact on the development of fungi [START_REF] Bryden | Mycotoxins and mycotoxicoses: Significance, occurrence and mitigation in the food chain[END_REF][START_REF] Paterson | How will climate change affect mycotoxins in food?[END_REF][START_REF] Tirado | Climate change and food safety: A review[END_REF]). In the long run, these new ways of detoxification, using plants and bacteria, have great potentials and could help the food safety sector, the livestock production and the crop disease management face these mycotoxins.

  vôtre compagnie : Pascal, Sabria, Julien, Julie, Joanna, Rhoda, Patricia, Yann, Stella, Anwar, HK, Stéphanie, Florian, Inès, Pauline, Duncan, Joanne, Leticia, Viviane, Sophal, Joya… j'espère que je n'oublie personne. Aux post-docs, Delphine, Selma et Imourane courage pour la suite et les concours !  Pendant cette thèse j'ai eu l'occasion de rencontrer des étudiantes supers (eh oui que des filles), dynamiques, intelligentes et courageuses, pour certaines prêtes à faire leurs études loin de leur famille. Les filles tout le meilleur pour la suite avec une super thèse, un super post-doc et un super échantillon à la clé… ! ;) THANKS Elisabeth, Marion, Yann et Sarah, merci pour tous les moments de rigolades partagés, les longues discussions…, les aprems détentes/jeux, tout le meilleur pour la suite ! Courage Marion et Sarah bientôt la fin pour vous aussi !;) Une pensée spéciale aux filles sublimes et exotiques! ;), qui m'ont soutenu, aidé avec plein d'entrain et de bonnes humeur! Joanna, Rhoda, Joanne, Joya et Christelle, merci de m'avoir fait découvrir la culture libanaise à travers vous. Christelle merci énormément pour tes corrections d'anglais de dernière minute ! Isaura, Amaranta, associées aux brésiliennes, merci de m'avoir fait découvrir un brin de votre culture et la joie de vivre des filles d'Amérique du Sud !;) Bianca, j'ai été ravie de découvrir ton beau pays ! Je tiens aussi à remercier et à citer Beyoncé pour son soutient (même si elle n'est pas au courant) pendant toutes ces soirées de travail et de rédaction… : « I'm a survivor, I'm not gonna give up, I'm not gon stop, I'm gon work harder… ».

Figure 1 -

 1 Figure 1 -Major mycotoxins produced by fungi and naturally found in several food products

Figure 2 -

 2 Figure 2 -Structural diversity of major mycotoxins

Figure 3 -

 3 Figure 3 -"Saint Antoine tentation" painted between 1512 -1516 by Grünewald (on the left). Ears of rye contaminated with ergot (on the right).

Figure 4 -

 4 Figure 4 -Global mycotoxin prevalence in surveyed regions (adapted from Schatzmayr and Streit 2013). Aflatoxins (AF), zearalenone (ZEA), deoxynivalenol (DON), fumonisins (FB), ochratoxin A (OTA).

Fig. 1 .

 1 Fig. 1. Chemical structure of the main mycotoxins present in pig feed.

-

Fig. 2 :

 2 Fig.2: Summary of the intestinal toxicity of the main mycotoxins present in feed pig.

Figure 1 -

 1 Figure 1 -Systematic definition of "modified mycotoxins"(Rychlik et al. 2014)

  (α-ZEL) β-zearalenol (β-ZEL) α-zearalenol-14-α-D-glucopyranoside (α-In bold: Masked mycotoxins in the strict sense

  strategies were then developed to manage mycotoxins and DON contamination, like physical (cleaning, sieving), chemical (ammoniation) and biological (binding agents, feed additives) treatments. Thereby, in the context of use of new strategies of detoxification, some metabolites are formed during the biological transformation of DON. In this context my work deals with the toxicity of biological degradation product of mycotoxins the deepoxy-deoxynivalenol (DOM-1) and the 3-epi-deoxynivalenol (3-epi-DON) from bacterial degradation and the deoxynivalenol-3-β-D-glucopyranoside (D3G) transformed into the plant. The general objective of this thesis was to assess the toxicity of three derivatives of DON: D3G, DOM-1 and 3-epi-DON.

  to assess the toxicity of the masked form of DON, D3G, and to determine its molecular mode of action using in vitro, ex vivo and in silico approaches (Chapter I, Part 1).  to assess the toxicity of two microbial derivatives of DON, DOM-1 and 3-epi-DON and to determine their mode of action using in vitro, ex vivo and in silico models. In the case of DOM-1, in vivo experiment was also performed to assess its toxicity and anorectic effect in piglets (Chapter I, Part 2 & Chapter II).  as DON was always included as a positive control, the experiments allowed us to get more insight on intestinal toxicity of DON, especially at the transcriptional level (Chapter I).The aim of this thesis was to assess the toxicity of several DON derivatives, formed either in the plant (D3G) or after microbial transformation (DOM-1 and 3epiDON). D3G is formed by a natural way of defense in plant, aiming to manage xenobiotics and their excretion.Microbial transformation, is the use of isolated and stabilized microorganism able to transform mycotoxin.The use of in silico, in vitro, and ex vivo models, allow the comparison of the intestinal and immune toxicity of D3G, DOM-1 and 3-epi-DON to the one of DON. In silico analysis allowed to draw molecules in three dimensions, to determine the interaction of the molecules and the ribosome and to understand the molecular mechanism of toxicity. In vitro model using cell line is a powerful enabling tool for the exploration of fundamental questions regarding cell and the study of drug delivery dynamics and kinetics.

  genes and biological pathways impacted by DON as well as demonstrating the absence of toxicity of the DON derivatives. secondary metabolites produced by various molds, such as Aspergillus,

  for the toxic activity of DON and a detoxification strategy targeting this part of the C3-OH, leading to the formation of 3-epi-DON, was recently proposed(Karlovsky 2011). Four bacterial strains, all isolated from soil, have been described to epimerize DON into 3-epi-DON(Karlovsky 2011; He et al. 2015a). Only one paper has investigated the effect of 3-epi-DON and demonstrated the lack of toxicity, both in vitro and in vivo, of this DON metabolite(He et al. 2015b).The aim of the current study was to assess the efficacy of microbial transformation through analysis of the intestinal toxicity of deepoxy-DON and 3-epi-DON. Using physiological, histological and transcriptomic analysis, we have observed reduced toxicity of deepoxy-DON and 3-epi-DON, both for human intestinal epithelial cells and pig intestinal explants. We have further demonstrated that these microbial metabolites of DON fit into the ribosome pocket but do not elicit ribotoxic stress or activate the MAPKinase pathway. Our paper provides the first molecular insight for the reduced toxicity of deepoxy-DON and 3-epi-DON.Experimental proceduresToxinsPurified DON was purchased from Sigma-Aldrich (St Louis, MO, USA). DOM-1 was obtained by transformation of crystalline DON (Romer Labs, Tulln, Austria), dissolved in medium10 (Caldwell and Bryant, 1966) at a concentration of 2 mg/ml, by inoculation with BBSH 797, Gen. nov. sp. nov. of family Coriobacteriaceae in sterile medium, at 37°C for six days. Biotransformation of DON to deepoxy-DON was confirmed by LC-MS/MS, and deepoxy-DON was purified by solid phase extraction and preparative HPLC (Schwartz-Zimmermann et al., 2014). The purity of the deepoxy-DON preparation was 99%, based on chromatograms recorded at 220nm. 3-epi-DON was produced by microbial transformation of DON (He et al. 2015a). Briefly, DON was co-incubated with the bacterial strain, Devosia mutans 17-2-E-8, in corn meal broth medium at 28 °C for 48 h. High-speed counter-current chromatography (HSCCC) and preparative high performance liquid chromatography (prep-LC) were applied to separate 3epi-DON. The obtained product was analyzed by liquid chromatography (LC) and identified by congruent retention time and UV/Vis spectrum and mass spectrometric (MS) data. Nuclear magnetic resonance (NMR) experiments such as correlation spectroscopy (COSY), heteronuclear single quantum coherence (HSQC) and nuclear overhauser effect (NOE) were conducted for structural characterization of 3-epi-DON. The 3-epi-DON used in the experiment had a purity of 96.8%. Toxins were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) and stored at -20°C until use.Caco-2 cell cultureCaco-2 cells (passages 99 -106) obtained from the TC7 were cultured in 75-cm 2 culture flasks (Cellstar cell culture flasks, Sigma-Aldrich) in Dulbecco's Modified Eagle Medium enriched with glutamine (Gibco, Cergy-Pontoise, France), supplemented with 10% of heat inactivated fetal bovine serum, 0.5% of gentamycine (Eurobio, Courtaboeuf, France) and 1%

  DON disturbed pathways related to immunity/inflammation, such as cytokines regulations (IL-17 axis, IL-10 signaling), leukocytes functions (diapedesis), iNOS and NFkB signaling. DON affected other pathways associated with cell cycle regulation, apoptosis and ER stress response. Moreover, the results underlined the effects of DON on PXR/RXR, FXR/RXR signaling pathways and mitochondrial L-carnitine Shuttle Pathways.

  panel A). Similar trends were observed in jejunal explants (Figure7panel B).The last step was to investigate the ability of DON and its bacterial metabolites, deepoxy-DON and 3-epi-DON, to bind to the 60S sub-unit inside the A-site of the peptidyl transferase center of the ribosome. The crystallographic data (4U53.pdb) obtained for DON and yeast ribosomes were used (Garreau deLoubresse et al. 2014). As shown in Figure8, (panel A) DON is able to fit in the pocket of the A-site of the ribosome 60S subunit. Within the pocket, the 3-hydroxyl group of DON is associated with a magnesium atom and stabilized by other nucleotides. In this position, DON forms 3 hydrogen bonds with the A-site. The first one is between the oxygen of the DON epoxy group on C12 and one hydrogen of the sugar of the uracil U2873; the second one is between the oxygen of the C15 group CH2OH and one hydrogen of the guanine basis G2403; and the last one is between the hydrogen of the C3 group and one oxygen of the uracil U2869. The in silico analysis revealed that both deepoxy-DON and 3-epi-DON were also able to fit into the pocket of the peptidyl transferase center of the ribosome. However, because of the absence of the epoxy group or the isomeric change, these two metabolites were only able to form 2 hydrogen bonds with the A sites of the peptidyl transferase center. Deepoxy-DON and 3-epi-DON didn't form the bond with U2873 and U2869, respectively.

  when compared to DON. The toxicity of DON and its bacterial metabolites, was first investigated on proliferating and differentiated Caco-2 cells. As already demonstrated, DON induced a significant decrease in Caco-2 cell proliferation, reduced their barrier function and altered their respiratory capacities(Alassane-Kempbi et al. 2013[START_REF] Akbari | Deoxynivalenol: A trigger for intestinal integrity breakdown[END_REF][START_REF] Bin-Umer | Elimination of damaged mitochondria through mitophagy reduces mitochondrial oxidative stress and increases tolerance to trichothecenes[END_REF]. This is the first investigation of the toxicity of deepoxy-DON on human intestinal epithelial cells, although the absence of toxicity of 3-epi-DON on Caco-2 cells has been recently demonstrated(He et al. 2015b).

Figure 1 .Figure 2 .

 12 Figure 1. Molecular structures of DON, deepoxy-DON and 3-epi-DON.

Figure 3 .

 3 Figure 3. Effects of deepoxy-DON and 3-epi-DON on trans-epithelial electrical resistance (TEER) of human intestinal epithelial cells. Differentiated caco-2 cells, were treated with 10µM of diluent (♦), DON (■), DEEPOXY-DON (▲) or 3-epi-DON (•) and TEER was measured. Results are expressed as mean ± SEM of 3-4 independent experiments, ***p<0.001.

Figure 4 .

 4 Figure 4. The acute effect of toxins on oxygen consumption rate (OCR) of cultured Caco-2 cells. After establishment of baseline oxygen consumption rate in proliferated Caco-2 cells seeded to 1.5x10 4 cells/well, diluent (♦), DON (■), deepoxy-DON (▲) or 3-epi-DON (•), was injected at final concentration of 10µM 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. DON treatment is significantly different from others treatments, ***p<0.001, n=5.

Figure 5 .

 5 Figure 5. Comparative effect of deepoxy-DON and 3-epi-DON and DON on morphology of intestinal explants. Jejunal explants from 4 different animals were exposed for 4 hours, to diluent or 10µM toxins and stained with HE for histological analysis. Normal villi lined with columnar enterocytes were observed on control explants (A), multifocal villi atrophy (arrow) and cell debris (arrowhead), apical necrosis (insert) on DON explants (B), histological aspects similar to control group on deepoxy-DON (C) or 3epi-DON (D) explants. Bar 100 µm; insert bar 20 µm.

Figure 6 .

 6 Figure 6. Gene expression profile of intestinal explants exposed to deepoxy-DON, 3-epi-DON or DON Jejunal explants from 4 different animals were exposed for 4 hours, to diluent or 10µM toxins and gene expression was analyzed with a 60K microarray. Groups are represented by different colors: DON in grey, Control in cyan, deepoxy-DON (or DOM-1) in pink and 3-epi-DON in dark blue. Panel A: Venn diagram illustrating the overlaps between the probes significantly up-or downregulated in response to DON, deepoxy-DON and 3-epi-DON treatment. Panel B: Principal Component Analysis of differentially expressed probes between DON/D3G and control (747 with BH adjusted p-value < 0.05). Panel C: Heat map representing differentially expressed probes between DON, deepoxy-DON, 3-epi-DON and control explant. Red and green colors indicate values above and below the mean (average Zscore) respectively. Black color indicates values close to the mean.

Figure 7 .

 7 Figure 7. Effects of deepoxy-DON or 3-epi-DON on activation of MAPK on human intestinal epithelial cells. Panel A: Caco-2 cells, differentiated on inserts. Panel B: Jejunal explants. Samples were treated for 1h with 10µM toxins and analyzed by western blot for expression of phosphorylated P38, phosphorylated JNK and GAPDH, used as a protein loading control. Representative immunoblots and normalized expression graph. Results are expressed as mean ± SEM of 3-4 independent experiments, *p<0.05, **p<0.01, ***p<0.001.

Figure 8 .

 8 Figure 8. Interaction between the ribosome 60S subunit binding site and DON, deepoxy-DON and 3-epi-DON.

Figure 1 -

 1 Figure 1 -Experimental design of the in vivo experiment

Figure 4 -

 4 Figure 4 -Histological intestinal samples from piglets submitted by gavage to control treatment (blue), DON (0.15 mg/Kg BW, red) or DOM-1 (0.14 mg/Kg BW, green) during 21 days. Two-way ANOVA, Bonferroni test as Post-hoc, p<0.05, n=6 to 8. A-Jejunum, control group. Villi with normal morphology. B-Jejunum, DON group. Villi fusion and lymphatic vessel expansion. C-Jejunum with Peyer patches, DOM-1 group. Villi with normal morpholy. D-Ileum, DON group. Villi atrophy, villi fusion and edema of lamina propria. Hematoxilin-eosin. Bar 100 µm.

Figure 5 -

 5 Figure 5 -The mRNA expression levels of markers of inflammation are affected by gavage to control treatment (blue), DON (0.15 mg/Kg BW, red) or DOM-1 (0.14 mg/Kg BW, green) during 21 days. At the end of the experiment, samples from different organs (A.jejunum, B.jejunal Peyer patches) were collected, and mRNA levels of inflammatory markers (IL1beta, IL6, IL8, IL1alpha, IL10, IL17A, IFNgamma, TNFalpha, TGFbeta, CCL20) were measured by RT-PCR. Results are expressed as the relative mRNA expression as means ± SEM; n=6 to 8 animals/group.*p<0.05, **p<0.01 and ***p<0.001, Two Away ANOVA with Bonferroni Post-hoc test.

Figure 6 -

 6 Figure 6 -Histological liver samples from piglets submitted by gavage to control treatment (blue), DON (0.15 mg/Kg BW, red) or DOM-1 (0.14 mg/Kg BW, green) during 21 days. One-way ANOVA, Bonferroni test as Post-hoc, p<0.05, n=6 to 8. A-Control group. Hepatocytes' trabeculae with normal arrangement. B-DON group. Hepatocyte apoptosis (arrows) and mild vacuolation of hepatocytes cytoplasm. C-DOM-1 group. Disorganization of hepatocytes' trabeculae and mild vacuolation of cytoplasm. D-DON group. Nuclear vacuolation of hepatocyte (arrow) and megalocytosis. Hematoxilin-eosin. Bar 30 µm (A), 20 µm (C), 10 µm (B,D).

Figure 9 -

 9 Figure 9 -IgG-antiOVA during the experiment per treatment control (blue), DON (0.15 mg/Kg BW, red) or DOM-1 (0.14 mg/Kg BW, green) during the experiment, following one vaccine injection against OVA at day 3 and one booster at day 8. Two-way ANOVA, Bonferroni test as Post-hoc, p<0.05, n=6 to 8.

paper

  Chapter I, Part 2), we found that DOM-1 didn't induce MAPKs activation and a proinflammatory response like DON. The results obtained in the present study indicate that DOM-1, not activating MAPKinase have the same effect on the immune response than DON that activates the MAPKinase. This suggests that all effects induced by DON, especially the ones on the immune response are not mediated by the way MAPKs works. This was already proposed by Wu et al. (2014) when investigating the effect on the immune response induced by D3G, another DON derivative that do not activate MAPKinase.

Table 1 .

 1 Regulation and recommendations for the main mycotoxins present in pigs feed and feedstuffs.

	(EC Directive 2002/32/EC, and EC Recommendations 2006/576/EC and 2013/165/EU)
	Mycotoxins	Pig feeds	Max. content mg/Kg (ppm)
	AFB1+ B2	Cereals	60
		Complete and complimentary
		feeding stuffs for pigs, horse,	0.5
		rabbit and pets
	OTA	Complete and complimentary feeding stuffs for pigs	0.05
	DON	Cereals	8
		(without maize by-products)	(12)
		Complete and complimentary feedstuffs for pigs	0.9
	ZEN	Cereals	2
		(without maize by-products)

Les mycotoxines "masquées" : un nouveau risque en production porcine ?

	1)INRA, UMR 1331, Toxalim, Research Center in Food Toxicology, 31300 Toulouse, France
	(2) Université de Toulouse, INP, UMR 1331, Toxalim, 31400 Toulouse, France
	(3) BIOMIN Research Center Technopark 1, 3430 Tulln, Austria
	Alix.Pierron@toulouse.inra.fr, Isabelle.Oswald@toulouse.inra.fr

Table 1 -

 1 Reglementation and recommendation of mycotoxins in pig feed: different type of feed and maximum levels (EC Directive 2002/32/EC, et EC Recommandations 2006/576/EC et 2013/165/EU) (adapted from Stoev, 2014).

Table 2 -

 2 Associated effects to mycotoxins' presence in pig feed

	Mycotoxins 1

1 Abbreviations : Aflatoxin B 1 (AFB 1 ), Aflatoxin B 2 (AFB 2 ), Ochratoxine A (OTA), Deoxynivalenol (DON), Zearalenone (ZEN), Fumonisin B1 (FB1), Fumonisin B2 (FB2), Toxin T2 (T2) and toxin HT2 (HT2).

2 +, ++, +++ : low effect, middle, and high of mycotoxin (s) on the parameter studied

Table 3 -

 3 Major

« modified » mycotoxins (adapted from Broaekart et al., 2015) "Native"mycotoxin "Modified" mycotoxin

Table 4 -

 4 Occurrence of trichothecenes and ZEN and occurrence of their "modified" forms in cereals (samples of wheat, barley, oat, maize and rice from different countries) (adapted from(Broekaert et al. 2015)

	Mycotoxins 1	Number of	Incidence,	Mean, µg/kg of
		samples 2	%	feed
	DON	5743	84	458
	3ADON	2227	22	14,7
	15ADON	686	31	36,6
	D3G	529	55	85
	NIV	3062	32	17,8
	ZEN	2158	12	39,6
	ZEN14G	36	25	37
	ZEN14S	12	25	6
	T2	321	45	16,7
	HT2	321	54	61
	T2-Glc	15	73	2,4
	HT2-Glc	15	80	5,1

Table 5 -

 5 "Modified" mycotoxin proportion compared to their "native" mycotoxins in raw materials naturally

	contaminated

1 Abbreviations: see tables 1 et 3.

Table 6 -

 6 Becoming of some "modified" mycotoxins along the digestive tract (adapted from De Boevre et al., 2015) 2

	Mycotoxins 1

2

In italic:% of "modified" mycotoxin found after oral ingestion in different parts of the diigestive tract. In normal character: % of "modified" mycotoxin found in his 'native" form in the different parts of the digestive tract. "Traces" if the molecule is slightly transformed

Table 7 -

 7 Pigs exposure estimation for certain mycotoxins ("native" accumulated forms and "modified") in two hypotheses (h) high and low (adapted from (EFSA 2014)1

						Level of exposure, µg/kg feed daily intake/day
	Age	Weig ht, kg	Feed daily intake (Kg/day)	ZEN "native" &"modified"	NIV "native" &"modified"	Toxins T2 + HT2 "native" &"modified"	Fumonisins "native" &"modified"
				h low	h high	h low	h high	h low	h high	h low	h high
	Piglet	20	1	0,7	1	0,53	2,07	0,3	1,43	3,7	10,3
	Feeder Pig	100	3	0,6	0,9	0,31	1,21	0,31	0,96	7,4	11,1
	Saw	200	6	2,2	2,5	0,32	1,26	0,33	0,92	4,6	11,9

1 Abbreviations: see tables

1, 3 and 4.

  3 (Cy3) labeled cRNA was prepared from 200 µg of total RNA using the One-Color Quick Amp Labeling kit (Agilent) according to the manufacturer's instructions, followed by Agencourt RNAClean XP (Agencourt Bioscience Corporation, Beverly, Massachusetts).

	About 600 ng of Cy3-labelled cRNA were hybridized on SurePrint G3 Porcine GE
	microarray (8X60K) following the manufacturer's instructions. Slides were scanned
	immediately, after washing on an Agilent G2505C Microarray Scanner with Agilent Scan
	Control A.8.5.1 software. All experimental details are available in the Gene Expression
	Omnibus	(GEO)	database	under	accession	GSE66918
	(http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=olixoosedvmdnqr&acc=GSE66918).
	The differentially expressed (DE) genes (adjusted p-value ≤ 0.05) were hierarchically
	clustered and visualized in heat maps. Functional analysis of DE genes was performed using

the Ingenuity pathway Analysis tool (IPA, http://www.ingenuity.com) to identify pathways and processes affected by toxins.

Table 1 . Primer sequences used for RT-qPCR analysis (F: Forward; R: Reverse)

 1

	640
	641

Table 2 . DON but not deepoxy-DON & 3-epi-DON up regulated mRNA relative expression levels of pro- inflammatory cytokines and chemokines in pig jejunal explants

 2

	Cytokines	Control	DON	Explant treatments Deepoxy-DON 3-epi-DON
	IL1B	1.00 ± 0.40 a	17.4 ± 5.1 b	0.7 ± 0.2 a	0.8 ± 0.3 a
	IL1A	1.00 ± 0.30 a	3.9 ± 1.4 b	0.9 ± 0.2 a	0.9 ± 0.2 a
	IL8	1.00 ± 0.20 a	4.5 ± 1.2 b	1 ± 0.1a	0.9 ± 0.2 a
	IL12p40	1.00 ± 0.31 a	2.3 ± 0.4 b	1.2 ± 0.2 a	0.9 ± 0.2 a
	IL17A	1.00 ± 0.50 a	15.8 ± 5.6 b	0.8 ± 0.1 a	1.3 ± 0.4 a
	IL22	1.00 ± 0.30 a	7.9 ± 1.3 b	1.3 ± 0.5 a	1.4 ± 0.5 a
	TNFA	1.00 ± 0.30 a	3.5 ± 0.5 b	1.1 ± 0.4 a	1.1 ± 0.3 a

Notes: results are expressed in arbitrary units relative to control group. Results are mean ±SEM of 6 animals.

Means in a row without a common letter differ (Newman-Keuls test, P<0.05).

Table 3 :

 3 Top scored differentially expressed genes in DON treated porcine jejunal explants 652

	Gene symbol Gene name

Table 4 .

 4 Ten top scored canonical pathways differentially regulated in 10µM DON treated porcine jejuna explants and list of genes in each pathway

	655
	a. Up-regulated pathways
	Ingenuity Canonical Pathways	-log (p-value)	Ratio		Molecule s
				CCL3,IL1B,MMP12,EZR,CCL20,CLDN4,CCL3L1/CCL3L3,SELE,MMP13,
	Granulocyte Adhesion and Diapedesis	1.18E01	1.1E-01	CXCL2,VCAM1,CXCL8, CXCR4, IL18,
				IL1RN,TNF,CXCR2,ICCL4,XCL1
				CCL3,IL1B,MMP12,EZR,CCL20,CLDN4,CCL3L1/CCL3L3,SELE,MMP13,
	Agranulocyte Adhesion and Diapedesis	1.13E01	1.04E-01	CXCL2,VCAM1,CXCL8,
				CXCR4,IL18,IL1RN,TNF,CXCR2,IL1A,CCL
				4,XCL1
				HSPA2,NFKB1,CCL3,CSF2,JAK2,IL1B,PLAU,SELE,NFKBIE,NFATC1,NF
	Glucocorticoid Receptor Signaling	1.11E01	7.69E-02	KBIA,VCAM1,CXCL8,
				FOS,IL1RN,DUSP1,SGK1,NR3C1,TNF,SMAD3,CDKN1A,IL10,F
				OXO3
	Differential Regulation of Cytokine Production in			CCL3,CSF2,IL1B,TNF,IL1A,IL17A,IL10,CC
	Intestinal Epithelial Cells by IL-17A and IL-17F	1.07E01	3.91E-01	L4,IL17F
	Communication between Innate and Adaptive Immune			CCR7,CCL3,CSF2,IL1B,CCL3L1/CCL3L3,CD40,CXCL8,CD83,IL18,IL1RN
	Cells	1.01E01	1.25E-01	,TNF,IL1A,IL10,CCL4
	Differential Regulation of Cytokine Production in			CCL3,CSF2,IL1B,TNF,IL17A,IL10,CCL4,IL
	Macrophages and T Helper Cells by IL-17A and IL-17F 1.01E01	4.44E-01	17F
				CCR7,NFKB1,IL1B,EDNRB,MMP13,CD40,VCAM1,CXCL8,IFNGR1,TNF,S
	Hepatic Fibrosis / Hepatic Stellate Cell Activation	9.55E00	1.03E-01	MAD3,IL1A,EDN1,IL4R,
				IL10,TIMP
				FOS,IL18,NFKB1,IL1RN,IL1B,SOCS3,TNF,IL1A,IL4R,IL10,NFK
	IL-10 Signaling	9.48E00	1.54E-01	BIE,NFKBIA
				GATA3,IFNGR1,BCL6,IL18,STAT4,TNF,ICOSLG/LOC102723996,IL17A,IL
	T Helper Cell Differentiation	9.41E00	1.67E-01	4R,IL10,CD40,IL17F
				NFKB1,IL1B,SELE,MMP13,CD40,VCAM1,CXCL8,CXCR4,IL18,IL1RN,TN
	Atherosclerosis Signaling	9.4E00	1.08E-01	F,IL1A,F3,TNFRSF12A,
				PLA2G4A
	656
	b. down-regulated pathways
		-log
	Ingenuity Canonical Pathways	(p-value)	Ratio		Molecules
	PXR/RXR Activation	2.28E00	2.17E-02	ABCC2, CPT1A

Table 1 -

 1 Primer sequences used for RT-qPCR analysis (F: Forward; R: Reverse)

  SCOOP. 2003. Collection of occurrence data of fusarium toxins in food and assessment of dietary intake by the population of eu member states. Directorate-general health and consumer protection. . http://europaeuint/comm/food/fs/scoop/ task3210pdf Accessed Fev 222012:1-606. Sergent T, Parys M, Garsou S, Pussemier L, Schneider YJ, Larondelle Y. 2006.

	Deoxynivalenol transport across human intestinal caco-2 cells and its effects on cellular
	metabolism at realistic intestinal concentrations. Toxicol Lett 164:167-176.
	Taranu I, Marin DE, Bouhet S, Pascale F, Bailly JD, Miller JD, et al. 2005. Mycotoxin
	fumonisin b1 alters the cytokine profile and decreases the vaccinal antibody titer in pigs.
	Toxicol Sci 84:301-307.
	Wu W, He K, Zhou H-R, Berthiller F, Adam G, Sugita-Konishi Y, et al. 2014. Effects of oral
	exposure to naturally-occurring and synthetic deoxynivalenol congeners on proinflammatory
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Cette revue recense les connaissances actuelles sur la toxicité des formes "modifiées" du déoxynivalénol, des toxines T2 et HT2, de la zéaralenone, de la fumonisine et de l'ochratoxine A pouvant se retrouver dans l'alimentation du porc. Nous nous attacherons à comparer le métabolisme et la toxicité des formes "modifiées" à celle de leurs précurseurs et à analyser la possible reconversion de ces formes "modifiées" par la flore intestinale ou les voies de métabolisation du porc.Masked mycotoxins: a new risk in pig production?Mycotoxins are secondary metabolites originating from mold, which contaminate many cereals and their by-products and so can be found in the pig's diet. Some recommendations and regulations for animal feed have been decreed in the EU for six mycotoxins for which the toxicity is well known. Recent detection methods have revealed new mycotoxins and new molecules that are derivates of these mycotoxins. They were originally called "Masked" mycotoxins because they are not detected by conventional analytical methods. Then, they are more generally called "Modified", and "masked" when they are metabolized by the plant.So because of the difficulty in detecting them, there is little information about the toxicity of these molecules and they are not included in the current regulation on mycotoxin contamination in pig feed. Moreover, a high proportion of these modified mycotoxins can be found in co-contamination with the mycotoxins. Pigs are really sensitive to mycotoxins, and their high cerealrich diet means that they are highly susceptible to mycotoxins and to these modified mycotoxins. These modified mycotoxins can potentially increase the amount of mycotoxins to which pigs are exposed if they are hydrolyzed in the animal. This review summarizes recent knowledge about the toxicity of the modified mycotoxins of deoxynivalenol, T2 and HT2 toxins, zearalenone, fumonisin and ochratoxin A, and presents recent studies about the metabolization and toxic effects on the animals of these modified mycotoxins, and their potential impact on their health.

Abbreviations : Aflatoxin B1 (AFB1), Aflatoxin B2 (AFB2), Ochratoxin A (OTA), Deoxynivalenol (DON), Zearalenone (ZEN), Fumonisin B1 (FB1), Fumonisin B2 (FB2), Toxin T2 (T2) and toxin HT2 (HT2).

Acknowledgment A. Pierron was supported by a fellowship from CIFRE (2012/0572, jointly financed by the BIOMIN Holding GmbH, Association Nationale de la Recherche Technique and INRA). This work was supported in part by the projects Liporeg (APP-ICSA 2015) and PHC STEFANIK 2016. Acknowledgments The authors are grateful to A.M. Cossalter, J. Laffitte and P. Pinton (INRA Toxalim) for sample collection and western-blot experiments. The authors thank Drs L. Huc (INRA, Toxalim), F. Smih-Rouet and M. Barutaut (I2MC INSERM UMR 1048) for Bioenergetic measurements. Thanks are also due to Catherine Anne Moll for language editing. A. Pierron and L.S. Murate were supported by fellowship from CIFRE (2012/0572, jointly financed by the BIOMIN Holding GmbH, Association Nationale de la Recherche Technique and INRA) and CAPES (Brazil) respectively. The authors declare no conflicts of interest during the realization of the experimental work submitted.

List of figures and tables

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Competing interests

The authors declare that they have no competing interests.

Authors' contributions

All authors agree fully to the content of the review.

Abstract (250 words)

The contamination of cereals with deoxynivalenol (DON), the most prevalent mycotoxin in the world, cannot be avoided however biotransformation can be used to reduce its toxicity.

Bacteria are able to de-epoxidize or epimerize DON to deepoxy-deoxynivalenol (deepoxy-DON or DOM-1) or 3-epi-deoxynivalenol , respectively. Using in silico, in vitro and ex vivo approaches, the intestinal toxicity of DON, deepoxy-DON and 3-epi-DON 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 10µM DON induced intestinal lesions not seen in explants treated with deepoxy-DON and 3epi-DON. A pan-genomic transcriptomic analysis was also performed on intestinal explants treated with DON and its biotransformation metabolites. 747 probes, representing 323 genes, involved in immune and inflammatory responses, oxidative stress, cell death, molecular transport and mitochondrial function, 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. In this position 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 deepoxy-DON modified their interaction with the ribosome, leading to an absence of MAPKinase activation and toxicity.

Supplementary materials

Chapter II

In vivo toxicity of purified deepoxydeoxynivalenol (DOM-1) in piglets

II. Material and Methods

A.

Experimental design

Animals

All animal experimentation procedures were carried out in accordance with the European Directive on the protection of animals used for scientific purposes (Directive 2010/63/EU). All the experimental design is summary in the Figure 1.

For the experiment, twenty-two 4-wk-old weaned castrated male pigs (PIC 410) were obtained locally (GAEC Calvignac, St Vincent d'Autejac, France). As previously described a greater effect of DON occurs on male pigs compared to female pigs (Cote et al. 1985;Marin et al. 2006). Animals were acclimatized for 2 weeks in the animal facility of the Toxalim Laboratory (INRA, Toulouse, France), prior to being used in experimental protocols. Eight pigs were allocated per box to each treatment on the basis of body weight for (1) control group and ( 2) DON group, and six pigs for (3) DOM-1 group. At the start of the experiment, there were no differences in body weights among all treatment groups including the control; piglets weighted 10.9±0.08Kg, p>0.05. Then they were weighed and observed daily, and the animal behaviour was noted. During the three weeks of experimental period, each group was given free access to water and feed. They were fed with a commercial feed adapted to their age, complete food for the first piglet age Belecla (Annex 1) and complete food for the second piglet age Stimiouti (Annex 2). A period of transition of 3 days was respected between the two foods. After the experimental period the animals were submitted to electrical stunning, and euthanized by exsanguination.

Toxins

Purified DON was purchased from Sigma-Aldrich (St Louis, MO, USA). DOM-1 was obtained by transforming the crystalline DON (Romer Labs, Tulln, Austria), dissolved in medium 10 (Caldwell and Bryant, 1966)

Gavage

During the experiment, animals were exposed to DON and DOM-1 as powder at 100%

and 63% of purity respectively (provides by Austria BIOMIN GmgH, Tulln). The two molecules were administered by gavage for 21 days, at the same molar concentration (0.15 mg/kg BW /day for DON and 0.14 mg/Kg BW/day for DOM-1). Toxins were diluted in water and black current syrup, to increase the palatability.

Sample collection

The experimental design used in this study was randomized with eight or six repetitions (each animal represented one repetition). To evaluate the vaccine response, all piglets were immunized by subcutaneous inoculation with 1 mg of ovalbumin (OVA) for the primary injection and 2 mg for the booster vaccine, respectively (Sigma, St Quentin Fallavier, France), dissolved in sterile PBS and mixed with incomplete Freund's adjuvant (Sigma). At weekly time intervals, blood samples were aseptically collected from the left jugular vein.

Blood was collected in tubes containing sodium heparin or EDTA (Vacutainer®, becton-Dickinson, USA) for blood culture or blood formula, respectively. Serum samples were obtained after centrifugation of blood and stored at -20°C for later analysis. After 21 days of exposure pigs were killed by electrical stunning and exsanguination.

III. Results

A. Comparative effect of DOM-1 and DON on animal performance

During the experiment, the mean body weight gained per animal was no significantly different between groups (Fig. 2), excepted on week 2, where DOM-1 group had a weight gain higher in comparison to other groups (Fig. 2).

In another study with similar design but higher dose of toxins (0.3 mg DON or DOM-1 /Kg BW for 2 weeks) we obtained similar results. Animals in the DOM-1 group had a significant higher weight gain on the second week compared to than animals in the control and DON groups, while animals in the DON group had a significant lower weight gain on the first week (Fig. 3). Morover 4 of 6 pigs receiving DON vomitted during the experiment.

B. Comparative intestinal toxicity of DOM-1 and DON

Ingestion of DOM-1 induced no significant change on intestinal morphology when compared to control. By contrast, animal fed with DON showed a significant increase in the lesional score of the jejunum (4.5 fold higher than control) and the ileum (3 fold higher than control). Villi atrophy and fusion with flattening of enterocytes and denuded villi were the main histological changes observed on the intestine (Figure 4) in animals receiving DON.

The effects of DON and DOM-1 on the expression of cytokines were assessed, on the jejunum without (Fig. 5, A.) and with Peyer patches (Fig. 5,B.). Whatever the intestinal part and the cytokine considered, no significant increase in mRNA level were observed in DOM-1 treated animals when compared to control ones. In DON treated animals, IL-8, IL-10 and IL-17A mRNA levels were significantly increased in the jejunum when compared to control and DOM-1-treated animals. In jejunum with Peyer's patches, IL1 beta and IL17A significantly increased in animals from the DON group when compared to control and DOM-1 groups (Fig.

5, B.

). In this organ, a slight but non significant increase of IL-6, IL8, IL1alpha, IL10 and IFNgamma mRNA levels was also observed in DON treated animals.

21 when compared to control (15.2 ± 2.9; 86.3 ± 25.9 and 82.3 ± 37.4 Arbitrary Units in DOM-1, DON and control animals respectively) (Fig. 9). IPA highlighted several genes and pathways already known to be impaired by DON in the intestine. For example we observed that in the intestine, DON modulates cytokines involved in the inflammatory pathway, tight junction signaling, p38MAPK Signaling, Protein Ubiquitination pathways, oxidative stress, protein degradation pathway as already described (Katika et al. 2012;Mishra et al. 2014;Osman et al. 2010;Shen et al. 2007;van Kol et al. 2011). Functional analysis pathway also brings new information on DON effects in the intestine. It's the case on metallothionein,

shown to be a marker of oxidative stress and mycotoxins contamination in rats (Vasatkova et al. 2009). Results also underline the effect of DON on genes of intestinal transporters (Bookstein et al. 1997;Smith et al. 2013;Videmann et al. 2007) and on the decrease of the unfolded protein response pro-apoptotic gene CHAC1 that seems to play a role in glutathione degradation (Kumar et al. 2012).

This study also shows the impact of DON on mitochondrial dysfunction.

The in vivo experimental protocol

During the experiments, purified mycotoxins were used. In cereals naturally contaminated, there are always co-contamination that doesn't allow to discriminate the effect of each mycotoxin (Schatzmayr and Streit 2013). Moreover, fungi contamination on cereals affects nutritional quality and change the final quality of pig feed. So the use of purified mycotoxins allows to keep the nutritional quality of feed and to attribute observed effect to the tested mycotoxins. In addition, gavage allowed to adjust precisely the dose given and to be sure that all animals received the calculated dose per Kg/BW.

A moderate dose was used in the perspective to assess the toxicity of probable quantity of DON in animals. Many studies performed with DON contaminated feed using moderate amounts corresponding to contamination doses that can be found in natural conditions (Poolman et a., 1985;Harvey et al., 1996;Trenholm et al., 1984;Friend et al., 1992;Rotter et al. 1994).

B. Toxicity assessment of biological strategies to reduce toxic effects of DON

Despite all preventive efforts and improvement of agricultural practices, important concentrations of mycotoxins still occur in agricultural products. Due to the difficulty to eliminate and detoxify DON, alternative effective strategies of detoxification are needed. the use of plants resistant to fungi infection or biological biotransformation to detoxify are new promising strategies [START_REF] He | Chemical and biological transformations for detoxification of trichothecene mycotoxins in human and animal food chains: A review[END_REF]. Indeed, biological methods are a good way to eliminate toxicity without acting on the grain quality or on the processed feed. There have been several transformation products of DON reported (Table 1), such as DON-sulfonate, DON lactone, norDON A, norDON B and norDON C, 3-acetyldeoxynivalenol (3-ADON), , diacetyl-DON, triacetyl-DON, iso-DON, 3-keto-deoxynivalenol (3-keto-DON), deepoxy-deoxynivalenol (DOM-1), DON-3-glucuronide,

II. Perspectives

Results obtained in the present thesis demonstrate the non toxicity of several derivatives of DON on numerous parameters. To completely evaluate the toxicity of each molecule, an in vivo experiment on D3G and 3-epi-DON would be necessary. Actually, as we have seen for DOM-1, an in vivo experiment can make reveal unexpected properties of the molecule. No studies evaluating the toxicity of D3G and 3-epi-DON on farm animal were done yet. In order to assess in a significant way all the parameters impacted by D3G and 3-epi-DON, a longer exposure could be interesting to asses chronic effect of the toxin. The use of more animals, even if it is difficult to handle, should reduce the important individual variability that we observed in our in vivo experiment.

Our results associated with previous studies assessing the effectiveness and toxicity of bacteria transforming DON into DOM-1, as well as the safety for pig with tolerance studies (He et al. 1993;Li et al. 2011;EFSA 2013), seems to show that the use of bacteria to transform DON into a less toxic compound is be an efficient approach. It will be thus interesting to extend this approach to other mycotoxins. Indeed, as described in the introduction, the detoxification methods have not the same efficacy according to the mycotoxin. The use of bacteria efficient to transform other mycotoxins in less toxic compounds is already tested (Grenier et al. 2011); the toxicity of all the transformed products obtained on many different parameters has not been completely investigated.

In this context, the explants model, was shown to be efficient to evaluate the intestinal toxicity.

Combined with others tools, such as microarray and functional analysis, it allows to deeply investigate the impact of a molecule on the tissue and to determine all the genes impacted by the exposure in the tested tissue. This model also allows to perform functional tests. For example, the evaluation of the barrier function can be measured by the TEER of jejunal explants placed in Ussing Chamber.

Nevertheless, to completely assess the impact of a mycotoxin on the organism, in vivo experiments are required. These type of experiment especially allow to assess zootechnical effects, systemic or specific immune responses as well as the metabolism of the ingested molecule. Indeed, as we saw for DOM-1, its effect on the immune response was not observed in vitro or ex vivo. Concerning the metabolization, as described for D3G, modified mycotoxins could be retransformed into its native form by the host or the microbiota. Thus, in vivo experiment cannot be completely substituted by the use of in vitro and ex vivo models. Livestock feeding studies (e.g. swine, goat and chicken) are recommended to confirm the detoxification of the transformed product and to determine the metabolism/toxicokinetic of this modified mycotoxin. The toxicological results are critical for future industrial application, but they also provide fundamental scientific information for toxicological research.

In order to assess, the metabolization of modified and masked mycotoxin into the organism, the pig constitutes a relevant model. The bacterial profil of pig matches quite well with human, which is important to investigate the metabolization of "modified" DON in pig. Herein, pig is a good model; beside is similar intestinal tract to those of man (Heinritz et al, 2013), it also possess a similar bacterial

CONCLUSION Conclusion

The thesis brings new data on modified forms issued from strategies to detoxify mycotoxins.

The general objective of this research was to evaluate the toxicity of three DON derivatives issued from biological transformation, DOM-1, 3-epi-DON and D3G. This thesis allowed to examine numerous parameters, through in silico, in vitro and ex vivo experiments. Results increased the knowledge on the toxicity and the mechanism of toxicity of DON, using pangenomic array on a jejunal tissue and in silico analysis. Finally, in vivo experiments allowed assessing the intestinal and immune toxicity of purified DOM-1 in comparison to DON, on piglets.

In silico, in vitro and ex vitro results showed that the tested DON derivatives were less toxic than DON; they were not able to induce a ribotoxic stress, a "keystone" of DON toxicity. Another important aspect that was not studied during this thesis is the metabolism of these derivatives in the organism. Certainly, each derivative and mycotoxin has its own behavior into organism, and in vitro and ex vivo assessment are not enough to give use a complete information on the molecule. In vivo studies allow investigating the organ toxicity of the tested molecules. In the present work we did not investigate the transformation of the derivatives, to the "native" mycotoxin into the intestine. Such a transformation, especially with "masked" mycotoxin, could lead to an underestimation of the risk.

In perspectives, assessing the in vivo toxicity of 3-epi-DON, D3G and the metabolisation of all the molecules on farm animals, will bring us all the information needed regarding the management of risk assessment for these molecules. 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-epideoxynivalenol . Moreover, some plants naturally own the capacity to glycosylate DON in the aim to detoxify it, forming the deoxynivalenol-3-β-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.

RESUME :

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 . 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-β-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 (mitogenactivated 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.

MOT-CLES