Abdullah Khan Khoshal
email: abdullah.khoshal@inra.fr

Barbara Novak
email: barbara.novak@biomin.net

Pascal G P Martin
email: pascal.martin@inra.fr

Timothy Jenkins
email: timothy.jenkins@biomin.net

Manon Neves
email: manon.neves@inra.fr

Gerd Schatzmayr
email: gerd.schatzmayr@biomin.net

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

Philippe Pinton
email: philippe.pinton@inra.fr

CONTAM: Panel On Contaminants in the Food Chain Cyclo: Cyclo-(Pro-L-Tyr

Keywords: Mycotoxines émergentes, DON, Toxicité, Aliments pour porcs, IPEC-1 Emerging Mycotoxins, DON, Toxicity, Pig Feed, IPEC-1, Liver Slices, Gene Expression ABCG-8: ATP Binding Cassette BEAS-2B: Human Normal Lung Epithelial Cells C-C Motif Chemokine Ligand global survey, finished pig feed, co-occurrence, emerging mycotoxins, DON, toxicity, combined toxicity, IPEC-1

Pour la plupart des mélanges, la cytotoxicité combinée était similaire à celle du DON seul. Pour ce qui concerne la santé des animaux, ces résultats ont montré que lorsque ces mycotoxines émergentes sont présentes avec le DON, elles n'exacerbent pas la toxicité du DON. Outre l'intestin, le foie est le principal site de détoxification des xénobiotiques, y compris des mycotoxines, et représente un organe cible des contaminants alimentaires. Nous avons donc mis au point un nouvel outil, les Precision Cut Liver Slices (PCLS), des explants de foie d'épaisseur définie. Elles ont été utilisées pour évaluer la toxicité du DON (3 et 10 µM) à différents temps d'incubation (0 à 20 h), en étudiant l'expression génique, le contenu en ATP et en protéines totales. Le milieu d'incubation a permis d'évaluer la qualité des PCLS en mesurant les marqueurs de dommage hépatique (phosphatase alcaline, lactate déshydrogénase, alanine aminotransférase, aspartate aminotransférase et protéines totales). Nous avons montré que ces marqueurs n'étaient impactés ni par le temps d'incubation, ni par le traitement. Les PCLS traitées avec 10 µM de DON pendant 4 h ou plus, montrent une altération de l'expression de certains gènes. Ces expériences préliminaires ont montré que les PCLS représentent un modèle prometteur pour évaluer la toxicité hépatique des mycotoxines ou d'autres contaminants alimentaires. Mots-clés:

Résumé Français

Les mycotoxines sont des métabolites secondaires toxiques produits par certains champignons filamenteux. Selon leur toxicité et leur occurrence, certaines d'entre elles dont le déoxynivalénol (DON), l'une des toxines les plus répandues dans l'alimentation humaine et animale, sont été réglementées au sein de l'Union Européenne. D'autres métabolites secondaires découverts récemment ou encore peu étudiés sont appelées mycotoxines émergentes et ne sont ni détectés en routine ni réglementés. Les denrées alimentaires destinées à l'homme et à l'animal peuvent être naturellement contaminées par plusieurs mycotoxines et le risque lié à une exposition à des mélanges de mycotoxines est préoccupant.

Parmi les animaux d'élevage, le porc est une espèce très sensible aux mycotoxines. De par son alimentation riche en céréales, il peut être exposé à de fortes concentrations de ces contaminants. 524 échantillons d'aliments complets pour porcs prélevés dans le monde entier ont été analysés par une technique de chromatographie en phase liquide couplée à la spectrométrie de masse en tandem (LC-MS/MS) pour plus de 800 métabolites. [START_REF] Glenn | Molecular phylogeny of Acremonium and its taxonomic implications[END_REF]

Summary

Mycotoxins are toxic secondary metabolites, produced by several filamentous fungi.

Depending on their toxicity and occurrence, some of them, including deoxynivalenol (DON), one of the most common toxin in food and feed, have been regulated in the European Union.

Other secondary metabolites, which neither routinely determined nor regulated, are called emerging mycotoxins because they have been recently discovered or poorly investigated. Food and feed can be naturally contaminated by several mycotoxins and concern about the hazard of exposure to mycotoxin mixtures is increasing.

Among farm animals, pig is one of the most sensitive farm animal to mycotoxins and it can be exposed, through its rich cereal diet, to high concentrations of mycotoxins. In total, 524 finished pig feeds samples from worldwide were analyzed for more than 800 metabolites using, LC-MS/MS (liquid chromatography tandem-mass spectrometry) method. Eighty-eight percent of the samples were co-contaminated with DON and other regulated and emerging mycotoxins.

The toxicity of DON and the 10 most common emerging mycotoxins was analyzed on the viability of porcine intestinal epithelial cells (IPEC-1) over a 48 h period. Among the emerging mycotoxins, 3 of them (brevianamide F, cyclo-(L-Pro-L-Tyr), and tryptophol) did not alter cells viability. The other mycotoxins were ranked in the following order of toxicity: apicidin> enniatin A1> DON> beauvericin> enniatin B> enniatin B1> emodin> aurofusarin.

The combined toxicity of DON and the 10 emerging mycotoxins was assessed based on their actual ratios found in pig feed. We observed that, despite the very high frequency of cocontamination, there was a poor correlation between the concentrations of DON and emerging mycotoxins. Thus, we assessed the toxic effects of three mixtures corresponding to situations to which animals may be exposed. Ratio #1 was calculated using the P25 (1 st quartile) concentration of the emerging mycotoxin and P75 (3 rd quartile) concentration of DON. Ratio #2 was calculated using the median (2 nd quartile) concentration of DON and each emerging mycotoxin. Ratio #3 was the reverse scenario of ratio #1.

Cytotoxicity analyses showed that, in most of the mixtures, the combined toxicity was similar to the one of DON alone. These results demonstrated that, when these emerging mycotoxins are present with DON, in terms of pig health, it does not exacerbate the problem of the toxicity of DON.

The work presented in this thesis would not have been possible without many people in so many ways. I take this opportunity to extend my sincere gratitude and appreciations to all those who made this Ph.D thesis possible.

First and foremost, I would like to extend my sincere gratitude and deepest appreciation to my Ph.D director "Dr. Isabelle Oswald", as well research guide and Ph.D supervisor "Dr. Philippe

Pinton" for their full support, expert guidance, understanding and encouragement throughout these three years research project. Without their incredible patience and timely wisdom and counsel, my thesis work would have been a frustrating and overwhelming pursuit. I would like to say a heartfelt thanks to both of them.

Second, I would like to express my deepest gratitude to "Dr. Gerd Schatzmayr" and "Ms.

Barbara Novak" for their coordination, and regular monitoring of my research work and taking time to talk with me on many occasions. I also greatly appreciate the support received through the collaborative work undertaken with the BIOMIN, for providing the raw data for our article.

Many of my statistical work would not have been completed without the assistance and cooperation of "Dr. Pascal G. P. Martin" and "Dr. Timothy Jenkins". I would like to thank them for statistical analysis and providing some figures and tables to our article.

Many of my experimental work would not have been completed without cooperation of "Ms.

Manon Neves", "Ms. Elodie Person" and "Mrs. Sandrine Bruel", I would like to thank for all of their time, help and kind cooperation. I am grateful to Team-2 Metabolism of Xenobotic (MeX) who gave me permission to work in their laboratory and use the facilities available for the liver slices.

My thanks also go out to the support I received from the collaborative work with my dear friends and colleagues: Joelle Laffitte and Sylvie Puel for their availability, introductory in technique, assistance and contribution to my data collection. I am also very grateful to all those our members of the team: Dr. Olivier Puel, Dr. Laura Soler Vasco, Dr. Chloe Terciolo, Dr.

Sophie Lorber, Tarek Lahjouji, Dr. Alix Pierron, Dr. Shuai Wang, Carine Al-Ayoubi, Su LUO ... who contribute to the perfect atmosphere of this laboratory so welcoming which I found each time with much pleasure. This PhD study would not have been possible without the corporation and support extended by the doctoral school "SEVAB". A very special thanks to Dominique Pantalacci and Professor Claud Maranges for their patience during the numerous focus group discussions as well for the administrative organization.

My deep appreciation also goes out to my M.Sc. internship supervisor, "Dr. Alain Pinton" for being so supportive, encouragement and his valuable advices to pursue my PhD.

I gratefully acknowledge the funding received towards my PhD from the BIOMIN GmbH and Région Occitanie "ANR grants "EmergingMyco" (ANR-18-CE34-0014) and "Newmyco" (ANR-15-CE21-0010). I am also grateful to the Campus France for providing the first 18 months Security Social Scholarship "BCS" to undertake my PhD.

Last but not least, the best outcome from these past three years is finding my best friend, soulmate, and my wife. There are no words to thank for accompanying me during my PhD, and staying away from the family and country. I would like to say a heartfelt thank you to my wife.

I would also like to especially thank my parents. My hard-working parents have sacrificed their lives for us and provided unconditional love and care.

Table of Contents

List of Figures (Excluding Figures of Article)

Introduction 1.Generalities on mycotoxins

In today's changing world, safety and security remains basic human needs. Ensuring food safety has been a major focus of national and international actions over the last years. Nowadays, international trade in agricultural commodities such as wheat, rice, barley, corn, sorghum, soybeans, groundnuts and oilseeds reaches hundreds of millions of tons each year (FAO 2019).

In 2014, worldwide feed production was 964 million tons and among them 153 million tons were produced just in Europe [START_REF] Kovalsky | Co-Occurrence of Regulated, Masked and Emerging Mycotoxins and Secondary Metabolites in Finished Feed and Maize-An Extensive Survey[END_REF]. As the occurrence of mycotoxins is widespread throughout the world, the global trade of agricultural commodities (e.g., animal feed) requires to monitor fungal toxins [START_REF] Kovalsky | Co-Occurrence of Regulated, Masked and Emerging Mycotoxins and Secondary Metabolites in Finished Feed and Maize-An Extensive Survey[END_REF]. The worldwide contamination of food and feed with mycotoxins is a significant problem [START_REF] Hussein | Toxicity, metabolism, and impact of mycotoxins on humans and animals[END_REF]. Recent surveys on the occurrence of mycotoxins have shown that 60 -88% of the world's cereal grains are contaminated with mycotoxins (Gruber-Dorninger et al. 2019;[START_REF] Eskola | Worldwide contamination of food-crops with mycotoxins: Validity of the widely cited 'FAO estimate' of 25%[END_REF].

Mycotoxins can resist to high temperature and to chemicals like acids, both during storage/milling and cooking process and they are stable during food/feed processing like brewing, melting, hot drying or ensiling (Rodríguez-carrasco et al. 2016). Their presence in food and feed products is an important threat to human and animal health [START_REF] Heshmati | Co-occurrence of aflatoxins and ochratoxin A in dried fruits in Iran: Dietary exposure risk assessment[END_REF].

Origin of mycotoxins

Mycotoxins are toxic secondary metabolites, produced by toxigenic moulds in the Aspergillus, Alternaria, Claviceps, Fusarium, Penicillium and Stachybotrys genera occurring in food and feed commodities both pre-and post-harvest [START_REF] Milićević | Real and Perceived Risks for Mycotoxin Contamination in Foods and Feeds: Challenges for Food Safety Control[END_REF] under appropriate environmental conditions [START_REF] Jestoi | Emerging Fusarium Mycotoxins Fusaproliferin, Beauvericin, Enniatins, And Moniliformin-A Review[END_REF]). Mycotoxins represent a potential health risk for humans and/or livestock. [START_REF] García | Emerging mycotoxins: enniatins (A, A1, B and B1) and beauvericin[END_REF]. The exact number of mycotoxins is not known, but the number of potential toxic fungal metabolites has been estimated to be in the thousands (Council for Agricultural Science and Technology 2003). Traditionally, toxigenic fungi contaminating agricultural grains have been conventionally divided into two groups:

those invading seed crops have been described as "field" fungi (e.g., Cladosporium, Fusarium, Alternaria spp.), which reputedly gain access to seeds during plant development, and those proliferating during storage, "storage" fungi, (e.g., Aspergillus; Penicillium spp) [START_REF] Ismaiel | Mycotoxins: Producing Fungi and Mechanisms of Phytotoxicity[END_REF][START_REF] Alshannaq | Occurrence, Toxicity, and Analysis of Major Mycotoxins in Food[END_REF]. Among the field occurring mycotoxins, Fusarium mycotoxins are the most frequently identified in grain and animal feed and it is important for farmers to manage cereals contamination by Fusarium species [START_REF] Steffen | MYCOTOXINS -THE HIDDEN DANGER IN FOOD AND FEED[END_REF].

Occurrence and contamination

Over the last years, mycotoxins have been regularly classified in the top ten and top one causes (European Union (EU) members and non-members countries, respectively) for the Rapid Alert System for Food and Feed (RASFF) notifications. RASFF is an European tool that ensures the inter-countries flow of information when risks to public health are detected in the food chain (RASFF 2018). The evidence suggests that mycotoxins are occurring increasingly in agricultural products including grains destined for human and animal consumption [START_REF] Steffen | MYCOTOXINS -THE HIDDEN DANGER IN FOOD AND FEED[END_REF]. Food can be contaminated with mycotoxins at various stages of the food chain, in field, during storage, milling or at later points (Bennett and Klich 2003). In fact, there is a notable length of time between the harvest of agricultural commodity at the exporting country and its arrival at the distribution center of the importing country. Furthermore, storage conditions at the farm level as well as during transport under adverse weather conditions may not always be satisfactory. Therefore, there is considerable opportunity for mycotoxin contamination of agricultural commodities to take place throughout the food system [START_REF] Fao | FOOD, NUTRITION AND AGRICULTURE[END_REF]. On the other hands, feeding animal with contaminated feeds can lead to contaminated animal products (eggs, meat, milk…). For example, aflatoxin B1 in cattle feed can be metabolized by cows into aflatoxin M1, which is then secreted in milk [START_REF] Younis | Determination of Aflatoxin M1 and Ochratoxin A in Milk and Dairy Products in Supermarkets Located in Mansoura City, Egypt[END_REF]. Furthermore, ochratoxin A in pig feed can accumulate in porcine tissues [START_REF] Rutqvist | Ochratoxin A as the Cause of Spontaneous Nephropathy in Fattening Pigs[END_REF].

Human exposure to mycotoxins also results from the consumption of several sources of food such as plant-derived foods, animal products (meat, eggs and milk) and/ or the exposure to contaminated air and dust (Council for Agricultural Science and Technology 2003).

Regulations

Regulations on mycotoxins have been established in many countries in order to protect the consumers from the harmful effects of mycotoxins. Two factors are mainly taken into account in the decision-making process of setting limits for mycotoxins: (i) the toxicity and (ii) the exposure [START_REF] Van Egmond | Regulations relating to mycotoxins in food: Perspectives in a global and European context[END_REF]. International survey on mycotoxins legislation in foodstuffs and feedstuffs have shown that approximately 100 countries (covering approximately 85% of the world's inhabitants) had specific regulations or detailed guidelines for some mycotoxins (FAO 2004), whereas no data were available for about 50 countries, many of them were in Africa (van Egmond 2002). In EU, mycotoxins regulation concern, ochratoxin A, patulin, deoxynivalenol (DON), zearalenone (ZEN), fumonisins (FUM) B1 & B2, T-2 & HT-2 toxin, aflatoxin B1 (AFB1), total aflatoxins (AFs) and patulin [START_REF] Lerda | Mycotoxins Factsheet-4th edition[END_REF]). In the following (Table 1), the maximum level of each above mentioned mycotoxins for pig feed is stated based on the EU recommendations.

Economical losses

The economic consequences of mycotoxins contamination are important. Commodities over the regulation should be destroyed. In addition, contaminated feed can induce losses in animal production. Due to the insufficient information, the economic costs of mycotoxins are impossible to determine accurately. However, the U.S. Food and Drug Administration (FDA)

has utilized a computer model to estimate the losses due to selected mycotoxins such as AFs, FUM and DON. Only for United States, the mean economic annual costs of crop losses from the above mentioned mycotoxins are estimated to be $932 million (Council for Agricultural Science and Technology 2003), whereas in UK they represent approximately £200/hectare and €450/hectare in Germany [START_REF] Steffen | MYCOTOXINS -THE HIDDEN DANGER IN FOOD AND FEED[END_REF].

Toxicity

Acute and chronic dietary exposure to mycotoxins can induce a variety of adverse health effects in humans and animals, making these chemically diverse substances highly relevant agricultural contaminants [START_REF] Eskola | Worldwide contamination of food-crops with mycotoxins: Validity of the widely cited 'FAO estimate' of 25%[END_REF]. Mycotoxins can exhibit acute toxicity as well as carcinogenic, mutagenic, teratogenic, immunotoxic, hepatotoxic, nephrotoxic, neurotoxic or estrogenic effects in human and animals [START_REF] Van Egmond | Regulations relating to mycotoxins in food: Perspectives in a global and European context[END_REF] Unlike bacterial toxins, fungal toxins are not proteins and are usually barely detectable by the immune system of humans and animals. Most illnesses caused by mycotoxins are not reported to the doctors, whereas low levels can be dangerous to humans health [START_REF] Steffen | MYCOTOXINS -THE HIDDEN DANGER IN FOOD AND FEED[END_REF]. On the other hands, diagnosis of mycotoxicoses in human and animals is difficult as they may be similar to diseases with other causations. This is even more difficult in cases where more than one mycotoxin is involved (Alassane-Kpembi et al. 2017b).

Mycotoxins are more thoroughly studied in animals. Studies on animals have demonstrated that among farm animals, pig is one of the most sensitive species to the deleterious effects of mycotoxins. It can be exposed to high concentration of mycotoxins due to its rich cereal diets (Pinton et al. 2010). Previous studies on pig species have shown that, mycotoxins affect intestinal function. The gastrointestinal tract is the first physiological barrier against food contaminants, as well as the first target for these toxicants (Pinton and Oswald 2014). Indeed, after oral intake, the gastrointestinal tract is the first possible site of interaction (Fraeyman et al. 2018). In pigs, mycotoxins can decrease the feed consumption, reduce weight gain and alter reproduction and immunity [START_REF] Korosteleva | Effects of Feedborne Fusarium Mycotoxins on the Performance, Metabolism, and Immunity of Dairy Cows[END_REF]. Ruminants are less susceptible to mycotoxins than monogastrics, because of the rumen microbiota. Ruminant's digestive system and especially resident bacteria and protozoa, are able to detoxify mycotoxins. Nevertheless, there is some evidence that ruminants can be poisoned by mycotoxins, causing lower animal production or even death of animals. Recent in vitro research showed that at conditions of rumen acidosis and lower microbial activity, a reduced detoxification in the rumen can take place [START_REF] Debevere | In Vitro Rumen Simulations Show a Reduced Disappearance of Deoxynivalenol, Nivalenol and Enniatin B at Conditions of Rumen Acidosis and Lower Microbial Activity[END_REF]. Recent insights have generated an entirely new perspective where a bi-directional relationship exists between mycotoxins and gut microbiota, thus suggesting that gut microbiota might be involved in the development of mycotoxicosis [START_REF] Jouany | Risk assessment of mycotoxins in ruminants and ruminant products[END_REF][START_REF] Liew | Mycotoxin: Its Impact on Gut Health and Microbiota[END_REF]. Moreover, most of the toxicant can be detoxified in the liver, which is the main site of metabolism and detoxification of toxins and also a target organ of mycotoxins [START_REF] Zain | Impact of mycotoxins on humans and animals[END_REF][START_REF] Pongratz | Hormone-Disruptive Chemical Contaminants in Food[END_REF]. Nevertheless, the contamination of animal feed can have a major impact on dairy farming. Mycotoxins can be found in dairy products from two origins, indirect contamination, which results when dairy cows ingest feed that contains mycotoxins which pass into the milk such as aflatoxin M1 and direct contamination, which results from accidental growth of moulds secreting aflatoxins [START_REF] Younis | Determination of Aflatoxin M1 and Ochratoxin A in Milk and Dairy Products in Supermarkets Located in Mansoura City, Egypt[END_REF].

Mitigation strategies

Mycotoxins are still considered unavoidable contaminants in foods and feeds, because agronomic technology has not yet advanced to the stage at which pre-harvest contamination of susceptible crops by fungi can be eliminated (Wood 1992). In order to avoid harmful effects of contamination of food and feed caused by mycotoxins, three principles could be implemented;

(i) prevention of contamination; (ii) decontamination of mycotoxin containing food and feed; and (iii) inhibition of absorption of mycotoxin from the digestive tract [START_REF] Karlovsky | Biological detoxification of fungal toxins and its use in plant breeding, feed and food production[END_REF]). There are three common strategies to mitigate toxicity of mycotoxins (Figure 1). One of the methodologies employed for mycotoxins detoxification is the physical strategy. Even though mycotoxins are stable compounds, some food processes including sorting, trimming, cleaning, milling, brewing, cooking, baking, frying, roasting, canning, flaking, nixtamalization and extrusion may affect their chemical structure [START_REF] Bretz | Thermal Degradation of the Fusarium Mycotoxin Deoxynivalenol[END_REF]. Another common mitigation strategy is biotransformation. It consists in the use of enzymes that have been found to be effective in transforming mycotoxins into less toxic metabolites or completely inactivating them [START_REF] Varga | Chemical, Physical and Biological Approaches to Prevent Ochratoxin Induced Toxicoses in Humans and Animals[END_REF]. For example, a mixed culture of two soil bacteria Pseudomonas sp and Lysobacter sp through formation of 3-keto-DON an enzymatic epimerization was capable of transforming DON into the non-toxic 3-epi-DON [START_REF] Zhai | Detoxification of Deoxynivalenol by a Mixed Culture of Soil Bacteria With 3-epi-Deoxynivalenol as the Main Intermediate[END_REF]. Likewise, a de-epoxidase first isolated from Eubacterium isolated from bovine rumen fluid, is capable of converting DON into the nontoxic compound DOM-1 (Pierron et al. 2016c;[START_REF] Loi | Mycotoxin Biotransformation by Native and Commercial Enzymes: Present and Future Perspectives[END_REF].

Patulin was converted by Saccharomyces cerevisiae into two isomers of ascladiol, E-ascladiol and Z-ascladiol which are nontoxic to human cell lines [START_REF] Tannous | Patulin transformation products and last intermediates in its biosynthetic pathway, E-and Z-ascladiol, are not toxic to human cells[END_REF][START_REF] Wang | Diversity-oriented synthesis based on the brevianamide scaffold for the development of physiologically active compounds, "PhD dissertaion[END_REF].

Detoxification of fumonisin B1 was carried out in two steps. Initial step was de-esterification reaction followed by deamination of the resulting hydrolyzed fumonisin B1 [START_REF] Heinl | Degradation of fumonisin B1 by the consecutive action of two bacterial enzymes[END_REF][START_REF] Grenier | The low intestinal and hepatic toxicity of hydrolyzed fumonisin B₁ correlates with its inability to alter the metabolism of sphingolipids[END_REF]. Furthermore, isothiocyanates are natural reactive compounds that were found to reduce the toxicity of some mycotoxins [START_REF] Varga | Chemical, Physical and Biological Approaches to Prevent Ochratoxin Induced Toxicoses in Humans and Animals[END_REF]). In addition, yeast cell wall, probiotics, prebiotics, fibers and protein ingredients have been used to reduce mycotoxins bioaccessibility in the gastrointestinal tract [START_REF] Mallebrera | Influence of pro-and prebiotics on gastric, duodenal and colonic bioaccessibility of the mycotoxin beauvericin[END_REF]) (Figure 1).

Control should begin as early as possible, starting at the farm level with primary agricultural production. Good agricultural practices (GAP) is the first line against contamination of cereals with mycotoxins [START_REF] Awad | Decontamination and detoxification strategies for the Fusarium mycotoxin deoxynivalenol in animal feed and the effectiveness of microbial biodegradation[END_REF]. Furthermore, preventive strategies in storage level such as; storage at low moisture levels and prevention of grain damage during processing and prevention of the formation of mycotoxins in feed help to reduce mycotoxins content [START_REF] Dawson | The application of yeast and yeast derivatives in the poultry industry[END_REF].

Co-contamination

Another issue that pays attention of researchers is the co-contamination of food and feed by several mycotoxins. Food commodities are commonly contaminated with various fungal species at a time. In addition, most fungi are able to simultaneously produce number of mycotoxins. Therefore feed commodities can be contaminated by several mycotoxins, and completed feed is made from various commodities (Streit et al. 2012). The simultaneous presence of mycotoxins in products intended for human consumption is of high importance, because mycotoxins could interact with each other, potentially enhancing their toxic effects [START_REF] Ruiz | Toxicological interactions between the mycotoxins beauvericin, deoxynivalenol and T-2 toxin in CHO-K1 cells in vitro[END_REF]). Maize is an example where several mycotoxins have been reported to occur simultaneously [START_REF] Gonçalves | Report on the 2016 Proficiency Test of the European Union Reference for Mycotoxins[END_REF].

There are reports of a combination of many mycotoxins, such as DON, AF (B1, B2 and M1), FUM (A, B1, B2 and B3), ZEN and other fungal secondary metabolites in maize seeds and Washing grains, as well as in animal feed formulated (Streit et al. 2013;Anjorin et al. 2016;Abdallah et al. 2017). DON co-occurs with other regulated mycotoxins as well as with emerging mycotoxins [START_REF] Borutova | Co-occurrence and statistical correlations between mycotoxins in feedstuffs collected in the Asia-Oceania in 2010[END_REF]FVaclavikova et al. 2013;Streit et al. 2013). Multimycotoxin studies have reported that 75%-100% animal feed was co-contaminated by DON and other mycotoxins (Streit et al. 2012;[START_REF] Kovalsky | Co-Occurrence of Regulated, Masked and Emerging Mycotoxins and Secondary Metabolites in Finished Feed and Maize-An Extensive Survey[END_REF]Novak et al. 2019). In food and feed ingredients sourced in Southern Europe, DON was found in 94% of the samples at maximum 365 µg/kg concentration (total number of samples 'n' = 416) [START_REF] Griessler | Translational Research in Pharmacology and Toxicology Using Precision-Cut Tissue Slices[END_REF].

Deoxynivalenol (DON)

Origin Deoxynivalenol (DON) is a regulated mycotoxin belonging to the group of type B trichothecenes (Knutsen et al. 2017), mainly produced by the fungi of the Fusarium genus, [START_REF] Lewczuk | Histological structure of duodenum in gilts receiving low doses of zearalenone and deoxynivalenol in feed[END_REF]. DON is one of the most common contaminants of wheat, corn, and barley worldwide. Second, DON is a very stable compound, during both storage and the processing/cooking of food, and does not degrade at high temperatures. On the other hands, DON is chemically also stable and to some extent resistant to food and feed processing (Table 2) (Wood 1992;[START_REF] Kabak | The fate of mycotoxins during thermal food processing[END_REF]. DON occurs in cereal grains alone or in combination with its most relevant acetylated derivatives, such as 3-acetyl-DON, 15-acetyl-DON and DON-3glucoside (10-20% of the DON-levels) and either with other fungal secondary metabolites so called emerging mycotoxins [START_REF] Eskola | Worldwide contamination of food-crops with mycotoxins: Validity of the widely cited 'FAO estimate' of 25%[END_REF]Schatzmayr and Streit 2013). It is produced in fields prior to harvest and its occurrence in food cannot be completely avoided due to the major impact of weather conditions as well as the high chemical and thermal resistance, both during storage/milling and processing/cooking of food [START_REF] Lewczuk | Histological structure of duodenum in gilts receiving low doses of zearalenone and deoxynivalenol in feed[END_REF]. DON is an undesirable substance in animal feed; in particular pigs were identified as the most sensitive animal species (EFSA 2013). In addition, DON can affect all animal species, with the following rank order of sensitivity: pigs > mice > rats > poultry ≈ ruminants [START_REF] Rotter | Invited Review: Toxicology of Deoxynivalenol (Vomitoxin)[END_REF].

Occurrence

DON is one of the most widely distributed trichothecene [START_REF] Sprando | Characterization of the effect of deoxynivalenol on selected male reproductive endpoints[END_REF]. It contaminates grains and cereal-based food and feed such as wheat, barley, oats, rye and maize, and less often in rice, sorghum and triticale [START_REF] Kabak | The fate of mycotoxins during thermal food processing[END_REF]. A worldwide ten years survey on more than 70,000 different commodities reported that DON was found in finished feed (70%), maize (67%), maize dried distillers grains with soluble (DDGS) (83%), maize silage (62%), soybean grains

2016).

Toxicity

DON is one of the least lethal trichothecenes, but at high dose, acute exposure to DON elicits abdominal distress, increased salivation, malaise, diarrhea, necrosis of bone marrow, lymphoid tissue and both kidney and heart lesion [START_REF] Pestka | Deoxynivalenol: mechanisms of action, human exposure, and toxicological relevance[END_REF][START_REF] Ruiz | Toxicological interactions between the mycotoxins beauvericin, deoxynivalenol and T-2 toxin in CHO-K1 cells in vitro[END_REF]. Based on adverse gastrointestinal effects of DON on Chinese people, the CONTAM identified that vomiting occurred within 30 minutes after an eating occasion and a non-observed adverse effect level (NOAEL) for acute effect was calculated, 26 µg DON/kg b.w for a single eating occasion. The highest acute dietary exposure to DON was identified in the young children and infants. Human outbreaks from acute exposure to DON have been repeatedly reported in Asia. Indeed, vomiting is a critical acute effect of DON in humans (Knutsen et al. 2017). The evidence of adverse health effects in humans due to chronic exposure to DON is lacking, but the CONTAM panel has identified reduced body weight gain in experimental animals as the critical chronic effect for human risk assessment.

Furthermore, the CONTAM Panel calculated a lowest observed adverse effect level (LOAEL)

and NOAEL of DON for pig. It is noted that naturally contaminated feed had a stronger effect on the feed intake and weight gain than pure DON, and this lead to the assumption that other toxic compound are present [START_REF] Efsa | Opinion of the Scientific Panel on Contaminants in the Food Chain on a request from the Commission related to Deoxynivalenol (DON) as undesirable substance in animal feed[END_REF]. Overall, NOAELs for reduced feed intake and reduced body weight gain (0.7-5.0 mg DON/kg feed) was observed overlapping with even a wider range of LOAELs (0.35-13 mg DON/kg feed) (Knutsen et al. 2017). Impaired immune response, reproductive, neurological, hematological, and molecular effects are also reported from in vivo or in vitro studies [START_REF] Sobrova | Deoxynivalenol and its toxicity[END_REF]. Previous findings have shown that, if the concentration of DON increases over than 12.5 mg/kg feed, it causes feed refusal and vomiting in animals (Fink-Gremmels 1999). However, the oral bioavailability and absorption of DON in animals depends on several parameters including species, age and gender. For example after oral ingestion, within 15-30 min, 7 % of DON was detected in ruminants (sheep and cow), 25

% in rat's, and up to 89 % in pig's blood [START_REF] Goyarts | Bioavailability of the Fusarium toxin deoxynivalenol (DON) from naturally contaminated wheat for the pig[END_REF]Payros et al. 2016). A major part of the ingested DON in pigs was absorbed quickly from the proximal segments of the small intestine [START_REF] Eriksen | Toxicological evaluation of trichothecenes in animal feed[END_REF][START_REF] Dänicke | On the effects of deoxynivalenol (DON) in pig feed on growth performance, nutrients utilization and DON metabolism[END_REF]). In mice, DON was rapidly distributed to the tissues, e.g. liver, kidney, spleen and heart, following oral exposure to a 25 mg/kg b.w dose, reaching the maximum concentrations at about the same time as in plasma.

Concentration of DON in liver was (12.1 -19.6 µg/g,), in kidney (7.6 -9.0 µg/g,) and in spleen was observed (7.9 µg/g) respectively [START_REF] Pestka | Immunochemical assessment of deoxynivalenol tissue distribution following oral exposure in the mouse[END_REF]. Since nutrients are absorbed in the small intestine, the gastrointestinal tract is the first barrier against food contaminants and it may be exposed to high concentration of mycotoxins (Pinton and Oswald 2014;Alassane-Kpembi et al. 2015;Fraeyman et al. 2018).

Effects of DON were assessed on human intestinal epithelium using in vitro approach and on porcine intestinal epithelium using in vitro and in vivo approaches. DON decreased the transepithelial electrical resistance (TEER) in a time and dose-dependent manner in Caco- DON decreased by 40% the amount of the tight junction protein claudin-4 (CLDN-4), when pigs received DON contaminated feed (2.85 mg/kg DON/kg feed) during 5 weeks compared to the control diet. Overall, the porcine intestinal epithelial cells IPEC-1 showed more sensitivity than human cells Caco-2 (Pinton et al. 2009).

Mode and mechanism of action

Concerning the mode of action, DON binds to ribosomes, leading to a ribotoxic stress and the inhibition of protein synthesis and subsequently also RNA and DNA synthesis [START_REF] Sobrova | Deoxynivalenol and its toxicity[END_REF]. This ribotoxic stress also activates different mitogen-activated protein kinases (MAPKs). Activation of MAPKs explains several effects of DON, such as apoptosis or survival of cells, inflammatory effect and oxidative stress. Two major mediators of DON-induced anorexia/emesis have been described, pro-inflammatory cytokines and secretion of satiety hormones, which activate receptors in the abdominal vagus afferent (Knutsen et al. 2017).

Furthermore, DON regulates tight junction proteins such as CLDN through signaling molecules as activation of MAPKs (Ghareeb et al. 2015).

Regulation

Exposure to DON usually originates from the consumption of contaminated plant commodities, but might occur also via a secondary route following the consumption of meat, milk and eggs, containing residual amounts of mycotoxins ingested by food-producing animal (Fink-Gremmels 1999). The highest exposure of DON comes from grain-based products, especially 'bread and rolls', 'fine bakery wares' and 'pasta (raw) (Knutsen et al. 2017) 3 andTable 4).

Fungal toxic secondary metabolites (Emerging Mycotoxins)

Emerging mycotoxins are fungal secondary metabolites that are neither routinely determined, nor legislatively regulated [START_REF] Vaclavikova | Emerging' mycotoxins in cereals processing chains: Changes of enniatins during beer and bread making[END_REF]. Emerging Mycotoxins are usually coproduced with other well-known mycotoxins [START_REF] Hussein | Toxicity, metabolism, and impact of mycotoxins on humans and animals[END_REF]. The most relevant and frequently occurring emerging mycotoxins are Fusarium toxins including Enniatins (ENNs), Beauvericin (BEA), Apicidin (API), Aurofusarin (AFN), Moniliformin and Fusaproliferin (Gruber-Dorninger et al. 2016;Jajić et al. 2019). ENNs and BEA belong to a group of cyclic hexadepsipeptides, AFN is a dimeric naphthoquinone, and API is a cyclic tetra peptide [START_REF] Frandsen | The biosynthetic pathway for aurofusarin in Fusarium graminearum reveals a close link between the naphthoquinones and naphthopyrones[END_REF][START_REF] Niehaus | Apicidin F: Characterization and Genetic Manipulation of a New Secondary Metabolite Gene Cluster in the Rice Pathogen Fusarium fujikuroi[END_REF]Rodríguez-Carrasco et al. 2016). Furthermore, other fungal species such as Alternaria, Aspergillus and Penicillium produce some emerging mycotoxins such as Brevianamid-F (BRV-F), Emodin (EMO) and Cyclo-Pro-L-Tyr (Cyclo) (Streit et al. 2013). In addition, Acremonium which is perceived to be a heterogeneous taxon, also produce some emerging mycotoxins such as Tryptophol (TRPT) (Glenn et al. 1996).

As these mycotoxins have only been discovered over the last few decades, they are to date poorly investigated (Springler et al. 2016b). Although recent sensitive analytical methods via LC-MS/MS has assisted the discovery of new fungal secondary metabolites (Malachová et al. 2014), but still the toxicology, toxicokinetics and toxicodynamics data of these metabolites are fragmentary [START_REF] Taevernier | Enniatin-containing solutions for oromucosal use : Quality-by-design ex-vivo transmucosal risk assessment of composition variability[END_REF].

Emerging mycotoxins can occur in high frequency and sometimes also in high concentrations in cereals and in other grain-based products (Rodríguez-Carrasco et al. 2016;Gruber-Dorninger et al. 2016). In addition, their presence was reported in mold of water-damaged houses in milder climate regions such as North America and Western Europe [START_REF] Taevernier | Blood-brain barrier transport kinetics of the cyclic depsipeptide mycotoxins beauvericin and enniatins[END_REF]. Thus, emerging mycotoxins gain growing interest due to their rapidly increasing presence across the food chain [START_REF] Lucioli | The food contaminant deoxynivalenol activates the mitogen activated protein kinases in the intestine: Interest of ex vivo models as an alternative to in vivo experiments[END_REF]Fraeyman et al. 2018). Although emerging mycotoxins are concerned, maximum permitted levels may not be proposed in the immediate future. This is primarily due to the lack of data related to their occurrence, contamination level, and toxicity.

In order to better assess the risk of these mycotoxins, regular surveillance is a prerequisite to understand their significance as natural contaminants in human and animal nutrition [START_REF] Jestoi | Emerging Fusarium Mycotoxins Fusaproliferin, Beauvericin, Enniatins, And Moniliformin-A Review[END_REF]). Among them, the ten most prevalent emerging mycotoxins in feed are presented.

Enniatins (ENNs)

Origin

ENNs (A, A-1, B and B-1) are cyclic hexadepsipeptides (Table 5) secondary metabolites that have been known since few decades [START_REF] Ivanova | Emodin -a secondary metabolite with multiple ecological functions in higher plants[END_REF] [START_REF] Kamyar | Investigation of the electrophysiological properties of enniatins[END_REF][START_REF] Fraeyman | Emerging Fusarium and Alternaria Mycotoxins: Occurrence, Toxicity and Toxicokinetics[END_REF]. Naturally occurring ENNs commonly consist of three d-2-hydroxycarboxylic acid residues linked alternatively to three l-N-methyl-amino acid residues (Table 5) [START_REF] Uhlig | Beauvericin and enniatins A, A1, B and B1 in Norwegian grain: a survey[END_REF]. Polar organic solvents (e.g., aqueous methanol, ethanol, chloroform) DMP, DMSO and poor solubility in water Chemical structure and characteristics of ENNs (ENN-A, A1, B and B1) [START_REF] Uhlig | Beauvericin and enniatins A, A1, B and B1 in Norwegian grain: a survey[END_REF][START_REF] Escrivá | Quantitation of enniatins in biological samples of Wistar rats after oral administration by LC-MS/MS[END_REF].

Occurrence

ENNs were detected in food (63%, n= 4,251), feed (32%, n = 3,640) and unprocessed grains (76%, n = 2,647) samples collected between 2010 and 2013 in 12 EU countries (EFSA 2014).

ENNs (A, A-1, B and B-1) were also detected in more than 90% of feed and feed raw materials (n=83) with maximum concentrations of 1,745 μg/kg, 2,216 μg/kg, 780 μg/kg and 2,690 μg/kg respectively (Streit et al. 2013). In pig feed (n=1,141, worldwide) samples, ENNs (A, A-1, B and B-1) were found in 50%, 77%, 82% and 82% with maximum concentration of 307 μg/kg, 549 μg/kg, 1,514 μg/kg and 1,846 μg/kg respectively. ENN-B and ENN-B1 were the most prevalent than of ENN-A and ENN-A1 as well the concentrations of ENN-B and ENN-B1 were higher than of ENN-A and ENN-A1 (Novak et al. 2019). ENNs (A, A-1, B and B-1) were also found in Egyptian feed (87%, 91%, 93% and 91%) samples (n=77) and maize (32%, 35%, 44% and 28%) samples (n=79) respectively. Concentration of ENNs was higher in feed (1.9 -25 µg/kg) rather than maize (<1 µg/kg). Since feeds are composed of several raw materials, therefore compound feeds might be higher contaminated than maize (Abdallah et al. 2017).

ENNs (A, A-1, B and B-1) were also found in Finland's grain samples (wheat, barley, rye and oats, n=38) collected in 2001 and 2002. The incidence of positive samples and the concentrations of ENNs were quite high in both years, especially for ENN-B and B1 that were detected in all samples (maximum concentrations of 18,300 μg/kg and 5,720 μg/kg respectively). ENN-A and A1 were detected in 68 % of the samples with the highest levels of 950 μg/kg and 2,000 μg/kg respectively [START_REF] Jestoi | Presence and concentrations of the Fusarium-related mycotoxins beauvericin, enniatins and moniliformin in finnish grain samples[END_REF]). ENN-B was also found in 70 % of the baby food samples at levels up to 1,100 µg/ kg and in 44 % of pasta samples at levels of up to 106 µg/kg while other authors reported contamination rates of between 50 -90 % of wheat, maize and barley samples with total concentrations of ENNs of up to 500 mg/kg [START_REF] Juan | Evaluation of beauvericin and enniatins in Italian cereal products and multicereal food by liquid chromatography coupled to triple quadrupole mass spectrometry[END_REF].

Toxicity

ENNs possess a wide range of biological properties, such as; cytotoxicity, hemolysis, permeability and skin damage and decrease of TEER. ENNs have ionophoric properties and can form complexes with cations (Kolf-Clauw et al. 2013;[START_REF] Taevernier | Human skin permeation of emerging mycotoxins (beauvericin and enniatins)[END_REF][START_REF] Fraeyman | Comparative Oral Bioavailability, Toxicokinetics, and Biotransformation of Enniatin B1 and Enniatin B in Broiler Chickens[END_REF]Springler et al. 2016b;[START_REF] Olleik | Comparative Structure-Activity Analysis of the Antimicrobial Activity, Cytotoxicity, and Mechanism of Action of the Fungal Cyclohexadepsipeptides Enniatins and Beauvericin[END_REF]. Cytotoxicity of ENNs (A, A1, B and B1) was assessed during 24 h incubation in both proliferating and differentiated IPEC-J2 cells. ENNs were ranked in the following order of decreasing toxicity ENN-A > > ENN-A1 > ENN-B1> > >ENN-B respectively (Fraeyman et al. 2018). In similar cell line, after 48 h incubation all ENNs (A, A1, B and B1) showed almost similar absolute IC50 values of 3.40 µM, 4.15 µM, 3.25 µM and 3.67 µM respectively (Novak et al. 2019). In a similar time of exposure, cytotoxic effect of ENNs (0 to 100 µM) were studied in different cell types of human origin such as Caco-2, human normal lung epithelial cells (BEAS-2B), human normal epidermal keratinocytes (HEK), human liver cell line HepG2, human normal endothelial cells (HUVEC) and human gastric cell line (N87). Effect of ENNs in different cell types was dissimilar (Table 6). Among them, N87 cells were more sensitive than other cell lines [START_REF] Olleik | Comparative Structure-Activity Analysis of the Antimicrobial Activity, Cytotoxicity, and Mechanism of Action of the Fungal Cyclohexadepsipeptides Enniatins and Beauvericin[END_REF]. Furthermore, hemolytic activity of ENNs was assessed on human erythrocytes. According to their hemolytic activity, ENNs were ranked in the following order of toxicity; ENN-A> ENN-A1> ENN-B1> ENN-B respectively [START_REF] Olleik | Comparative Structure-Activity Analysis of the Antimicrobial Activity, Cytotoxicity, and Mechanism of Action of the Fungal Cyclohexadepsipeptides Enniatins and Beauvericin[END_REF].

In another study performed on Caco-2 cells, ENN-B decreased cell viability from 2.5 to 10 µM up to 30% 24 h whereas at 48 and 72 h it decreased the viability from 1.25 to 10 µM up to 50%

and over 90% respectively. The obtained IC50 were of 9.2 µM, 6.9 µM and 5.09 µM after 24 h, 48 h and 72 h respectively (Fernández-Blanco et al. 2016). For tumor cells (neuronal origin), ENN-B was highly toxic. In a short term treatment (8 h) and at a very low concentration, ENN-B (0.1-1 µM) had tumor promoting functions based on growth stimulation [START_REF] Dornetshuber | Enniatin Exerts p53-Dependent Cytostatic and p53-Independent Cytotoxic Activities against Human Cancer Cells[END_REF]).

Taking into account the above results, ENNs are toxic in vitro, whereas the in vivo studies that have been carried out in rodent and mice, have shown very low or no toxicity. Oral doses of 0.5-1 g/kg body weight (b.w) per day over 6 days to mice and single oral doses of up to 50 mg/kg b.w/day in rats did not produce toxic effects (Kolf-Clauw et al. 2013). In a study of (Rodríguez-carrasco et al. 2016), 5 mg/kg b.w ENN-B administrated to the mice for 9 days did not affect body weight, food intake and behavior of animals. In another study, 20.91 mg/kg b.w/day of ENN-A was administrated in the feed to a 2-month-old female Wistar rats, during the 28 day experiment and no adverse effect were seen in organ weight and histology of duodenum [START_REF] Manyes | A preliminary study in Wistar rats with enniatin A contaminated feed[END_REF]. In similar treatment period 465 mg/kg feed, ENN-A was assessed in rats, no significant difference in feed intake and no gross illness was observed [START_REF] Juan | Toxicon Evaluation of immunologic effect of Enniatin A and quantitative determination in feces , urine and serum on treated Wistar rats[END_REF].

Concerning the metabolism of ENNs, it depends on the species or compound. A toxicokinetic study was performed in one piglet, in order to investigate the simultaneous detection and quantification of ENN-A, A1, hB and B1 in animal plasma. The pig received a single oral intra-gastric bolus of 0.05 mg/kg b.w of all these mycotoxins including BEA. Blood samples were collected before (0 min) and at 10, 20, 30 and 40 min, 1, 1.5, 2, 3, 4, 6, 8, 10 and 12 h postadministration. The authors claimed that despite of their similar chemical structure, there was a big difference in oral absorption between the different ENNs. ENN-B seemed to have the highest oral absorption, followed by ENN-B1, A1 and finally ENN-A. The maximal plasma concentrations (Cmax) for ENN-B, B1, A1 and A, were 73.4, 35.2, 11.6 and 6.8 After IV administration, ENN-B1 was distributed and eliminated in accordance with oral administration [START_REF] Devreese | Pilot toxicokinetic study and absolute oral bioavailability of the Fusarium mycotoxin enniatin B1 in pigs[END_REF]).

Beauvericin (BEA)

Origin

BEA is a cyclic hexadepsipeptide (Table 7) synthesized by several Fusarium species, including F. subglutinans, F. proliferatum, F. oxysporum, F. moniliforme, F. anthophilum and F.

beauvrecin [START_REF] Fotso | Evaluation of Beauvericin Toxicity with the Bacterial Bioluminescence Assay and the Ames Mutagenicity Bioassay[END_REF]. BEA was firstly isolated from Beauveria bassiana [START_REF] Hamill | The structure op beauvericin, a new depsipeptide antibiotic toxic to Artemia salina[END_REF]) and reported for its insecticidal properties and its toxicity to Artemia salina [START_REF] Randazzo | Proliferin, a new sesterterpene from Fusarium proliferatum[END_REF]. BEA is known to exert a broad spectrum of biological effects such as, antibiotic, anti-inflammatory activities, as well as anticancer effects in various cancer cell lines [START_REF] Heilos | The Natural Fungal Metabolite Beauvericin Exerts Anticancer Activity In Vivo: A Pre-Clinical Pilot Study[END_REF]). Soluble in: Polar organic solvents (e.g., aqueous methanol, ethanol, chloroform) DMP, DMSO and poor solubility in water Chemical structure and characteristics of BEA [START_REF] Logrieco | Beauvericin Production by FusariumSpecies[END_REF][START_REF] Liuzzi | Enniatin and Beauvericin Biosynthesis in Fusarium Species: Production Profiles and Structural Determinant Prediction[END_REF].

Occurrence

BEA was reported in food (80%, n=732), feed (79%, n=861) and unprocessed grains (46%, n=554) samples collected between 2010 and 2013 in 12 EU countries. For food, the highest mean concentrations were measured in dried fruits (29.5 µg/kg), followed by oilseeds (8.86 µg/kg) and cereal-based food for infants and young children (8 µg/kg). For feed and unprocessed grains, BEA was present in maize gluten (EFSA 2014). In a worldwide survey of pig feed, BEA was reported in 68% of total samples (n=1,141) with maximum concentration of 413 µg/kg (Novak et al. 2019). BEA was found in high prevalence (98%, n=83) with maximum concentration of 2,326 µg/kg in feed and feed raw materials collected in EU (Streit et al. 2013).

Furthermore, occurrence of BEA was reported from different countries in various commodities.

BEA was found 33-95% in several food commodities, such as wheat based products, rice, maize based products and barley based products, collected in Italy, Spain, Morocco and Tunisia (n=265), with maximum concentrations 844 µg/kg, 57.4 µg/kg, 73.9 µg/kg and 82 µg/kg respectively [START_REF] Serrano | Co-occurrence and risk assessment of mycotoxins in food and diet from Mediterranean area[END_REF]. In Italian corn (n=94) and oat (n=7) samples BEA was found 27-57% in maximum concentrations 41 µg/kg [START_REF] Ritieni | Occurrence of Fusaproliferin, Fumonisin B1, and Beauvericin in Maize from Italy[END_REF][START_REF] Juan | Evaluation of beauvericin and enniatins in Italian cereal products and multicereal food by liquid chromatography coupled to triple quadrupole mass spectrometry[END_REF]. All the wheat samples (n=13), from Finland and all the rice samples (n=70) from morocco were contaminated by BEA with maximum concentration 3,500 µg/kg and 12,810 µg/kg respectively. Even, 33% of baby food (n=68) were contaminated by BEA with maximum concentration of 10,600 µg/kg in Morocco (Logrieco et al. 2002b;[START_REF] Meca | Further data on the presence of Fusarium emerging mycotoxins enniatins, fusaproliferin and beauvericin in cereals available on the Spanish markets[END_REF][START_REF] Sifou | First report on the presence of emerging Fusarium mycotoxins enniatins (A, A1, B, B1), beauvericin and fusaproliferin in rice on the Moroccan retail markets[END_REF]). In addition, BEA was reported in Egyptian maize (63%, n=79) and feed (88%, n=77).

Concentration of BEA in feed (88 µg/kg) was higher than maize (63 µg/kg) (Abdallah et al. 2017).

Toxicity

Acute exposure to BEA is not a concern to human health (EFSA 2014). However, in human and mammalian cell lines, BEA demonstrated toxic effects as it induces apoptosis, increases cytoplasmic calcium concentration and leads to DNA fragmentation (Logrieco et al. 2002a;[START_REF] Luz | Occurrence, toxicity, bioaccessibility and mitigation strategies of beauvericin, a minor Fusarium mycotoxin[END_REF]. The toxicity of BEA is primarily regarded to its ionophoric properties that alters the normal physiological concentrations of cations in and out of cells, acting as an ion carrier through the plasma membrane [START_REF] Hilgenfeld | Structural chemistry of natural and synthetic inoophores and their complexes with cations[END_REF][START_REF] Fotso | Evaluation of Beauvericin Toxicity with the Bacterial Bioluminescence Assay and the Ames Mutagenicity Bioassay[END_REF]. It also induces lipid peroxidation and alters kinetics in heart metabolism [START_REF] Ruiz | Toxicological interactions between the mycotoxins beauvericin, deoxynivalenol and T-2 toxin in CHO-K1 cells in vitro[END_REF].

Cytotoxic effect of BEA was assessed on different human cell lines: Caco-2, BEAS-2B, HEK, HepG2, HUVEC and N87 for 48 h. They were ranked based on the following order of decreasing sensitivity: HUVEC> HepG2> Caco-2> HEK> BEAS-2B>N87, with IC50 of 2.4 µM> 3.4 µM> 3.9 µM> 5.4 µM> 6.3 µM> 27.5 µM respectively [START_REF] Olleik | Comparative Structure-Activity Analysis of the Antimicrobial Activity, Cytotoxicity, and Mechanism of Action of the Fungal Cyclohexadepsipeptides Enniatins and Beauvericin[END_REF]). In addition, BEA revealed toxic effect on human fibroblast-like cell line (MRC-5) After 24 h, the IC50 was of 5.0 µM and 1.1 µM via Alamar Blue TM and BrdU assay respectively [START_REF] Ivanova | Emodin -a secondary metabolite with multiple ecological functions in higher plants[END_REF]. Furthermore, a toxicogenomic study was performed to investigate gene expression changes triggered by BEA exposure (1.5, 3 and 5 μM) during 24 h in Jurkat cells, a human lymphoblastic T cell line, through RNA-sequencing and differential gene expression analysis.

The results demonstrated BEA-induced mitochondrial damage affecting the respiratory chain and pointing to apoptosis through the caspase cascade. The most significantly altered pathways genes, involved in the respiratory chain, were significantly down-regulated. These results bring greater relevance to mitochondria as a target site for BEA induced cytotoxicity in cellular models (Escrivá et al. 2018).

Nevertheless, BEA is identified toxic in vitro, but is reported to be not toxic in vivo. Toxicity and pharmacological behavior of BEA was investigated in mice during 9 days, no effect on body weight, food intake and behavior was observed at 5 mg/kg b.w (Rodríguez-carrasco et al.

2016

). However, in a brain influx study performed in mice, BEA crossed the blood-brain barrier [START_REF] Taevernier | Blood-brain barrier transport kinetics of the cyclic depsipeptide mycotoxins beauvericin and enniatins[END_REF]. Therefore, no conclusion could be drawn with respect to chronic exposure due to the lack of relevant in vivo toxicity data.

Metabolization of BEA was studied in pig. A toxicokinetic study was performed in one piglet, in order to investigate detection and quantification of BEA in plasma. The pig received a single oral intra-gastric bolus of 0.05 mg/kg b.w of BEA along with ENNs (ENN-A, A1, B and B1).

Blood samples were drawn before (0 min) and at 10, 20, 30 and 40 min, 1, 1.5, 2, 3, 4, 6, 8, 10 and 12 h post-administration. The authors claimed that for BEA, no plasma concentration-time profile was designed as the plasma concentrations were above the limit of quantification (LOQ) level at only 2 time points, i.e. 0.51 ng/mL at 30 min and 0.82 ng/mL at 40 min postadministration. Thus, BEA revealed a lower oral bioavailability compared to ENNs [START_REF] Devreese | Quantitative determination of the Fusarium mycotoxins beauvericin, enniatin A, A1, B and B1 in pig plasma using high performance liquid chromatography-tandem mass spectrometry[END_REF].

Aurofusarin (AFN)

Origin

AFN is a dimeric naphthoquinone belonging to the naphthoquinone group of polyketides (Table 8) [START_REF] Frandsen | The biosynthetic pathway for aurofusarin in Fusarium graminearum reveals a close link between the naphthoquinones and naphthopyrones[END_REF]. AFN is a yellow-red pigment produced by Fusarium head-blight disease caused by several species of Fusarium, such as F. graminearum, F. culmorum, and F. crookwellense [START_REF] Kim | GIP2, a Putative Transcription Factor That Regulates the Aurofusarin Biosynthetic Gene Cluster in Gibberella zeae[END_REF]. Physical state Dependent on the pH value of the solvent, ranging from golden yellow in acidic solvents to red/purple in alkaline solvents Soluble in: Polar organic solvents (e.g., aqueous methanol, ethanol, chloroform) DMP, DMSO and poor solubility in water Chemical structure and characteristics of AFN (Tanaka and Tamura 1961).

Occurrence

AFN has been identified in feed ingredients in particular in contaminated grains and known as a new crop pollutant [START_REF] Dvorska | Protective effect of modified glucomannans against aurofusarin-induced changes in quail egg and embryo[END_REF]. AFN was found in feed raw materials 84% (n=83, Europe), finished feed 80.7% (n=1,141, worldwide), 73% (n=77, Egypt) and maize 9% (n=79, Egypt) with maximum concentration of 17,659 µg/kg, 85,360 µg/kg, 3,005 µg/kg and 1,858 µg/kg respectively (Streit et al. 2013;Abdallah et al. 2017;Novak et al. 2019). In addition, AFN was found in settled dust of Polish poultry farms (n=13), among 27 other detected chemicals, with the highest concentration of 281.44 µg/kg [START_REF] Skóra | Evaluation of Microbiological and Chemical Contaminants in Poultry Farms[END_REF].

Toxicity

AFN was investigated on several endpoints of toxicological importance such as cytotoxicity, genotoxicity and oxidative stress. AFN at 10 µM significantly decreased the cellular proliferation of colon adenocarcinoma cell line (HT29) and non-tumorigenic colon cells (HCEC-1CT) after 72 h of exposure at 12% and 7% respectively. Exposure to AFN 5 μM for 24 h significantly reduced mitochondrial activities of HT29 and HCEC-1CT (24 % and 23 %), whereas AFN 10 μM reduced up to 30 % and 40% respectively. Furthermore, the genotoxic impact of AFN (5 μM and 10 μΜ) after 1 h was investigated in the comet assay. AFN significantly increased a tail intensity of DNA damage. The authors claimed significant DNA damage indicating the potential involvement of other mechanisms in the genotoxic impact of AFN. Overall, AFN was found to be cytotoxic in both tumorigenic and non-tumorigenic colon cells [START_REF] Jarolim | The secondary Fusarium metabolite aurofusarin induces oxidative stress, cytotoxicity and genotoxicity in human colon cells[END_REF]. Likewise, AFN 10 µM reduced the mitochondrial activity of Caco-2 cells [START_REF] Dvorska | Effect of the mycotoxin aurofusarin on the antioxidant composition and fatty acid profile of quail eggs[END_REF]). AFN showed similar potential toxicity as DON, in terms of mitochondrial activity determined by the WST-1 assay. Although the results obtained with 1 and 5 µM has indicated that AFN may show a steeper dose response curve compared to DON [START_REF] Vejdovszky | Non-synergistic cytotoxic effects of Fusarium and Alternaria toxin combinations in Caco-2 cells[END_REF].

AFN was either toxic in vivo to poultry and reduced the nutritional quality of quail meat and eggs. A dose 26.4 mg AFN /kg feed administrated to 45 days old quails caused a significant decrease of vitamins A and E, total carotenoid, lutein and zeaxanthin concentrations in the meat and significantly increased egg yolk susceptibility to lipid peroxidation. AFN also compromised immune function of laying chickens and quail and decreased fertility and hatchability [START_REF] Dvorska | Protective effect of modified glucomannans against changes in antioxidant systems of quail egg and embryo due to aurofusarin consumption[END_REF]. Accumulation of AFN in egg yolk stimulated lipid peroxidation directly, causing a decrease in antioxidant concentration [START_REF] Dvorska | Aurofusarin, a newly described Fusarium graminearum mycotoxin: oxidative stress and protective effect of MycosorbTM[END_REF]) and also caused a significant decrease in vitamins A and E and fatty acids in egg yolk [START_REF] Kim | Functional Characterization of Genes Located at the Aurofusarin Biosynthesis Gene Cluster in Gibberella zeae[END_REF].

Another possibility proposed to explain the detrimental effect of AFN on antioxidants is a disruption of nutrients absorption in intestine [START_REF] Dvorska | Aurofusarin, a newly described Fusarium graminearum mycotoxin: oxidative stress and protective effect of MycosorbTM[END_REF].

In spite of a great variety of toxic effects, the molecular mechanisms of action are poorly understood and information about AFN is still limited [START_REF] Dvorska | Effect of the mycotoxin aurofusarin on the antioxidant composition and fatty acid profile of quail eggs[END_REF]).

Apicidin (API)

Origin

API is a cyclic tetrapeptide built up of four amino acids; N-methoxy-L-tryptophan, Lisoleucine, D-pipecolic acid, and L-2-amino-8-oxodecanoic acid (Table 9) produced by some Fusarium species [START_REF] Niehaus | Apicidin F: Characterization and Genetic Manipulation of a New Secondary Metabolite Gene Cluster in the Rice Pathogen Fusarium fujikuroi[END_REF]. It was firstly isolated in 1996 from F. pallidoroseum (later known as F. semitectum) as a new agent showing histone deacetylase inhibiting activity in apicomplexan parasites such as Plasmodium berghei [START_REF] Singh | Apicidins: Novel Cyclic Tetrapeptides as Coccidiostats and Antimalarial Agents from Fusarium paUidoroseum[END_REF][START_REF] Von Bargen | Structure Elucidation and Antimalarial Activity of Apicidin F: An Apicidin-like Compound Produced by Fusarium fujikuroi[END_REF]. Derivatives of API have been identified, but their structure, mode of action and biological activities are almost similar. The most frequent derivatives of APIs are API-E, API-D2 and API-B [START_REF] Jin | Functional characterization and manipulation of the apicidin biosynthetic pathway in Fusarium semitectum[END_REF]. Polar organic solvents (e.g., aqueous methanol, ethanol, chloroform) DMP, DMSO and poor solubility in water Chemical structure and characteristics of API [START_REF] Singh | Apicidins: Novel Cyclic Tetrapeptides as Coccidiostats and Antimalarial Agents from Fusarium paUidoroseum[END_REF].

Occurrence

API was found in feed raw materials 66% (n=83 Europe), finished feed 52% (n=1,141 worldwide) and 17% (n=77 Egypt) with maximum concentrations of 160 µg/kg, 1,568 µg/kg and 5.4 µg/kg respectively (Streit et al. 2013;Abdallah et al. 2017;Novak et al. 2019).

Toxicity

API is known as a potential toxic agent [START_REF] Zhang | Apicidin Inhibited Proliferation and Invasion and Induced Apoptosis via Mitochondrial Pathway in Non-small Cell Lung Cancer GLC-82 Cells[END_REF]. API revealed strongest cytotoxic effect on IPEC-J2 with an absolute IC50 value of 0.52 μM during 48 h (Novak et al. 2019). In the same cell line, API 2.5 µM decreased the TEER over 93%, after 24 h of exposure (Springler et al. 2016b). API inhibited proliferation of different human cancer cell lines (MCF-7, a human breast cancer cell; HBL-100, a human breast cancer cell line, HeLa, a human cervix cancer cell line; and AGS, a human gastric adenocarcinoma cell line) dose dependently. The half maximal effect (IC50) were calculated; 1.17, 0.57, 0.51 and 0.13 µM after 48 h respectively. In the same study, API showed toxicity in mouse cancer cell lines (v-ras-transformed NIH3T3, a mouse fibroblast cell line and Colon 3.1-M26, a mouse colon carcinoma cell line). The IC50 were obtained 0.18 and 0.17 µM respectively [START_REF] Han | Apicidin, a histone deacetylase inhibitor, inhibits proliferation of tumor cells via induction of p21WAF1/Cip1 and gelsolin[END_REF]. Human pancreatic cancer cell lines (Capan-1 and Panc-1) were exposed to API for 48 h. For concentrations above 100 nM, the cytotoxicity and a sustained loss of cell viability were significantly increased (Bauden et al. 2015). API showed potent inhibitory activity against lung carcinoma (GLC-82) cells with the IC50 value of 6.94 μM after 48 h. API 14.0 μM induced apoptosis via mitochondrial pathway in GLC-82 cells by 7.2 %, 16.6 %, 26.4 % and 37.8 %, after 12 h, 24 h, 36 h, and 48 h respectively [START_REF] Zhang | Apicidin Inhibited Proliferation and Invasion and Induced Apoptosis via Mitochondrial Pathway in Non-small Cell Lung Cancer GLC-82 Cells[END_REF]. API was incorporated into complete rat diets during 14 days at concentrations of 0.5 and 1 g/kg feed and fed to 21 day-old female rats. API induced toxic effects including body weight loss, hemorrhage in the stomach, intestine and bladder and finally death. API at 0.5 g/kg caused death after 10 to 14 days of exposure and at 1 g/kg animal died within 7 days [START_REF] Park | A hemorrhagic factor (apicidin) produced by toxic Fusarium isolates from soybean seeds[END_REF].

Emodin (EMO)

Origin

EMO, an orange-red crystalline compound, is a 1,3,8-trihydroxy-6-methylanthraquinone, extracted from Aspergillus wentii isolated from weevil-damaged Chinese chestnuts (Table 10) [START_REF] Wells | Emodin, a Toxic Metabolite of Aspergillus wentii Isolated from Weevil-Damaged Chestnuts[END_REF]. It belongs to the anthraquinones group, mainly produced by several Aspergillus species, including A. wentii, A. flavus and A. ochraceus [START_REF] Nandani | Isolation and identification of quercetin and emodin from Cassia tora L[END_REF]Streit et al. 2013). Chemical structure and characteristics of EMO [START_REF] Wells | Emodin, a Toxic Metabolite of Aspergillus wentii Isolated from Weevil-Damaged Chestnuts[END_REF].

Occurrence

EMO was originally reported as being more common in bark and roots, but it is clear now that EMO occurs in vegetative organs (stem, foliage) as well in reproductive organs (flower, fruit, seeds and pods). EMO is occurring in tropical and subtropical plant families as well in the plant families from temperate regions (Izhaki 2002). EMO was found in 89% sample of feed and feed raw material (n=83, Europe), finished feed 69% (n=1141, worldwide) and 92% (n=79, Egypt)

and maize 57% (n=77, Egypt) with maximum concentrations of 1,570 µg/kg, 1,170 µg/kg, 76 µg/kg and 66.6 µg/kg respectively (Streit et al. 2013;Abdallah et al. 2017;Novak et al. 2019).

Toxicity

The toxicity of EMO has been very poorly investigated either in vitro or in vivo. Cytotoxic effect of EMO was assessed on the viability of IPEC-J2 over 48 h. The maximum toxicity of EMO was reached up to 50 µM and IC50 was obtained 13.09 µM (Novak et al. 2019). The cytotoxic effect of EMO was assessed on mouse embryos at the blastocyst stage, subsequent embryonic attachment and outgrowth in vitro, and in vivo implantation by embryo transfer.

Blastocysts treated with EMO 25-75 µM exhibited significantly increased apoptosis and a corresponding decrease in total cells number. Furthermore, EMO in drinking water of mouse led to apoptosis and decreased cell proliferation in embryo and inhibited early embryonic development to the blastocyst stage [START_REF] Chang | Emodin induces embryonic toxicity in mouse blastocysts through apoptosis[END_REF]. EMO was also toxic, when it was given orally to redwing blackbirds (Agelaius phoeniceusin) and starlings (Sternus vulgaris). The oral administration of EMO to cockerels caused moderate diarrhea and mortality within 5 days of ingestion (LD50 = 3.7 mg/ kg) [START_REF] Wells | Emodin, a Toxic Metabolite of Aspergillus wentii Isolated from Weevil-Damaged Chestnuts[END_REF]).

Brevianamide-F (BRV-F)

Origin

BRV-F is a precursor of the biosynthetic pathway of fumitremorgins and the tryprostatins, produced by Penicillium brevicompactum, Aspergillus versicolor and A. fumigatus (Table 11) [START_REF] Frisvad | Metabolomics of Aspergillus fumigatus[END_REF]. Polar organic solvents (e.g., aqueous methanol, ethanol, chloroform) DMP, DMSO Chemical structure and characteristics of BRV-F [START_REF] Wilson | Production, Isolation, and Preliminary Toxicity Studies of Brevianamide A from Cultures of Penicillium viridicatum[END_REF].

Occurrence

BRV-F was found in feed and as well in feed raw materials. The proportion of the contaminated samples were 65.2% (n= 1,141, worldwide), 69% (n=83, Europe), 86% (n= 77, Egypt), maize 6% (n= 79, Egypt) and processed cassava 79.6% (n=373, Nigeria) with maximum concentration of 1,170 µg/kg, 2,043 µg/kg, 353 µg/kg , 5 µg/kg and 44 µg/kg respectively (Novak et al. 2019;Streit et al. 2013;Abdallah et al. 2017;[START_REF] Abass | Occurrence of Regulated Mycotoxins and Other Microbial Metabolites in Dried Cassava Products from Nigeria[END_REF].

Toxicity

The toxic effect of BRV-F is poorly investigated. In a recent study, effect of BRV-F was examined on the viability of IPEC-J2 cells over 48 h, but did not show negative effect up to 150 µM (Novak et al. 2019). Likewise, a preliminary toxicity study of BRV (BRV-A3) showed no acute toxicity in mice upon oral or intraperitoneal administration. However, the molecule induced cytotoxicity and inflammatory lung response in intratracheally exposed mice (Wauters 2015).

Cyclo-(L-Pro-L-Tyr) (Cyclo)

Origin

Cyclic dipeptide Cyclo-(L-Pro-L-Tyr) is a diketopiperazine formed by the fusion of tyrosine and proline (Table 12), reported as a secondary metabolite of fungi [START_REF] Capon | Citromycetins and Bilains A-C: New Aromatic Polyketides and Diketopiperazines from Australian Marine-Derived and Terrestrial Penicillium spp[END_REF]. Cyclo was firstly isolated from Alternaria alternata decades ago [START_REF] Stierle | Maculosin, a host-specific phytotoxin for spotted knapweed from Alternaria alternata[END_REF]. Chemical structure and characteristics of Cyclo [START_REF] Holden | Quorum-sensing cross talk: isolation and chemical characterization of cyclic dipeptides from Pseudomonas aeruginosa and other Gram-negative bacteria[END_REF].

Occurrence

Occurrence of Cyclo is reported in finished feed 87.6% (n=1,141 worldwide), 100% (n=77, Egypt) and maize 43% (n=79, Egypt) with a maximum concentration of 34,910 µg/kg, 4,244 µg/kg and 21 µg/kg respectively (Abdallah et al. 2017;Novak et al. 2019).

Toxicity

Toxicity of Cyclo is not reported yet. In a recent study, effect of Cyclo was assessed up to 150 µM on the viability of IPEC-J2 over 48 h, but was not toxic (Novak et al. 2019).

Tryptophol (TRPT)

Origin TRPT (indole-3-ethanol) is an aromatic alcohol (Table 13) produced by Acremonium species and formed as the end product of tryptophan catabolism [START_REF] Kosalec | Genotoxicity of Tryptophol in a Battery of Short-Term Assays on Human White Blood Cells in vitro[END_REF]Streit et al. 2013).

TRPT was firstly isolated from cucumber seed as a plant growth regulator [START_REF] Rayle | Isolation and Identification of Indole-3-Ethanol (Tryptophol) from Cucumber Seedlings[END_REF]. However, it is an endogenous plant constituent and growth regulator, but is also produced as a secondary metabolite by certain bacteria, yeast and fungi (Kosalec et al. 2011).

Occurrence

The occurrence of TRPT is reported in feed and feed raw materials. TRPT was found in 75% (n=1,141) of animal feed collected worldwide, with maximum concentration of 10,270 µg/kg (Novak et al. 2019). Another study, carried out on Egyptian animal feed and maize, reported the presence of TRPT in 90% (n=77) and 16% (n=79) of the samples at concentrations of 10,878 µg/kg and 101 µg/kg respectively (Abdallah et al. 2017). In addition, occurrence of TRPT was reported in 71% (n=83, Europe) of feed and feed raw materials samples in mean concentration of 267 µg/kg (Streit et al. 2013).

Toxicity

TRPT was not cytotoxic in vitro for IPEC-J2 exposed to 150 µM for 48 h (Novak et al. 2019).

Whereas, in vivo studies have reported that TRPT is a highly lipophilic compound that freely penetrates cell membranes. TRPT was injected into laboratory mammals and chicken and induced a sleep-like state and lethargy accompanied with alteration of body temperature. (Kosalec et al. 2011). When administered to laboratory animals, it was rapidly distributed to the brain and other tissues. TRPT also readily penetrates into lymphoid tissues and its immunosuppressive effects have been observed in mice [START_REF] Ackerman | The effects of tryptophol on immune responses and its implications toward trypanosome-induced immunosuppression[END_REF]. TRPT induces apoptosis in human lung (lymphoblast) U937 cell line via activation of caspase-8 followed by caspase-3 [START_REF] Inagaki | Tryptophol Induces Death Receptor (DR) 5-Mediated Apoptosis in U937 Cells[END_REF]. Despite interesting biological functions, the overall toxicity profile of TRPT has not yet been adequately explained. Only a few studies have dealt with mutagenicity and genotoxicity of TRPT (Kosalec et al. 2011).

Structure and function of the small intestine

The intestine 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 (Pierron et al. 2016a). It is the first barrier to food contaminants. Following the ingestion of mycotoxin contaminated food or feed, intestinal epithelial cells can be exposed to high concentrations of toxicants, potentially affecting intestinal functions (Pinton and Oswald 2014;[START_REF] Bouhet | The effects of mycotoxins, fungal food contaminants, on the intestinal epithelial cell-derived innate immune response[END_REF]. Indeed, the intestine is a privileged immune site, where immunoregulatory mechanisms simultaneously defend the body against pathogens, but also preserve tissue homeostasis to avoid immune mediated pathology in response to environmental challenges (Pinton and Oswald 2014).

The small intestine is a specialized abdominal tubular structure in length of 4 to 7 m (Thomson and Shaffer 1992) and its epithelium is a single-cell layer that constitutes the largest and most important barrier against the external environment. The intestinal epithelium has two critical functions: (i) it acts as a selective filter, allowing the translocation of essential dietary nutrients, electrolytes, and water from the intestinal lumen into the circulation; (ii) it acts as a barrier to prevent the passage of harmful intraluminal entities, including foreign antigens, microorganisms, and their toxins [START_REF] Groschwitz | Intestinal barrier function: Molecular regulation and disease pathogenesis[END_REF]. These functions are supported by the unique structure of layers of the gastrointestinal tract (GT). The GT is divided into several layers: the mucosa, the submucosa, the muscularis propria, and the serosa (Figure 2) [START_REF] Rao | Intestinal Architecture and Development. Morgan & Claypool Life Sciences 217. RASFF[END_REF]). The mucosa consists of an epithelial lining, a laminal propria of loose connective tissue, and the muscularis mucosae, which is composed of two layers of smooth muscle fibers, separating the mucosa from the submucosa. The submucosa is a layer of irregular loose connective tissue, containing large blood and lymphatic vessels, glands, lymphatic tissue, and a submucous (Meissner's) nerve plexus. The muscularis externa is formed of thick bundles of smooth muscle fibers arranged into two sublayers: circular in the internal sub layer and longitudinal in the external sublayer. Serosa is a thin layer of loose connective tissue. It is continuous with adjacent tissue in the esophagus. Serosa replace the adventitia in the gastrointestinal tract, which is composed of a layer of mesothelium and the underlying loose connective tissue, rich in blood and lymphatic vessels and adipose cells [START_REF] Zhang | Digestive System[END_REF].

Mucosa is also referred to as a mucous membrane, as mucus production is a characteristic feature of gut epithelium. The membrane consists of epithelium, which is in direct contact with ingested food, and the lamina propria, a layer of connective tissue analogous to the dermis [START_REF] Betts | Anatomy & Physiology[END_REF]. The small intestinal mucosa is constructed to act first as an absorptive surface and second as a barrier to potentially pathogenic substances and microorganisms. The intestinal epithelium is composed of absorptive cells such as enterocytes and protective cells, such as goblet cells, paneth cells and endocrine cells(Table 14) [START_REF] Greaves | Histopathology of Preclinical Toxicity Studies: Interpretation and Relevance in Drug Safety Evaluation[END_REF]. The normal gastrointestinal tract is a finely integrated system geared to carry out the assimilation of ingested foodstuffs. Assimilation (the process by which ingested foods reach body fluids and cells) takes place in the small intestine [START_REF] Thomson | First principles of gastroenterology[END_REF]. Thus, the intestine is potentially highly exposed to the food contaminants (Pinton and Oswald 2014).

In order to investigate the effect of food contaminants such as mycotoxins in the intestine, [START_REF] Goodwin | In vitro assays of angiogenesis for assessment of angiogenic and anti-angiogenic agents[END_REF].

several
The toxic effect of DON and emerging mycotoxins on intestinal cells using in vitro model are summarized in Tables 15 and16. This part is mainly focusing on in vitro studies, thus in vivo and ex vivo has not been included.

Structure and function of the liver

The porcine liver consists of five lobes, the left lateral and medial, right lateral and medial, and caudate lobes (Figure 3) [START_REF] Mulaikal | Liver anesthesiology and critical care medicine[END_REF]. The external anatomy is described from gross landmarks including the gallbladder, the vena cava and the hepatic ligaments. The internal anatomy is defined by the vascular structures and eight functionally independent segments, each segment with its individual blood supply and biliary drainage [START_REF] Mulaikal | Liver anesthesiology and critical care medicine[END_REF]Sibulesky, 2013). Moreover, the liver is composed of a rich population of specialized cells that allow it to carry out complex functions. They can be grossly characterized as "parenchymal" cells (hepatocytes) and "nonparenchymal cells" [START_REF] Mulaikal | Liver anesthesiology and critical care medicine[END_REF]. The parenchymal cells, make up 60 -80% of liver cells and carry out the metabolic, detoxification, and synthetic functions of the liver, such as metabolism of amino acids, fats, carbohydrates, vitamins, byproduct wastes, and toxins (Table 17). Whereas the nonparenchymal cells makes 20 -40% of the liver [START_REF] Sheth | The liver as an immune organ[END_REF][START_REF] Racanelli | The liver as an immunological organ[END_REF]. The liver is a unique anatomical and immunological site in which antigen-rich blood from the gastrointestinal tract is pressed through a network of sinusoids and scanned by antigen presenting cells and lymphocytes [START_REF] Racanelli | The liver as an immunological organ[END_REF]. Of the total hepatic blood flow (100-130 ml/min per 100 g of liver, 30 ml/min/kg/b.w), only one fifth to one third is supplied by the hepatic artery. The rest of blood about two thirds is supplied by portal vein.

This blood contains oxygen and many nutrients brought to the liver from the intestines for processing [START_REF] Lautt | Hepatic Circulation: Physiology and Pathophysiology[END_REF]. Any blood antigen or contaminant can target the liver [START_REF] Sheth | The liver as an immune organ[END_REF]. Liver is also the main site of detoxification for xenobiotics, including (CYP1, CYP2 and CYP3). Phase II reactions are conjugation reactions, for example with glucuronic acid, sulfate, glutathione and/or amino acids [START_REF] Slobodchikova | Characterization of Phase I and Glucuronide Phase II Metabolites of 17 Mycotoxins Using Liquid Chromatography-High-Resolution Mass Spectrometry[END_REF]. The majority of mycotoxin biomonitoring is performed using urine since it is non-invasive and accessible in relatively large volumes. These methods of analysis can be performed with and without B-glucuronidase treatment. B-glucuronidase catalyzes hydrolysis of conjugated mycotoxins, such as sulfate and glucuronide conjugates. Thus, the use of enzymatic hydrolysis can provide an appropriate alternative to direct metabolite monitoring for at least those mycotoxins which are predominately metabolized to phase II conjugated forms such as DON [START_REF] Vidal | Humans significantly metabolize and excrete the mycotoxin deoxynivalenol and its modified form deoxynivalenol-3-glucoside within 24 hours[END_REF].

The liver's lymphocyte population is selectively enriched in natural killer T cells which play critical roles in first line immune defense against invading pathogens, modulation of liver injury and recruitment of circulating lymphocytes [START_REF] Racanelli | The liver as an immunological organ[END_REF]. The high local blood flow results in a high rate of interaction with foreign antigens, and confers immunologic responsibility on the liver. Because the liver acts as a detoxification center for the body, it can be regenerated if injured [START_REF] Sheth | The liver as an immune organ[END_REF]. Lastly, the liver is the site of cholesterol synthesis and therefore crucial in the genesis of endogenous steroid hormones such as cortisol, aldosterone, and testosterone. While these hormones are synthesized in the adrenal gland, their precursors have an hepatic origin [START_REF] Shen | Sex Hormones and Their Receptors Regulate Liver Energy Homeostasis[END_REF].

Despite the similarity of digestive physiology between human and pig, their liver are different from the morphological point of view. Human liver consists of four lobes, the left, right, quadrate, and caudate lobes. Whereas, porcine liver consists of five lobes (Figure 3). Another difference is that inferior vena cava is divided into supra-and infra-hepatic in humans, whereas in porcine dissection between the liver and vena cava is very difficult [START_REF] Kim | Anatomy of the pig[END_REF]. [START_REF] Mulaikal | Liver anesthesiology and critical care medicine[END_REF] We have reviewed the effects of DON and emerging mycotoxins on liver and hepatic cells extensively in vitro and in vivo (Tables 18, 19 and20). Their acute and chronic dietary exposures can induce a variety of adverse health effects in humans and animals [START_REF] Eskola | Worldwide contamination of food-crops with mycotoxins: Validity of the widely cited 'FAO estimate' of 25%[END_REF]. International enquiry's on existing mycotoxins legislation in foodstuffs and animal feedstuffs have been carried out several times (FAO 2004).

Nevertheless, so far, only few mycotoxins are regulated worldwide. In European Union, only DON, ZEN, ochratoxin A, patulin, FUM, T-2 and HT-2 toxins are regulated [START_REF] Lerda | Mycotoxins Factsheet-4th edition[END_REF]). In addition to those regulated mycotoxins, other fungal secondary metabolites have been discovered which are neither routinely determined, nor legislatively regulated and have been defined as emerging mycotoxins [START_REF] Vaclavikova | Emerging' mycotoxins in cereals processing chains: Changes of enniatins during beer and bread making[END_REF]. They are usually co-produced with other well-known toxins [START_REF] Eskola | Worldwide contamination of food-crops with mycotoxins: Validity of the widely cited 'FAO estimate' of 25%[END_REF][START_REF] Hussein | Toxicity, metabolism, and impact of mycotoxins on humans and animals[END_REF].

The toxicity of mycotoxins when present together, cannot always be predicted based upon their individual toxicities. Multi-exposure may lead to additive, synergistic or antagonistic toxic effects (Alassane- Kpembi et al. 2015). Therefore, analysis of the combined toxicity of mycotoxins is highly important. However, at the present very little is known about the health risk from combined exposure to mycotoxins.

In this study, we aimed to provide additional information about the occurrence of emerging mycotoxins and their combined effects when present in mixture with DON. DON is one of the most widely distributed type B trichothecene contaminants found in food and animal feed [START_REF] Sprando | Characterization of the effect of deoxynivalenol on selected male reproductive endpoints[END_REF], whereas emerging mycotoxins have been identified but less investigated so far [START_REF] Vaclavikova | Emerging' mycotoxins in cereals processing chains: Changes of enniatins during beer and bread making[END_REF]. In order to do so, pig finished feed samples were collected worldwide market. Then combined toxicity of DON and the 10 most prevalent emerging mycotoxins was assessed on porcine intestinal cell line (IPEC-1), using their concentration observed in the feed.

The second objective of this thesis is driven by the fact that the intestine is not the only target for food contaminants. Indeed, liver which is the main site of metabolism and detoxification of xenobiotics is also a target organ for mycotoxins [START_REF] Zain | Impact of mycotoxins on humans and animals[END_REF]. Therefore, we developed a tool, called precision cut liver slices (PCLS), in order to assess the toxicity of mycotoxins on this ex vivo model and the tests performed during this thesis were focused on DON.

Contribution of this doctoral thesis

Among food/feed contaminants, mycotoxins have been regularly classified in the top ten and top one causes of alert notification for RASFF in EU members and non-members countries respectively (RASFF 2018). So far, numerous studies have been carried out on the toxicity of well-known mycotoxins such as DON, ZEN, FUM, AFs whereas, emerging mycotoxins are still poorly investigated.

So far, most of the studies on the occurrence of mycotoxins are performed in cereal grains and raw materials. However, presence and concentration of mycotoxins in compound feed is not always similar as in raw materials (Streit et al. 2012). It is important to investigate mycotoxins in compound and finished feed. Furthermore, most of the studies and surveys on the occurrence of mycotoxins are limited to one region or one country (Streit et al. 2013;Abdallah et al. 2017).

Occurrence of mycotoxins and contamination levels across the globe is different and it is important to have a global view on the worldwide occurrence of mycotoxins in feed samples.

The aim of this study is to investigate the occurrence of emerging and regulated mycotoxins in finished pig feed worldwide.

Among farm animals, pig is one of the most sensitive species to the deleterious effects of mycotoxins. It can be exposed to high concentration of mycotoxins due to its rich cereal diets (Pinton et al. 2010). Following ingestion of contaminated feed, the gastrointestinal tract is the first physiological barrier against food contaminants, as well as the first target for these toxicants (Pinton and Oswald 2014).

The toxicity of known mycotoxins are quite well investigated, the knowledge on the potential toxicity of emerging mycotoxins is still scarce. Therefore, this study aimed at investigating and characterizing the potential toxicity of the 10 most prevalent and DON co-occurring emerging mycotoxins using a model of porcine intestinal epithelial cells. The risk for human and animal health related to the presence of combinations of mycotoxins should not be neglected. In this context, most of the published studies on mixtures have focused on iso-toxicity or combination of identical ratios (1:1), regardless of their ratios in food or feed [START_REF] Ficheux | Co-exposure of Fusarium mycotoxins: In vitro myelotoxicity assessment on human hematopoietic progenitors[END_REF]Prosperini et al. 2014b;Fernández-Blanco et al. 2016) . To the best of our knowledge, there is no study investigating the combined toxicity based on the ratios concerning the occurrence in the food or feed. Herein, this study will investigate combined toxicity of emerging mycotoxins and DON based on the actual ratios determined in pig finished feed worldwide.

Some of the mycotoxins such as DON can cross the intestinal barrier, reach into the blood circulation and finally reach to the liver. Of the total hepatic blood flow (100-130 ml/min per 100 g of liver, 30 ml/min/kg/b.w), only one fifth to one third is supplied by the hepatic artery.

The rest of blood (about two thirds) is supplied by portal vein. This blood contains oxygen and many nutrients brought to the liver from the intestines for processing [START_REF] Lautt | Hepatic Circulation: Physiology and Pathophysiology[END_REF]. Therefore, not only the intestine can be a target of mycotoxins, the liver can be also a target organ to mycotoxins. Most of the published studies on the effect of DON on liver are performed either in vivo or in vitro [START_REF] Nielsen | Potential of deoxynivalenol to induce transcription factors in human hepatoma cells[END_REF][START_REF] Sahu | Comparative hepatotoxicity of deoxynivalenol in rat, mouse and human liver cells in culture[END_REF]Wu et al. 2014a;[START_REF] Gerez | Deoxynivalenol alone or in combination with nivalenol and zearalenone induce systemic histological changes in pigs[END_REF].

Although, both models are used in experimentation, but none of them are considered an ideal model. Since in vitro assays do not allow assessing more than one cell at a time and in vivo assays require large number of animals and is quite costly. In the context of reducing the number of experimental animals, ex vivo models represent a powerful tool as many samples can be obtained from one animal (Kolf-Clauw et al. 2013). In the ex vivo models, the cytoarchitecture should be retained as well as many of the intercellular connections and interplays.

In order to investigate the toxicity of mycotoxins in liver, the precision cut liver slices (PCLS), ex vivo explants with well-defined thickness represent a promising model [START_REF] Graaf Ia De | Preparation and incubation of precisioncut liver and intestinal slices for application in drug metabolism and toxicity studies[END_REF].

They contain all cell types of the tissue in their natural environment and natural architecture, with intercellular and cell-matrix intact interactions [START_REF] Zimmermann | Improved reproducibility in preparing precision-cut liver tissue slices[END_REF]). To the best of our knowledge, PCLS have never been used to investigate the toxicity of mycotoxins (Graaf et al. 2010). This study will aim developing this new tool in order to assess the toxicity of mycotoxins using an ex vivo model of liver explants.

Article

Co-Occurrence of DON and Emerging Mycotoxins in Worldwide

Finished Pig Feed and Their Combined Toxicity in Intestinal Cells

Introduction

Mycotoxins are low molecular weight fungal secondary metabolites that trigger a detrimental response when ingested by humans and animals. They are mainly produced by filamentous fungi belonging to Aspergillus, Fusarium and Penicillium species [1]. Mycotoxin contamination can occur all along the food chain from field to storage, including the food process. This depends upon the requirements of fungi, and Fusarium mostly occurs in the field, whereas Aspergillus and Penicillium mostly occurs during storage. Because of their toxicity and occurrence, deoxynivalenol (DON), zearalenone, aflatoxins, ochratoxin A, patulin, fumonisins and T-2/HT-2 toxins are regulated in Europe. For example, the maximum recommendations limits that are set up for complete piglet feed are 0.9, 0.1, 0.05, 5 and 0.25 mg/kg feed for DON, zearalenone, ochratoxin, fumonisins and T2 + HT2, respectively [2,3]. However, in addition to regulated mycotoxins, many other fungal secondary metabolites are being identified in food and feed [4,5]. Metabolites that are neither routinely determined, nor legislatively regulated, have been defined as 'emerging mycotoxins' [6], while the derivatives of regulated mycotoxins that are undetectable using conventional analytical techniques due to their modified structure, are defined as 'modified/masked mycotoxins' [4,7]. Recent findings showed that more than 70% of the world's cereal grains are contaminated by mycotoxins [8,9], often in a mixture [10].

Among regulated mycotoxins, DON very frequently contaminates cereals (wheat, barley, oats, rye and maize, and less frequently rice, sorghum and triticale) and cereal-based food and feed. DON belongs to the group of B-trichothecenes, and is one of the most widely distributed contaminants in human food and animal feed. In a total of more than 25,000 samples collected from 28 European countries between 2007 and 2014, DON was found in 47% of 4000 feed samples and 45% of 1621 unprocessed grains with no defined end use, respectively [11]. Even though DON is considered as a non-carcinogenic compound [12], the maximum level of this toxin in food and feed have been set up in different countries. For example, in complete piglet feed, the maximum limits are 0.9, 1 and 5 mg/kg feed in Europe, Canada and the USA, respectively [2,13]. Exposure to high concentrations of DON is associated with diarrhea, vomiting (emesis), leukocytosis and gastrointestinal bleeding. Chronic exposure affects growth, immunity and intestinal barrier function in animals [14][15][16]. This toxin interacts with the peptidyl transferase region of the 60S ribosomal subunit, inducing 'ribotoxic stress', resulting in the activation of mitogen-activated protein kinases (MAPKs) and their downstream pathways [14,17].

Among emerging mycotoxins, those that occur most frequently are enniatins (ENNs), beauvericin (BEA), apicidin (API), aurofusarin (AFN), culmorin, butenolide, fusaric acid, moniliformin, fusaproliferin and emodin (EMO). They are produced by Fusarium species except EMO, which is produced by Aspergillus species [6,18]. ENNs and BEA were detected in food (63% and 80%), feed (32% and 79%), and unprocessed grains (24% and 46%) collected between 2010 and 2014 in 12 European countries [19]. AFN, API, brevianamide-F (BRV-F), EMO and tryptophol (TRPT) were also found in pig feed (80%, 52%, 65%, 63% and 75%) [20], Egyptian animal feed (73%, 17%, 86%, 98% and 90%) [21] and feed raw materials (84%, 55%, 5%, 74%, 59%) [22].

Multiple mycotoxins are frequently present in food and feed [10]. The co-occurrence of DON, aflatoxins, fumonisins, zearalenone and other fungal secondary metabolites in maize seeds and grains, as well as in animal feed, has been reported [21][22][23]. The presence of different fungi on the same raw material, the ability of fungal species to produce several toxins, as well as the various commodities present in completed feed, can explain this multiple contamination [24,25]. Compound feed is particularly prone to multiple contaminations, as it typically contains a mixture of several raw materials.

The co-occurrence of mycotoxins is challenging for at least two reasons: (i) The toxicity of mycotoxins when present together cannot always be predicted based upon their individual toxicity and (ii) the risk assessment is performed on a chemical-by-chemical basis [24,25]. Scientific interest in the toxicity of these mixtures of mycotoxins is currently increasing rapidly [26][27][28][29]. Several studies have investigated the combined toxicity of regulated mycotoxins on the intestine [30][31][32][33], but the combined effect of regulated and other mycotoxins is poorly documented [34,35].

Among farm animals, pig is one of the most sensitive species to mycotoxins [36]. As feed raw materials are potentially contaminated by several fungi at a time, and completed feed is made from various commodities, pig can be exposed, through its rich cereal diet, to high concentrations of mixtures of mycotoxins [10,37]. The sanitary and economic losses due to mycotoxin contamination are important in the pig industry, even if they are hard to estimate precisely [38]. The aims of this study were thus to (i) determine the prevalence and concentration of mycotoxins present in finished pig feed and (ii) assess the intestinal combined toxicity of DON in mixture with the 10 most prevalent emerging mycotoxins present in pig feed using realistic ratios.

Results

Occcurrence and Abundance of Emerging Mycotoxins and DON in Finished Pig Feed

A total of 524 finished pig feed samples collected worldwide were analyzed, and more than 235 different metabolites were detected, including regulated mycotoxins, emerging mycotoxins and modified/masked mycotoxins. Table 1 lists the 60 most prevalent fungal metabolites that contaminated more than 20% of the finished pig feed samples. Among regulated mycotoxins, DON was detected in 463 samples (88%), mostly in the Northern Hemisphere and in relatively similar concentrations in samples from all countries (median concentration 206 µg/kg) (Figure 1A,B). All DON-contaminated samples were co-contaminated by other mycotoxins. The distribution of the samples was checked. Table 1 gives the descriptive statistics for DON and the most abundant metabolites that co-occur with DON. The 60 mycotoxins found in more than 20% of the 524 samples of finished pig feed. Their concentrations in the three quartiles (P25, P50 and P75) are expressed in µg/kg of feed. The correlation of their concentration with DON and the associated P-value was calculated using Spearman's rank correlation coefficient.

From this list, the most prevalent emerging mycotoxins co-occurring with DON and which were commercially available were selected for the toxicological studies. Because of its high toxicity [39],

Toxins 2019, 11, 727 5 of 17 apicidin (API) was included in the list. The worldwide distributions of these compounds are presented in Supplementary Figure S1. Except for API, which was detected in only 67% of DON-contaminated samples, the selected emerging mycotoxins were present in more than 87% of them (Table 1). Three compounds API, emodin (EMO) and beauvericin (BEA) were detected in a median concentration range of 5 to 10 µg/kg feed. The median concentration of four metabolites, enniatins A1, B, B1 (ENN-A1, B, B1) and brevianamide F (BRV-F), was in the range of 15-40 µg/kg feed, and the last three compounds aurofusarin (AFN), cyclo-(L-Pro-L-Tyr) (Cyclo) and tryptophol (TRPT) had median concentrations close to 200 µg/kg feed, like DON (Supplementary Figure S2). Despite their high co-occurrence with DON, with the exception of AFN, and to a lesser extent BEA, the concentration of these mycotoxins showed limited correlation with DON concentration (Table 1 and Supplementary Figure S3).

Intestinal Toxicity of Emerging Mycotoxins Found in Pig Feed, alone or Combined with DON

Individual Toxicity of DON and Emerging Mycotoxins

The individual intestinal toxicity of 10 selected emerging mycotoxins, as well as that of DON, was first analyzed at a wide range of concentrations (Supplementary Figure S4). As shown in Supplementary Figure S4, all tested metabolites exhibited dose-dependent toxicity toward intestinal epithelial cells, except BRV-F, Cyclo and TRPT, that were not toxic for this porcine cell line. AFN, EMO and ENN-B1 reduced the viability of IPEC-1, but their toxicity was less than that of DON. The toxicity of BEA and ENN-B was close to that of DON, whereas API and ENN-A1 were more toxic than DON. As shown in Figure 2, low doses of API (0.01-0.3 µM) significantly stimulated the proliferation of IPEC-1. When the doses leading to a 50% reduction in the cell viability (IC 50 ) of these emerging mycotoxins were compared with the dose of DON, then BRV-F, Cyclo and TRPT were classified as non-toxic metabolites, while EMO, AFN and ENN-B1 were given as moderately toxic metabolites, and finally API, ENN-A1, ENN-B and BEA as highly toxic metabolites (Table 2). Data are the mean ± the standard error of the mean (SEM) of three biological replicates.

Combined Toxicity of DON and Emerging Mycotoxins

Next, the combined toxicity of DON and the selected emerging mycotoxins was assessed. As mentioned above, the concentration of these secondary metabolites was not correlated with the concentration of DON (Table 1 and Supplementary Figure S3). Thus, to account for the different situations to which pigs can be exposed, three ratios were tested (Supplementary Table S1). Ratio 1 was calculated using the P25 (1st quartile) concentration of the emerging mycotoxin and P75 (3rd quartile) concentration of DON. Ratio 2 was calculated using the median (2nd quartile) concentration of DON and each emerging mycotoxin. Ratio 3 was calculated using the P75 concentration of the emerging mycotoxin and P25 concentration of DON. For each ratio, serial dilutions were tested to obtain a dose-effect curve that encompassed the realistic concentrations of the mixture of DON and the tested metabolites.

Combined Toxicity of DON and the Non-Toxic Secondary Metabolites (BRV-F, Cyclo and TRPT)

First, the combined toxicity of DON and the 'non-toxic' secondary metabolites BRV-B, Cyclo and TRPT were analyzed. As shown in Figure 3, whatever the ratios tested, the toxicity of the combination of DON and the compound being tested was similar to the toxicity of DON alone.

Combined Toxicity of DON and the Moderately Toxic Secondary Metabolites (AFN, EMO and ENN-B1)

Next, the combined toxicity of DON and the moderately toxic metabolites AFN, EMO and ENN-B1 was assessed (Figure 4). The toxicity of AFN, EMO and ENN-B1 was minimal when used at ratio 1. In these conditions, the toxicity of the combination of DON and emerging toxins was similar to the toxicity of DON alone. Ratio 2 reached toxic concentrations of AFN and ENN-B1, but the toxicity of the mixture was similar to the toxicity of DON alone, except at the highest concentration of ENN-B1 (4.1 µM), where the toxicity of the mixture (ENN-B1 4.1 µM + DON 50 µM) was higher than the toxicity of ENN-B1 alone, but lower than the toxicity of DON alone. When cytotoxicity was tested at ratio 3, the toxicity of AFN + DON was identical to the one of DON alone except at the concentrations DON 0.6 µM + AFN 1.6 µM and DON 1.9 µM + AFN 4.7 µM, when the toxicity of the mixture was slightly lower than the toxicity of DON alone. At this ratio, EMO alone was still not toxic, and even induced proliferation (up to 130% of treated cells).

The combined toxicity of DON and EMO was similar to the toxicity of DON alone except at the highest concentration of EMO, when the mixture of DON (50 µM) and EMO (5 µM) was still less toxic than DON alone. The combined toxicity of ENN-B1 + DON was the same as the toxicity of DON alone, except at the highest concentrations of ENN-B1 (6 and 18 µM), when the toxicity of the mixture was the same as the toxicity of ENN-B1 alone, but lower than the toxicity of DON alone.

In conclusion, our data showed that the toxicity of the combination of DON and emerging mycotoxins such as AFN, EMO and ENN-B1 was similar to or lower than the toxicity of DON alone, whatever the ratio used. The combined toxicity of DON and highly toxic compounds (ENN-B, BEA, ENN-A1 and API) was also analyzed at different ratios (Figure 5). At the first tested ratio (ratio 1), ENN-B, BEA, ENN-A1 and API were not toxic, and their combined toxicity in the presence of DON was similar to the toxicity of DON alone. Ratio 2 reached toxic concentrations of ENN-B, ENN-A1 and API. of DON (50 µM) + ENN-B (3.6 µM) was similar to the toxicity of ENN-B alone, but lower than the toxicity of DON alone, whereas the combined toxicity of DON (50 µM) + ENN-A1 (1.7 µM) was higher than the toxicity of ENN-A1 alone, but still lower than the toxicity of DON alone. The combined toxicity of API and DON was similar to the toxicity of DON only for the higher doses. For the doses lower than DON (5.6 µM) + API (0.09 µM) the combined toxicity was lower than the toxicity of DON alone, but higher than API alone.

At ratio 3, the combined toxicity of DON and ENN-B displayed a different characteristic. At a high concentration, the combined toxicity of DON (50 µM) + ENN-B (17.2 µM) was lower than the toxicity of either DON alone or ENN-B alone. At this ratio, the toxicity of the mixture of DON and BEA was similar to the toxicity of DON alone, except at the highest concentration of BEA (2.3 µM) when mixed with DON (50 µM), where the toxicity was higher than that of BEA alone, but lower than that of DON alone. The combined toxicity of DON (16.7µM) + ENN-A1 (2.2 µM) at ratio 3 was similar to the toxicity of ENN-A1 alone, but lower than the toxicity of DON alone; the toxicity of DON (50 µM) + ENN-A1 (6.5 µM) was higher than the toxicity of ENN-A1 alone, but lower than the toxicity of DON alone. Finally, the combined toxicity of DON and API, at concentrations lower than DON (5.6 µM) + API (0.3 µM), was always similar to API alone, but lower than DON alone, whereas at higher concentrations, it was similar to the one of DON alone and higher than the one of API alone. As mentioned above, a proliferation of IPEC-1 cells was observed at low concentrations of API.

In conclusion, our data showed that the toxicity of the combination of DON and highly toxic emerging mycotoxins such as ENN-B, BEA, ENN-A1 and API, whatever the ratio used, was similar to or lower than the toxicity of DON alone.

Discussion

Progress in analytical methods enabled the discovery of numerous fungal secondary metabolites that are the subject of increasing attention today due to their prevalence in human food and animal feed [19,20]. In the present study, 524 samples of finished pig feed were analyzed. In addition to regulated mycotoxins such as DON, zearalenone and fumonisin B1, less known secondary metabolites were detected.

As already described in other surveys [20][21][22], BRV-F, Cyclo, TRPT, ENNs, EMO, BEA and AFN, culmorin, and moniliformin were highly prevalent emerging mycotoxins detected in more than 85% of pig feed samples. The diversity of the metabolites detected is very likely related to the wide range of fungal species that contaminate the raw materials used to make pig feed. Indeed, Fusarium species produce ENNs (A1, B and B1), BEA, AFN, API, culmorin and moniliformin, while Penicillium species produce BRV-F and Cyclo, Aspergillus EMO and Acremonium TRPT [22,40].

The toxicity of these new poorly documented metabolites was also investigated in the present study. The results of our analyses showed that, even at high concentrations of up to 300 µM, BRV, Cyclo and TRPT are not toxic to intestinal cells. Similar results were recently obtained using another porcine intestinal cell line, IPEC-J2, and a different readout, cellular protein content [20]. Interestingly, at a much higher concentration (2 mM), TRPT induced DNA damage in HepG2, A549 and THP-1 cells [41]. AFN and EMO were identified as moderately toxic compounds at relative IC 50 values of 19.1 µM and 19 µM, respectively. These emerging mycotoxins were found to be more toxic for IPEC-J2 with relative IC 50 of 9.3 µM and 13.1 µM, respectively [20]. On the other hand, human multiple myeloma blood cells were less sensitive to EMO (IC 50 38 µM) [42]. According to their IC 50 , ENNs were ranked in the following order of toxic potency ENN-A1 > ENN-B > ENN-B1. Similar ranking was reported for HT-29 [43] and IPEC-J2 [44]. ENN-A1 is also more toxic than ENN-B1 for Caco-2 and HepG2, but ENN-B displayed no toxicity at all [43]. The mechanism of toxicity of ENNs is related to their ionophoric properties [19] that facilitate the transport of mono-or divalent cations such as K + or Ca 2+ across membranes, but the relative sensitivity of the different cell lines to the different ENNs is still not understood. The high toxicity of BEA has already been reported in other cell lines of human and porcine origin, including Caco-2, HT-29 and IPEC-J2 [20,43]. BEA also has ionophoric properties and increase ions permeability (Na + K + and Ca 2+ ) in biological membrane, this mechanism of action participates to the toxicity of this mycotoxin [6,19,44].

Data on the toxicity of API are very limited. We confirmed the toxicity of this metabolite when used at high concentration (> 0.5 µM) [20,39]; we also observed that low concentrations of API (<0.1 µM) stimulate the proliferation of porcine intestinal cells. The proliferative effect of low doses of some mycotoxins has already been described. For example, up to 200% proliferation has been observed in lymphocytes and splenocytes exposed to low doses (> 0.1µM) of DON, nivalenol, aflatoxin B1 or fumonisin B1 [45]. Because of its high prevalence and its low IC 50 , it would be of great interest to deepen our knowledge of API. The toxicity of other very prevalent metabolites, such as culmorin and moniliformin, could not be evaluated in this study because they were not commercially available in the quantities required for cellular experiments.

Different studies have described the co-occurrence of mycotoxins in cereals and other raw materials used for animal feed in several regions of the globe [21,46,47]. In the present study, we investigated the co-occurrence of mycotoxins in pig feed. We identified 60 metabolites that co-occur with DON in more than 20% of samples, confirming the prevalence of co-contamination, and as a result, the exposure of animals to a mixture of mycotoxins. Our results are in accordance with those of previous surveys of raw materials and finished feed [20,46]. Although the exact composition of the analyzed pig feed is not known, the main component of the majority of the samples is maize. The extraction efficiencies are between 85%-95% determined in seven different matrices [48]. However, the focus of our study is to provide a general picture of the worldwide contamination of pig feed and to test realistic ratios of fungal compounds in vitro.

Despite the very high frequency of co-contamination by DON and emerging mycotoxins, the correlation between the concentrations of DON and emerging mycotoxins was very low. The correlation between DON and emerging mycotoxins is poorly documented. In winter wheat, Blandino and co-workers [49] addressed the correlation between DON and other mycotoxins produced by Fusarium graminearum and F. culmorum, and showed that correlations between DON and either culmorin or moniliformin were significant (0. 94 and 0.42, respectively). By contrast, the correlations between DON and AFN or BEA were not significant (0.2 and -0.14, respectively). Correlations between the concentration of DON and its modified forms enabled EFSA to calculate ratios between these different toxins [11].

In the present study, the absence of correlation between the concentrations of the emerging mycotoxins and DON could be explained by the diversity of fungi that produce the various metabolites via different biosynthetic pathways. Furthermore, pigs feed is made of different raw materials, which also explains the lack of correlation between the amounts of the different fungal metabolites involved.

The main objective of the present study was to assess the combined toxicity of DON and emerging mycotoxins. As no correlation was found, to encompass the situations to which animals may be exposed, different plausible ratios were tested. These ratios were based on the P25, the median and the P75 concentrations of DON and emerging mycotoxins observed in pig feed, because these summary statistics are robust to extreme outlier values. In most cases, we observed that when the non-toxic metabolites (BRV, Cyclo and TRPT) were present in mixture with DON, whatever the doses or the ratio, the effect was driven by DON. We observed a similar trend for the combined toxicity of DON with moderate and highly toxic metabolites ENNs, BEA, API, AFN and EMO. The effect of the mixtures was mostly similar to the effect of DON alone. The only exception was when very high concentrations of DON were used, in which cases surprisingly, the toxicity of the mixture was lower than the toxicity of DON alone.

To the best of our knowledge, this is the first study to investigate the interactions of different mycotoxins using plausible ratios. Most published studies used toxins of equal toxicity, regardless of their concentration in food or feed. For example, exposure of Caco-2 or IPEC-1 cells to low doses of DON, combined with isotoxic concentrations of nivalenol and/or their acetylated derivatives, led to a synergistic effect [31,32]. Similarly, mixtures of ENNs as well as mixtures of DON, ENNs and alternariol managed to induce synergistic cytotoxicity in Caco-2 cells [50,51]. A synergistic inflammatory effect was also observed in porcine intestinal explants co-exposed to DON and nivalenol [33].

In the future, these original data on the intestinal toxicity of realistic mixtures of DON and emerging mycotoxins should be completed by toxicity studies on other types of cells to account for all target organs. In vivo experiments are also needed to confirm the in vitro data. More widely, the effects of other mixtures of mycotoxins or of mycotoxins with other food contaminants should be investigated at realistic doses. As the pig is a good model for human toxicity studies of food contaminants [52], the results would be useful to estimate the effects of similar mixtures of toxins on human health. Better knowledge of the occurrence and toxicity of the real mixtures present in food is a precondition for the assessment of health risk [29].

Conclusions

This global survey of finished pig feed confirmed that such feed is co-contaminated by DON and emerging mycotoxins. However, despite the high percentages of co-occurrence, no correlation was found between the concentration of DON and most of these emerging mycotoxins. Using ratios based on the concentration of DON and emerging mycotoxins in feed, we observed that the toxicity of most of the mixtures was similar to the toxicity of DON alone. This demonstrates that, when these emerging mycotoxins are present together with DON, the toxicity of the mixture is not exacerbated.

Materials and Methods

Extraction and Analysis of Metabolites

A total of 524 finished pig feed samples were collected from 2014 to 2018 on the world market, but most in Europe (76.5%) and North America (15.8%) and fewer in Asia (3.2%), South Africa (1.5%), Australia (2 samples) and some of unknown origin (2.5%) (Figure 1 and Supplementary Figure S1), and more than 800 analytes, including fungal and bacterial secondary metabolites, were sought. Samples were provided by the BIOMIN Mycotoxin Survey, and the analyses were performed using the Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) multi-mycotoxin method described by Malachova [5].

A QTrap5500 LC-MS/MS System (Applied Biosystems, Foster City, CA, USA) equipped with a TurboIonSpray electrospray ionization (ESI) source and a 1290 Series ultra high performance liquid chromatography (UHPLC) System (Agilent Technologies, Waldbronn, Germany) was used for detection and quantification of the analytes (Supplementary Figures S5 andS6). Chromatographic separation was performed on a Gemini ® C18-column (150 × 4.6 mm i.d., 5 µm particle size) equipped with a C18 security guard cartridge (4 × 3 mm i.d.) (all from Phenomenex, Torrance, CA, USA) at 25 • C. Elution was carried out in binary gradient mode. Both mobile phases contained 5 mm ammonium acetate (NH 4 CH 3 CO 2 ) and were composed of methanol/water/acetic acid in a ratio of 10:89:1 (v/v/v; eluent A) or 97:2:1 (v/v/v; eluent B). After an initial time of 2 min at 100% A, the proportion of B was increased linearly to 50% within 3 min. Further linear increase of B to 100% within 9 min was followed by a hold-time of 4 min at 100% B and 2.5 min column re-equilibration at 100% A. The flow rate was 1000 µL/min. ESI-MS/MS was performed in the scheduled multiple reaction monitoring (sMRM) mode both in positive and negative polarities in two separate chromatographic runs. The sMRM detection window of each analyte was set to the respective retention time ± 27 s and ± 42 s in positive and in negative mode, respectively. The target scan time was set to 1 s. Confirmation of positive analyte identification is obtained by the acquisition of two sMRMs per analyte (with the exception of moniliformin and 3-nitropropionic acid, that each exhibit only one fragment ion), which yields 4.0 identification points according to commission decision 2002/657/EC (EU, 2002).

A total of 235 mycotoxins and other fungal secondary metabolites were detected and quantified in the samples of finished pig feed analyzed. The threshold of relevant concentrations was set at > 1.0 µg/kg or the limit of detection, whichever was higher. Samples were collected only by trained staff, or after the instruction of untrained staff according to a protocol. A minimum of 500 g homogenized sample was sent to the laboratory of the Institute of Bioanalytics and Agro-Metabolomics at the University of Natural Resources and Life Sciences Vienna (BOKU) in Tulln, Austria. Samples were milled and extracted with a mixture of acetonitrile, water and acetic acid (79:20:1, per volume) on a shaker for 90 min. The solution was centrifuged, after which the supernatant was diluted and injected into an LC-MS/MS system (electrospray ionization and mass spectrometric detection using a quadrupole mass filter). Quantification was done by comparing an external calibration using a multi-analyte stock solution. During the period 2014 to 2018, the number of substances measured using this method increased each year, and more substances were included in the survey. Nevertheless, the list of compounds investigated in this manuscript were those measured in 2014. All concentration data were collected in a single file, and sample information such as sampling year and month, country and region of origin and sample matrix were added for subsetting. Data were imported and analyzed in R v 3.5.1 mainly using tidy-verse packages (www.tidyverse.org). Spearman correlation coefficients and associated p-values were calculated with the corr.test function from the psych R package. Data were plotted (including maps) using ggplot2.

Toxins

For the cytotoxicity test, toxins were purchased from Sigma (St Quentin Fallavier, France): deoxynivalenol (DON) (purity > 98%), tryptophol (TRPT) (purity > 97%), apicidin (API) (purity > 98%) and emodin (EMO) (purity > 90%). Enniatins (ENNs) (A1, B, B1, purity > 99%), cyclo-(L-Pro-L-Tyr) (Cyclo) (purity > 98%), and brevianamide-F (BRV-F) (purity > 95%) were purchased from BioAustralis (Smithfield, Australia), beauvericin (BEA) (purity > 95%) and aurofusarin (AFN), (purity > 97%) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). All mycotoxins were dissolved in dimethyl sulfoxide (DMSO) (Sigma) to prepare stock solutions stored at -20 • C. Working dilutions were freshly prepared in cell culture medium for each experiment.

To convert the concentration in the pig feed into the concentration to which intestinal cells might be exposed, we assumed, as already done in previous studies [53], that mycotoxins were ingested in one meal, diluted in 1 L of gastrointestinal fluid and were entirely bioaccessible. Next, the ratio of DON to emerging mycotoxins was calculated based on three plausible scenarios according to the concentration of DON and emerging mycotoxins in the feed (Supplementary Table S1).

Several 3-fold dilutions of each individual toxin and mixtures at different ratios were performed to account for the concentrations present in feed.

Cell Culture and Cytotxicity Assay

IPEC-1, derived from the small intestine of a newborn unsuckled piglet were maintained in complete medium (Dulbecco's modified Eagle's medium (DMEM)/Ham's F12 medium (Sigma) plus 1% Penicillin Streptomycin (Eurobio, Courtaboeuf, France), 5% fetal bovine serum (FBS) (Eurobio), 1% L-glutamine (Eurobio), 5 µg/L epidermal growth factor (EGF) (Becton-Dickinson, Le Pont de Claix, France), and 1% ITS solution (insulin (5 µg/mL), transferrin (5 µg/mL), selenium (5 ng/mL), (Sigma Aldrich)) at 39 • C under 5% CO 2 , as previously described [54].

For the cytotoxicity experiments, cells were seeded in 96-white-well flat-bottom cell culture plates (Costar, Cambridge, MA, USA) at a rate of 10 4 cells per well in 100 µL culture medium. After 24 h, the medium was replaced by complete medium without FBS containing the mycotoxins and incubated for a further 48 h. Toxicity was then assessed using the CellTiter-Glo ® Luminescent Cell Viability Assay (Promega, Charbonnières-les-Bains, France) that determine the number of viable cells based on the quantitation of adenosine triphosphate (ATP). The luminescent signal produced by the luciferase reaction, reflecting the presence of metabolically active cells, was read using a multiplate reader (TECAN, Lyon, France). The results were obtained by calculating the percentage of viability obtained by calculating the ratio of the luminescence in treated samples and the luminescence in non-treated samples.

Statistical Analysis

The reported values on viability are expressed as the means ± the standard error of the mean (SEM) of at least three biological replicates, with duplicate wells for each dose. The IC 50 value, the dose of each toxin leading to 50% viability, was determined using CompuSyn statistical software (CompuSyn Version-1 Inc. Paramus, NJ, USA). Significant differences between groups were analyzed using the Bonferroni multiple comparison test in GraphPad (GraphPad Prism 4 La Jolla, CA, USA).

Supplementary Materials:

The following are available online at http://www.mdpi.com/2072-6651/11/12/727/s1, Figure S1: Worldwide contamination of emerging mycotoxins. Figure S2: Abundance of emerging mycotoxins in finished pig feed. Figure S3: Scatter plot of the correlation between DON and 10 selected emerging mycotoxins. Figure S4: Dose-effect curves of the individual toxicity of DON and 9 selected fungal emerging mycotoxins Table S1: Concentration of the metabolites in 3 quartiles (P25, P50 and P75) and ratios between the concentration of DON and 10 selected emerging mycotoxins. Figure S5: LC-ESI(+)-MRM chromatograms of one pig feed sample. Figure S6: Overlay of the extracted ion chromatograms of both quantifier as well as qualifier of the 11 investigated compounds in one pig feed sample.

Author Contributions: A.K.K., I.P.O. and P.P. designed the experiment, A.K.K. and M.N. performed the experiments, B.N. and G.S. contributed to provide raw data on finished pig feed, T.J. analyzed raw data on finished pig feed, P.G.P.M. performed statistical analysis on correlation, A.K.K. analyzed the cytotoxicity results, A.K.K., P.P. and I.P.O. wrote the paper, B.N. and P.G.P.M. reviewed the paper.

Cell viability (% of control cells) Concentration of metabolites (µM)

Supplementary Figure S4. Dose effect curve of individual toxicity of 9 selected emerging mycotoxins (BRV-F, Cyclo, TRPT, AFN, EMO, ENN-B1, ENN-B, BEA, ENN-A1) and DON. Data are mean ± SEM of three biological replicates. S1. Concentrations of DON and emerging mycotoxins converted in µM and the corresponding different ratios. The concentrations corresponding to the quartiles P25, median, P75 of emerging mycotoxins in µg/kg in pig feed were converted in µM assuming their dilution in 1 L of gastrointestinal fluid. Ratio 1 was calculated using the P25 (1 st quartile) concentration of emerging mycotoxin and P75 (3 rd quartile) concentration of DON. Ratio 2 was calculated using the median (2 nd quartile) concentration of DON and each emerging mycotoxin. Ratio 3 was calculated using the P75 concentration of emerging mycotoxin and P25 concentration of DON.

Supplementary Table

Metabolites

Development of an ex vivo model for studying the effects of mycotoxins on liver: the Precision Cut Liver Slices

Liver is an important organ for the uptake, biliary excretion and metabolism of xenobiotics and food contaminants. The main target organ for toxicity and carcinogenicity is the liver [START_REF] Peraica | Toxic effects of mycotoxins in humans[END_REF]. In most of the cases, toxicity of liver go unnoticed due to the absence of good predictive models and specific toxicity biomarkers. In vivo methods imply the use of a large number of animals. Therefore, more attention has been given to design and validate alternative models for toxicity testing that lead to reduction and eventually replacement of animal use.

Thus, there is interest in developing reliable liver models for early detection of toxic effects of toxins [START_REF] Starokozhko | Viability, function and morphological integrity of precision-cut liver slices during prolonged incubation: Effects of culture medium[END_REF]. In order to explore liver toxicity of mycotoxins, we developed precision-cut liver slices (PCLS). PCLS are viable ex vivo explants of tissue with well-defined thickness. PCLS technique is easily transferable to mouse, mini-pig, monkey or pig liver, and human liver. Slices can be remained viable up to 96 h when incubated under carbogen (95% O2/5% CO2) and appropriate conditions [START_REF] Graaf Ia De | Preparation and incubation of precisioncut liver and intestinal slices for application in drug metabolism and toxicity studies[END_REF].

PCLS contain all cell types of the tissue in their natural environment and natural architecture, with intercellular and cell-matrix interactions remaining intact [START_REF] Zimmermann | Improved reproducibility in preparing precision-cut liver tissue slices[END_REF]). In addition, PCLS has already been proven to be functional and efficient in numerous metabolism and toxicity studies [START_REF] Elferink | Microarray analysis in rat liver slices correctly predicts in vivo hepatotoxicity[END_REF]. They are mainly used to study the metabolism and toxicity of xenobiotics, but they are suitable for many other purposes, such as endogenous metabolism, biotransformation and its induction and transport of drugs, as well as for toxicological studies, and for assessing the efficacy of drugs in diseased tissues.

The main objective of the development of this model was first to determine whether liver slices could be incubated and maintained viable during a reasonably long time of incubation and then, to expose pig liver slices to DON and assess its toxic effect through different markers.

We measured at different time points, the total protein and ATP contents in the liver slices. We have also collected the incubation medium in order to measure different liver damage markers.

Ultimately, we assessed the effect of DON on the expression of selected genes.

Materials and Methods

Liver slices experiments were performed on pig and the first aimed to optimize the conditions of cutting and incubation of slices. Three piglets (5 weeks -old) were slaughtered in consecutive days. Liver was sampled and a piece of the right lateral lobe (Figure 4) was perfused with cold sodium chloride (NaCl 0.9%) (Sigma St Quentin Fallavier, France) using a 50 mL syringe.

Perfused liver was placed under the cylinder-shaped coring tool and liver cores were prepared using a drill and a tissue coring tool (Ø 10 mm) (Figure 5a). Then, cores were transferred to the cylindrical core holder of the Krumdieck slicer (Figure 5b-d). Slices were in thickness of 250-350 μm and average weight was 50-70 mg (Figure 5e) [START_REF] Carol | A New Instrument for the Rapid Preparation of Tissue Slices 155[END_REF]. Coring and slicing was carried out into "Krebs-Henseleit" buffer supplemented with NaHCO3 (Sigma).

Krebs was bubbled in advance with carbogen. Good quality slices were round, equally thick at all sides and had smooth edges (Figure 5f). Slices were transferred to 12-well plates using a spatula to avoid damages (Figure 5g-h) [START_REF] Graaf Ia De | Preparation and incubation of precisioncut liver and intestinal slices for application in drug metabolism and toxicity studies[END_REF]. In parallel, William medium E (WME) (Sigma) supplemented with 0.5% Gentamycin (10 mg/ml) and 1% Glutamine (200 mM) (Eurobio, Courtaboeuf, France) was also bubbled in carbogen, 1 h in advance.

Slices were maintained in Krebs buffer under carbogen bubbling for 1 h of regeneration phase, to maintain the quality and remove the debris. Surface and edges of the slices were smoother after regeneration. Then slices were immersed individually in 12 wells plate containing 2 mL WME containing or not DON (Sigma) dissolved in DMSO (1%) (Sigma). Control slice were incubated with same concentrations of vehicle in each incubation time. Plates were incubated at 39°C for 2, 4, 6, 8 and 20 h in presence of DON or without DON (control) under orbital shaking. In the space between wells, few mL of medium were added and bubbled with carbogen in order to maintain high humidity and oxygen concentration. Incubation medium was refreshed after 8 h in order to maintain quality of medium. At each incubation time, incubation medium was collected to assess liver damage markers such as: alkaline phosphatase (ALP), aminotransferases such as aspartate transaminase (AST), and alanine transaminase (ALT), lactate dehydrogenase (LDH) and total protein over the incubation. PCLS were also collected in microtubes, snap frozen in liquid nitrogen and stored at -80 °C for assessment of gene expression, total protein and ATP contents.

Statistical Analysis

One-Way Analysis of Variance (ANOVA) non-parametric test was used to find significant difference between the mean group across the incubation time or treatment and significant differences between the control and treated samples were analyzed via Bonferroni's multiple comparison tests, using statistical software GraphPad (La Jolla, CA, USA).

Analysis of Liver damage markers in culture medium

The main goal of this experiment was to assess quality of PCLS through several sensitive liver damage markers such as ALP, AST, ALT, LDH and total proteins, in order to know whether incubation (time) or treatment (DON 10 µM) influence the quality of PCLS.

Methods

Incubation medium was collected in different time (2, 4, 6, 8 and 20 h). Medium was renewed after 8 h of incubation. Samples were analyzed by GenoToul-Anexplo platform (Toulouse, France) using a Pentra 400 Clinical Chemistry benchtop analyzer (Horiba, Les Ulis, France).

The reported values were expressed as means ± SEM of the three biological replicates.

Results and discussion

Overall, we observed that the release of enzymes was numerically but not statistically higher in the treated samples (DON 10 µM) than the control samples. Release of enzymes was gradually increased from 2 h up to 8 h. Medium was renewed at 8 h. Thus, similar result was obtained at 8 h and 20 h, whereas the release of AST and LDH was lower at 20 h compared to 8 h. Overall, no significant difference between the control and treated samples was observed whatever the duration of incubation (Table 21). The level of ALP, AST, ALT, total protein, LDH was not significantly impacted by time or DON treatment. PCLS could be damaged during preparation or incubation. This potentially causes the release in the incubation medium of some enzymes ALP, ALT, AST, LDH as well total proteins [START_REF] Pratt | Evaluation of Abnormal Liver-Enzyme Results in Asymptomatic Patients[END_REF]Limdi 2003). These markers are sensitive indicators of hepatocellular injury [START_REF] Pratt | Evaluation of Abnormal Liver-Enzyme Results in Asymptomatic Patients[END_REF]. ALP originates mainly from two sources, liver and bone. The physiological role of these enzymes is not entirely clear but their production increases in tissues undergoing metabolic stimulation (Limdi 2003). LDH is a cytosolic enzyme mainly present in periportal hepatocytes and released when the cells are lysed by hepatotoxins. The amount of released enzyme is proportional to the extent of damage caused to the cell [START_REF] Naik | Protection of liver cells from ethanol cytotoxicity by curcumin in liver slice culture in vitro[END_REF]. ALT, a cytosolic enzyme is found in its highest concentrations in the liver and is a more specific indicator to liver injury. AST is found, in decreasing order of concentration, in liver, cardiac muscle, skeletal muscle, kidneys, brain, pancreas, lungs, leukocytes, and erythrocytes [START_REF] Pratt | Evaluation of Abnormal Liver-Enzyme Results in Asymptomatic Patients[END_REF].

AST, ALT, ALP, LDH, and total protein are used to evaluate patients with known or suspected liver disease [START_REF] Dufour | Diagnosis and Monitoring of Hepatic Injury. I. Performance Characteristics of Laboratory Tests[END_REF][START_REF] Starokozhko | Viability, function and morphological integrity of precision-cut liver slices during prolonged incubation: Effects of culture medium[END_REF]. These markers are also used to evaluate the liver slices. After 5 days of incubation, the protein content and the hepatic markers AST and ALT were modestly decreased (17-22%) relatively to their initial levels at day 0. In contrast, the levels of the ALP increased substantially (3-fold) and LDH also increased up to 25-28% [START_REF] Behrsing | Extended rat liver slice survival and stability monitored using clinical biomarkers[END_REF]. In another study, human liver slices were incubated and LDH release into the medium after 120 h of incubation was assessed in two different incubation medium, WME and RegeneMed®. LDH release was about 9-11% after 24 h. Thereafter it remained relatively stable (4-6% per day). The elevated LDH release during the first hours of incubation can be explained by the damage of the outer cell layers during the slicing and handling procedures. No difference was observed between slices incubated in WME and RegeneMed® [START_REF] Starokozhko | Viability, function and morphological integrity of precision-cut liver slices during prolonged incubation: Effects of culture medium[END_REF]. When we assessed the LDH release at different incubation time (2 h -20 h), we observed a maximum release at 8 h, whereas after 8 h release of LDH remained stable.

Analysis of total Proteins and ATP content in PCLS

The main goal of this experiment was to assess effect of DON on total proteins and ATP contents in PCLS during short (2 h) and longer (20 h) exposure period.

Methods

Tubes with ceramic beads (Bertin Technologies, St. Quentin en Yvelines, France) containing PCLS were taken out from the freezer -80 ⁰C. Then, 1 mL of ice-cold CelLytic™ MT buffer (Sigma) was added. All the tubes were transferred into FastPrep-24™ 5G Classic Homogenizer (MP Biomedicals). Tissues were immediately homogenized without thawing for 40 seconds (speed 6.0 m/s). Homogenates were placed on ice and centrifuged at 10000 g (gravitational acceleration) at 4 °C for 10 min to remove cellular debris. In total 0.85-0.9 ml of supernatant was transferred into a clean microtube. Proteins contents were measured directly by spectrophotometer using Nanodrop ND1000 (Labtech International) directly at 280 nm. The concentrations were determined by using a bovine serum albumin (BSA) standard curve (0.1 -1.2 mg/mL), and the proteins content was expressed as mg/mL per slice. Data was obtained as mean ± SEM of three biological replicates.

ATP contents were also assessed in the same lysis extraction buffer from a single slice using 96-white well flat-bottom cell culture plates (Costar, Cambridge MA, USA). Serial ratios of lysed samples and CellTiter-Glo® (CTG) Luminescent (Promega, Charbonnières-les-Bains, France) were prepared for the measurement of ATP. Ratio 1 was 1/1 (100 µL CTG + 100 µL lysis sample). Ratio 2 was, 1.66/1 (125 µL CTG + 75 µL lysed solution), ratio 3 was 3/1 (150 µL CTG + 50 µL lysed solution) and ratio 4 was 7/1(175 µL CTG + 25 µL lysed solution)

respectively. The aim of testing serial dilutions was to verify whether the amount of CTG in the mixture was limiting confirm that test work correctly and respect to their dilution factor, the final value of diluted or not diluted samples was almost similar. ATP was measured by luminescence using spectrophotometer (Tecan Infinite M200 ProTM equipped with the Tecan iControl TM software). Blank was subtracted from all the wells. Luminescence of treated slices was compared with untreated one.

Results and discussion

We first investigated the effect of DON and incubation time on total proteins. As shown in This result demonstrated that incubation had no effect on the total protein contents of PCLS, whereas DON 10 µM in longer incubation (20 h) significantly decreased total protein contents.

Mainly, at the cellular and subcellular level, DON binds to the ribosome, inhibits protein and nucleic acid synthesis and triggers ribotoxic stress, leading to the activation of mitogenactivated protein kinases (MAPKs) and their downstream signaling pathways (Pierron et al. 2016b). Thus, decrease of proteins in PCLS confirms that DON inhibits synthesis of proteins, and inhibition was clearly observed at longer incubation.

We then analyzed the effect of DON and incubation on ATP content. Results demonstrated that a short DON exposure (2h) did not have any effect on ATP content. By contrast a long incubation time (20h) with or without DON decreased ATP contents by more than 70% (Figure 7). Overall, total protein contents of PCLS was reduced just due to the treatment, whereas unlikely to protein, ATP was highly reduced due to the incubation (20 h).

Total ATP contents in PCLS

The viability of PCLS is usually assessed via ATP and proteins content. Some studies have demonstrated that in longer incubation, a decrease of weight, thickness, total proteins and ATP was observed. In rat PCLS incubated for 24 h the wet weight significantly decreased from 17.6 mg per slice to 9.0 mg (~51%), thickness was reduced from 218 μm (fresh slices) to 184 μm (~84%) and total proteins content was reduced significantly from 1.58 mg per slice to 1.03 mg(~66%). However, ATP was slightly decreased (17%) compared to the one of control [START_REF] Granitzny | Maintenance of high quality rat precision cut liver slices during culture to study hepatotoxic responses: Acetaminophen as a model compound[END_REF]. In addition to the treatments, incubation medium also play a role in the degradation of ATP. When human liver slices were incubated in two different mediums, WME and RegeneMed® after 120 h, the amount of ATP was dissimilar. Slices incubated in RegeneMed® had a consistently lower ATP level over time compared to WME. However, the incubation time had no effect on ATP levels. These lower ATP levels in slices incubated in RegeneMed® medium may be explained by the formation of the large necrotic regions in the inner part of the slices [START_REF] Starokozhko | Viability, function and morphological integrity of precision-cut liver slices during prolonged incubation: Effects of culture medium[END_REF]. Furthermore, ATP level was measured in rat and mouse PCLS incubated in WME and DMEM medium for 72 h. Both mouse and rat slices lost more than 60% ATP level within 72 h in WME medium, however in DMEM with growth factors and hormones ATP level instead increased within 72 h [START_REF] Koch | Murine precision-cut liver slices (PCLS): a new tool for studying tumor microenvironments and cell signaling ex vivo[END_REF].

Our results on the total proteins and ATP contents are not in accordance with already published results. We incubated slices up 20 h, but we did not see any effect of incubation on total protein contents, whereas we observed the significant effect of incubation on the ATP. Previous stated results are from human, rats and mouse, whereas we assessed the behavior of pig liver slice.

These variations might be dependent to the animal species.

Assessment of the time and dose dependent effects of DON on the expression of selected genes

The main objectives were (i) to assess the effect of DON 10 µM on the expression of genes of PCLS at different time of exposure (2 h, 4 h, 6 h, 8 h and 20 h) and (ii) to assess the doses effect of dose (DON 3 µM and 10 µM) at 20 h. Treated samples were compared to corresponding controls at each incubation time.

Methods

Extraction

A single PCLS was lysed in 1 mL Extract-all reagent (Eurobio) in a microtube with ceramic beads (Bertin Technologies, St. Quentin en Yvelines, France) using FastPrep-24™ 5G Classic Homogenizer (MP Biomedicals). Rapidly, the lysate was transferred into a new microtube.

Then, 200 µL of chloroform (Sigma) were added into lysate, shacked vigorously several times and incubated for 5 minutes at room temperature. Afterward, microtubes were centrifuged for 15 min in 13,000 g. The solution was separated into aqueous and organic phases. RNA remaining in the aqueous phase was collected and recovered by precipitation with isopropyl alcohol and washed with ethanol 75% (Sigma) before dilution in RNase-free water. RNA concentration and purity with A260/A280 and A260/A230 ratios >1.95 were determined using spectrophotometer Nanodrop ND1000. In addition, quality of PCLS RNA was assessed via RNA integrity number (RIN) using Agilent 2100 Bioanalyzer (Agilent Santa Clara, CA USA).

An aliquot of total RNA (1 μL) was analyzed using RNA Nano LabChips (Agilent). RIN value is determined by an algorithm for assigning integrity values to RNA measurements using the 28S-to-18S-Ribosomic RNA ratio. Quantitative real time polymerize chain reaction (qRT-PCR) was performed to assess the relative mRNA expression levels. Each sample was tested in duplicate and the potential genomic DNA contamination was verified through a non-template control (NTC). For one reaction, 2.5 µL of SyberGreen Power® SYBR Green qPCR master MIX (Sigma-Aldrich) and 1.5 µL of Primers (reverse and forward) and 1 µL of cDNA (5 ng/ µL) was prepared. The specificity of qPCR products was assessed at the end of the reactions by analyzing the dissociation curves using QuantStudio -TM (Realtime PCR Software v. 1.1, 2014). Data were exported into an Excel file. LinRegPCR v2014.3 program was used for qPCR data analysis to calculate N0 (the initial RNA concentration) and amplification efficiency [START_REF] Ruijter | .X) Analysis of quantitative RT-PCR data 224[END_REF] and then the data were exported back as an Excel file. For quantification and statistical analysis, target genes mRNA expression was normalized with the housekeeping genes (HKG) using N0 (=10 intercept ) with LinRegPCR. Three HKG, ribosomal protein L32 (RPL32), Beta-2-Microglobulin (B2-microglobulin) and TATA-Box Binding Protein (TBP) were used for data normalization. The best housekeeping genes were determined using Normfinder program which calculates a stability value based on the combined estimate of both intra-and intergroup values [START_REF] Andersen | Normalization of Real-Time Quantitative Reverse Transcription-PCR Data: A Model-Based Variance Estimation Approach to Identify Genes Suited for Normalization, Applied to Bladder and Colon Cancer Data Sets[END_REF]. In brief, NormFinder and LinRegPCR are the best approaches for reference gene selection and efficiency determination, respectively.

Results and discussion

Quality of the RNA

The assessment of RNA integrity is a critical first step before the following steps of RT and qPCR. RNA-Integrity (RIN) was obtained from each control slice. Analysis of the Bioanalyzer profile of total RNA indicated that the 28S and 18S rRNA bands were visible for each individual in each incubation time except for one sample at 20 h (Figure 8). The RIN for an RNA sample ranges from 1 to 10. A RIN of 10 is fully intact and a RIN of 1 is completely degraded [START_REF] Fleige | RNA integrity and the effect on the real-time qRT-PCR performance[END_REF]. Average RIN of 3 liver slices (biological replicates) were calculated. Initial RIN (sample without incubation) was 7.5 whereas; the final RIN (20 h incubation) was obtained 6.2. This moderate decrease demonstrates that the quality of PLCS were maintained stable after different time of incubation (Figure 9). Thus, the RIN we obtained allowed us to go further and perform the qPCR and perform assessment of gene expression. Quality of RNA were evaluated in different species (human, rat and mouse). Initial RIN for human, rat and mouse PCLS were 8.9, 8.3 and 8.2, whereas after 3 days of incubation the final RIN were 7.8, 6.8 and 7.3 respectively (Niehof et al. 2017). RIN is usually higher in cell lines rather than PCLS. For example, RIN were always ≥ 8.1 for tissue and ≥ 9.5 for cells during 4 h incubation [START_REF] Delgado-Ortega | Innate immune response to a H3N2 subtype swine influenza virus in newborn porcine trachea cells, alveolar macrophages, and precision-cut lung slices[END_REF].

It should be kept into account, the RIN number is not a tell-all with regard to downstream applications. For example, depending upon primer design, RT-PCR can be performed successfully when starting with partially degraded RNA when the amplicon is short. Similarly, RIN numbers are not particularly useful for nucleic acids that have been purified from archived pathology specimens. RNA recovered from formalin-fixed paraffin-embedded tissues and tissue sections also exhibit some degree of degradation [START_REF] Farrel | RNA Methodologies: Laboratory Guide for Isolation and Characterization[END_REF].

Assessment of gene expression

Next, we selected several genes involved in different cellular function and processes (Table 22) including 3 HKG. Selection of the genes was based on the literature [START_REF] Nielsen | Potential of deoxynivalenol to induce transcription factors in human hepatoma cells[END_REF][START_REF] Sahu | Comparative hepatotoxicity of deoxynivalenol in rat, mouse and human liver cells in culture[END_REF][START_REF] Kouadio | Lipid metabolism disorders, lymphocytes cells death, and renal toxicity induced by very low levels of deoxynivalenol and fumonisin B 1 alone or in combination following 7 days oral administration to mice[END_REF][START_REF] Osselaere | Deoxynivalenol Impairs Hepatic and Intestinal Gene Expression of Selected Oxidative Stress, Tight Junction and Inflammation Proteins in Broiler Chickens, but Addition of an Adsorbing Agent Shifts the Effects to the Distal Parts of the Small Intestine[END_REF]Wu et al. 2014b;[START_REF] Liu | The metabolic responses of HepG2 cells to the exposure of mycotoxin deoxynivalenol[END_REF][START_REF] Luo | Specific intestinal toxicity of deoxynivalenol and cadmium: analysis on pig jejunal explants[END_REF]. The goal of the first experiment was to determine the quality of the PCLS over the incubation time. Therefore, we selected genes already investigated in previous studies to confirm those results and hence to evaluate the relevance of the model of PCLS. First, we analyzed the effects of DON 10 µM on the gene expression at different incubation time (2,4,6,(8)(9)(10)(11)(12)(13). Genes were modulated after 4 h when PCLS were treated with DON 10 µM at different exposure time. Hereby, inflammatory cytokines were upregulated time dependently until 8 h. Most of the genes were upregulated time dependently compared to the controls one. CCL-20, IL-1b, IL-8 and IL-10 were significantly upregulated until 8 h of exposure, but at 20 h, their expression was decreased. TNF-alpha was upregulated only at 6 h of exposure. None of the tested inflammatory cytokines was expressed at 2 h (Figure 10). Obtained results were consistent to the previous results. DON at 2.5 mg/kg BW markedly upregulated TNF-a, IL-1b, IL-6, CXCL-2, CCL-2 and CCL-7 after 2 and 6 h in spleen (Wu et al. 2014b). DON at 24 µM also induced inflammatory cytokines IL-1b, IL-1a, IL-8, and TNFa when jejunal explants were treated for 4 h [START_REF] Luo | Specific intestinal toxicity of deoxynivalenol and cadmium: analysis on pig jejunal explants[END_REF]. Altogether, DON induces inflammation in liver as in other organs.

Among the tested genes involved in cellular function of lipid metabolism (ABCG8 and SREBP2) were significantly upregulated and LXR was significantly downregulated time dependently (Figure 11). Our obtained results were in accordance with previous results. DON significantly affected lipids metabolism in HepG2 cells when treated with DON at 10 µM for 4 h -12 h periods. [START_REF] Liu | The metabolic responses of HepG2 cells to the exposure of mycotoxin deoxynivalenol[END_REF]. Likewise, DON revealed disorders in lipid metabolism, when Swiss mice were orally administered with low doses of DON (45 μg/kg bw/day) during 7 days [START_REF] Kouadio | Lipid metabolism disorders, lymphocytes cells death, and renal toxicity induced by very low levels of deoxynivalenol and fumonisin B 1 alone or in combination following 7 days oral administration to mice[END_REF]. In liver, disorders of lipid metabolism, may result in deposit of lipids in the walls of blood vessels, which can lead to atherosclerosis (a disease characterized by abnormal thickening and hardening of the walls of the arteries). In the consequence of gene Genes involved in the cellular function of oxidative stress were also modulated time dependently except SOD1. All three genes NFKB, CAT and CCS were upregulated significantly compared to the corresponding control (Figure 12). These results confirm that DON induces oxidative stress in liver. Obtained results were in accordance with previous results. DON at 10 µM for 4 h-12 h induced oxidative stress in HepG2 cells [START_REF] Liu | The metabolic responses of HepG2 cells to the exposure of mycotoxin deoxynivalenol[END_REF].

Likewise, similar cells were exposed to DON at 25 µM for 6h-24 h, and was associated with oxidative stress [START_REF] Sahu | Comparative hepatotoxicity of deoxynivalenol in rat, mouse and human liver cells in culture[END_REF]). Oxidative stress is traditionally defined as an imbalance between pro-oxidant compounds and antioxidant defenses (Kunsch Charles and Medford Russell M. 1999). Oxidative stress is thought to play a role in the genesis of hepatic encephalopathy found in patients who have liver failure [START_REF] Webb | Oxidative stress and liver disease[END_REF].

DON had moderately effect on gene modulation involved junctional protein of PCLS. Only E-Cadherin was significantly up regulated in longer incubation time (20 h) (Figure 13). Obtained results were consistent and confirmed previous results. DON at 7.5 mg/kg feed, fed during 3 weeks to broiler chickens, up-regulated CLDN-5 in the jejunum, while no effect was seen on genes involved in cells junction [START_REF] Osselaere | Deoxynivalenol Impairs Hepatic and Intestinal Gene Expression of Selected Oxidative Stress, Tight Junction and Inflammation Proteins in Broiler Chickens, but Addition of an Adsorbing Agent Shifts the Effects to the Distal Parts of the Small Intestine[END_REF]. It is well known that in eukaryotic cells, at the cellular and subcellular level, DON binds to the ribosome, and inhibits protein synthesis (Pierron et al. 2016b). Tight junction (TJ) proteins exert their functional role as integral proteins in forming barriers in the liver. A causative role of TJ proteins has been established in the pathogenesis of colorectal cancer and gastric cancer. Among the best characterized roles of TJ proteins in liver disease biology is their function as cell entry receptors for HCV (hepatitis C virus)-one of the most common causes of hepatocellular carcinoma [START_REF] Zeisel | Tight junction proteins in gastrointestinal and liver disease[END_REF]. et al. 2003;[START_REF] Islam | p38 mitogen-activated protein kinase mediates IL-8 induction by the ribotoxin deoxynivalenol in human monocytes[END_REF].

Level of expression

Time of incubation

Discussion on PCLS

The model of tissue slices was invented in 1923 to measure cell metabolism and oxygen consumption in tumor tissue and then explored to study amino acid metabolism in various organs and species including human [START_REF] Li | Precision-cut intestinal slices: alternative model for drug transport, metabolism, and toxicology research[END_REF]. PCLS have been used by many researchers because they represent an organ mini-model that closely resembles the organ from which it is prepared, with all cell types present in their original tissue-matrix configuration [START_REF] Graaf Ia De | Precision-cut tissue slices as a tool to predict metabolism of novel drugs[END_REF]. Before, PCLS were used for a dynamic organ culture system as incubation system [START_REF] Dogterom | Development of a simple incubation system for metabolism studies with precision-cut liver slices[END_REF]. Whereas now, PCLS have been shown to be a valuable tool to predict metabolism of novel drugs, metabolism and toxicity of xenobiotics and as well for many other purposes [START_REF] Graaf | Empirical validation of a rat in vitro organ slice model as a tool for in vivo clearance prediction[END_REF][START_REF] Graaf Ia De | Precision-cut tissue slices as a tool to predict metabolism of novel drugs[END_REF]. This model has been developed long time ago.

We developed the PCLS model in order to assess toxicity of mycotoxins through several biomarkers, such as ATP and total proteins contents, enzyme release such as ALP, AST, ALT and LDH, and as well gene expression. Although we used piglet liver, this model it is easily transferable to mouse, guinea pig, monkey, dog, and as well to human [START_REF] Graaf Ia De | Preparation and incubation of precisioncut liver and intestinal slices for application in drug metabolism and toxicity studies[END_REF]. We found that liver slices is a good model for long incubation time under constant presence of carbogen. On the opposite, certain organs such as intestine cannot be incubated for longer incubation [START_REF] Graaf Ia De | Preparation and incubation of precisioncut liver and intestinal slices for application in drug metabolism and toxicity studies[END_REF].

Our results of liver damage markers, ALP, AST, ALT, LDH and total protein contents in the incubation medium were not affected either by incubation or treatments. We chose a time of exposure of 20 h in order to maintain quality of RNA in PCLS and avoid its degradation. The RNA molecule is inherently fragile in the natural environment and constantly degrade into smaller molecules through hydrolysis, preventing the faithful reproduction [START_REF] Spurway | Genetics and Molecular Biology of Muscle Adaptation[END_REF][START_REF] Fiore | The Origin and Early Evolution of Life: Prebiotic Chemistry of Biomolecules, 1st edn[END_REF].

Then, we assessed effect of DON 3 µM and 10 µM on gene expression of PCLS. Genes were selected based on literature involved in different cellular functions or processes. We confirmed that the effect of DON on the gene expression was dose and time dependent. In longer incubation and in high concentration of DON, regulation was significant compared to their corresponding controls. Most of the genes were modulated time dependently compared to the controls. Modulation was mainly pronounced after 2 h of incubation. Most of inflammatory cytokines, i.e. CCL-20, IL-1b, IL-8 and IL-10, were significantly upregulated until 8 h of exposure. Among the tested genes involved in cellular function of lipid metabolism, ABCG8 and SREBP2 were significantly upregulated and LXR was significantly downregulated time dependently. Genes involved in the cellular function of oxidative stress (NFKB, CAT and CCS)

were also modulated time dependently except SOD1. DON had a moderate effect on gene modulation involved in junctional protein of PCLS. Only E-Cadherin was significantly up regulated at longer incubation times.

To our knowledge, this is the first study that assess the effect of DON on the PCLS. However, effect of DON have been addressed on hepatic cells in vivo and in vitro [START_REF] Sahu | Rat liver clone-9 cells in culture as a model for screening hepatotoxic potential of food-related products: hepatotoxicity of deoxynivalenol[END_REF][START_REF] Grenier | Individual and combined effects of subclinical doses of deoxynivalenol and fumonisins in piglets[END_REF][START_REF] Osselaere | Deoxynivalenol Impairs Hepatic and Intestinal Gene Expression of Selected Oxidative Stress, Tight Junction and Inflammation Proteins in Broiler Chickens, but Addition of an Adsorbing Agent Shifts the Effects to the Distal Parts of the Small Intestine[END_REF][START_REF] Bracarense | Deoxynivalenol in the liver and lymphoid organs of rats: effects of dose and duration on immunohistological changes[END_REF]. Effect of low dose (commonly found in crops) of DON was examined on five weeks old piglets. DON contaminated diets induced significant liver lesions when compared to animals fed control diet [START_REF] Grenier | Individual and combined effects of subclinical doses of deoxynivalenol and fumonisins in piglets[END_REF]. Effects of 1.75 mg/kg or 11.4 mg/kg of DON in contaminated diet was assessed in rats exposed for 7, 14 or 30 days, using histological and immunohistochemical analyses on liver. Ingestion of contaminated feed induced a significant increase in the lesional score in the liver. The main histological findings observed were cytoplasmic vacuolization and hepatocellular megalocytosis. A significant increase in hepatocyte proliferation was observed in rats that received 1.75 mg/kg of DON. The results of the morphological and immunohistochemical analyses suggest that the ingestion of DON can induce functional hepatic impairment and immunosuppression in a dose-and time-dependent manner (Bracarense et al.

2017).

In conclusion, the liver slices appear as a promising model to study the effects of food contaminants on liver tissue. It allows the assessment of different markers into the tissue or released in the culture medium. This tool may also prove useful for identification of genes in liver-resident cells that support cancer or invasion in liver. Moreover, this tool can partially supplant in vivo pig experiments for prescreening liver diseases.

Discussion on Experimental models

A model is "a representation of a real or actual subject". In the field of toxicology, experimental scientific models are used to understand toxicological mechanisms. Toxicology is an experimental laboratory science that has traditionally utilized various animal models to study the effects of chemicals. Historically, toxicology studies relied heavily on various histopathological and biochemical endpoints in whole animals to draw conclusions on the effects of a chemical on living systems [START_REF] Choudhuri | From Classical Toxicology to Tox21: Some Critical Conceptual and Technological Advances in the Molecular Understanding of the Toxic Response Beginning From the Last Quarter of the 20th Century[END_REF]. However, the general adoption of the principles of 3R (Replace, Reduce, Refine) provided the impetus for the development of in silico, ex vivo and in vitro models in toxicity testing (Figure 15). Their relative contributions to the scientific and animal welfare implications as well the opportunities they offer in the research are different. As each of them facilitated research in one way, on the other hand they have disadvantages and limitations. properties of the compounds and their biological effect [START_REF] Cronin | Silico Toxicology: Principles and Applications[END_REF]. In silico toxicity is assessed using a wide variety of tools (i.e., methods, algorithms, software, data, etc) to analyze beneficial or adverse effects of a compound [START_REF] Yap | In Silico Identification of Novel Inhibitors[END_REF]. In silico models are The building of in silico models is an exciting field as it draws on so many areas of science and is dynamic and continually evolving. Looking to the future, there are many opportunities for further development of this field. This will include the use of more successful computational techniques to gain insight into fundamental molecular and chemical processes (Cronin and Madden 2010).

In vitro models

The term in vitro presents "the technique of performing a given procedure in an artificial environment outside the living organism". The major aims of in vitro systems are to develop, simulate and predict biological reactions to materials when placed into or on tissue in the body.

The in vitro methods not only assist in hazard identification and risk assessment but also in understanding dose response relationship and evaluating a new product before its market launch or supplying to researchers or pharmacological companies [START_REF] Dhawan | Vitro Toxicology[END_REF].

Although, the in vitro studies can be criticized for being very different from the natural environment, they present several advantages. Despite of many advantages of ex vivo models, they also has some limitations as well.

In vitro models

Pros

Ex vivo models

Pros

In vivo models

In vivo models are defined as "living organisms with an inherited naturally-acquired or induced pathological process that closely resembles the same phenomenon in human" [START_REF] Chow | Using Animal Models in Biomedical Research: A Primer for the Investigator[END_REF]. Their application in biomedical research, and pathophysiological and toxicological studies, provide the opportunity to determine pharmacokinetics of toxic compounds and to observe other clinical symptoms such as feed and water intake. In order to interpret and validate the results of other experimental models (in vitro, in silico, ex vivo), it is essential to perform in vivo assays. In vivo models are complex and integrated models that includes ingestion, absorption, metabolization and excretion processes. Following are briefly pointed out the main pros and cons of this model:

In vivo model

Pros Cons

 Presence of all metabolic process of host et al. 2011). Animal models are used as preclinical tools for potential drug screening processes and translation into clinical trials. Preclinical testing includes testing of all materials and prototypical devices before testing or using in humans. This testing is conducted in order to provide an assurance of device safety prior to use in humans in a clinical trial [START_REF] Belma | ANIMAL MODELS IN MODERN BIOMEDICAL RESEARCH[END_REF].

The overall effect of the experiment on a living organism can be observed in in vivo models.

Thus, in vivo experiments are more realistic than in vitro experiments with respect to the phenomenon and toxicity which comes out in the consequence of a toxicological experiment.

Their main advantages are that they allow studying the toxicity of mycotoxins in presence of host defense mechanisms and integrated (ingestion, digestion, and excretion) system. In addition, in vivo assays provide the opportunity to assess the toxicity of mycotoxins through several endpoints simultaneously. For instance, suitable biomarkers for DON and ZEN exposure in porcine plasma, urine and feces and for DON, AFB1 and OTA in plasma and excreta of broiler chickens were identified [START_REF] Lauwers | Biomarkers for Exposure as A Tool for Efficacy Testing of A Mycotoxin Detoxifier in Broiler Chickens and Pigs[END_REF]. The effects of DON and FUM alone or in combination during 5 weeks diet on hematological and biochemical blood parameters as well histopathological effects on the liver, lungs and kidneys were investigated [START_REF] Grenier | Individual and combined effects of subclinical doses of deoxynivalenol and fumonisins in piglets[END_REF]. A sub-chronic toxicity study of NIV was conducted in male and female rodents for 90 days to assess histopathology of the hematopoietic and immune organs, body weight gain, white blood cell and red blood cell counts as well the hemoglobin concentration [START_REF] Takahashi | A 90-day subchronic toxicity study of nivalenol, a trichothecene mycotoxin, in F344 rats[END_REF].

So far, numerous experimental models have be introduced. Each model has its justification at some points when applying to investigate toxicity (Sande 1999). Thus, a number of animal and non-animal models are used in research over the last years. It is established worldwide that in vitro testing models are of extreme importance for screening as well as to elaborate the mechanism and set of causes of various diseases [START_REF] Jain | Models and Methods for In Vitro Toxicity[END_REF].

Among all the model previously presented, first we chose a cell culture model in order to investigate individual and combined toxicity of DON and emerging mycotoxins in porcine intestinal cells in vitro. Then, we developed precision-cut liver Slices (PCLS) model "ex vivo" in order to assess hepatotoxicity of DON in liver slice, as briefly discussed their implication, pros and cons as following ahead.

Use of intestinal cells to assess the toxicity of emerging mycotoxins

In our study, first we used an in vitro model of intestinal cells, the first target of ingested contaminants, to characterize the potential toxicity of emerging mycotoxins. We used the porcine intestinal epithelial cells (IPEC-1) and this in vitro model allowed us to assess serial doses-toxic effects of mycotoxins either alone or in mixture. In the following part, we focused on the pro and cons of this cell culture model.

Intestinal Epithelial Cell culture model

Pros

2019

). On the base of iso-dose effect, the combined toxicity of several ENNs (A, A1, B and B1) were assessed individually or in combination using Caco-2 cells (Prosperini et al. 2014a).

Binary ENN+DON (1:1) and quaternary mixture of ENNS (A, A1, B, B1) were tested in ratios 1:1:1:1 in Caco-2 cells (Prosperini et al. 2014a). Likewise, binary mixture of ENN+BEA, DON+BEA and DON+ZEA were tested in Caco-2 cells using in vitro [START_REF] Ficheux | Co-exposure of Fusarium mycotoxins: In vitro myelotoxicity assessment on human hematopoietic progenitors[END_REF]Fernández-Blanco et al. 2016).

Development of PCLS model to assess toxicity of mycotoxins

Precision-cut liver slices (PCLS) are viable ex vivo explants of tissue with a reproducible, well defined thickness. They represent a mini-model of the organ under study and contain all cells of the tissue in their natural environment, leaving intercellular and cell-matrix interactions intact, and are therefore highly appropriate for studying multicellular processes (de [START_REF] Graaf Ia De | Preparation and incubation of precisioncut liver and intestinal slices for application in drug metabolism and toxicity studies[END_REF]. We used a liver slice model to assess the toxicity of DON. We aimed to develop the PCLS model in order to make screening and assess toxicity through several markers such as ATP, total protein contents and gene modulation. With an increasing public demand to limit the number of animals used in research and to reduce the distress to the experimental animals (3R concept), PCLS represent a promising method with numerous advantages for toxicological studies.

PCLS model

Pros

General Conclusion

Maintaining a safe global food and feed supply is a critical issue facing society. Cereal grains and their processed food products are frequently contaminated with mycotoxins. The diverse nature of mycotoxigenic fungi contribute to the worldwide occurrence of mycotoxins. Recent finding and surveys have shown that 60 -80% of food, feed and cereal grains are contaminated by well-known as well as by emerging mycotoxins (Gruber-Dorninger et al. 2019;[START_REF] Eskola | Worldwide contamination of food-crops with mycotoxins: Validity of the widely cited 'FAO estimate' of 25%[END_REF]Abdallah et al. 2017;Streit et al. 2013).

We collected more than 500 finished pig feed samples from the worldwide market during five years and analyzed them via LC-MS/MS method. We found that more than 95% of the samples were contaminated by at least one mycotoxin and 88% of them were naturally co-contaminated by DON and other regulated and emerging mycotoxins. The co-occurrence of mycotoxins represents the reality of fungal contamination of cereals and cereal-derived food products [START_REF] Lee | Worldwide Occurrence of Mycotoxins in Cereals and Cereal-Derived Food Products: Public Health Perspectives of Their Co-occurrence[END_REF].

We analyzed the prevalence and concentration of mycotoxins in compound feed which exact composition was unknown. The composition of feed might have an influence on the prevalence and as well on the concentration of mycotoxins. Although, more than one mycotoxin, produced by a single or several fungal species, may occur in various combinations in a given sample or food. Thus, compound feed that are made of several and various commodities, could be more contaminated rather than the raw materials (Streit et al. 2012). However, some authors have claimed that the concentrations of mycotoxins tend to be lower in processed food products; the incidences varied depending on the individual mycotoxins, possibly due to the varying stability during processing and distribution of mycotoxins [START_REF] Lee | Worldwide Occurrence of Mycotoxins in Cereals and Cereal-Derived Food Products: Public Health Perspectives of Their Co-occurrence[END_REF].

DON is one of the most wide distributed regulated mycotoxins across the world (Knutsen et al. 2017;Gruber-Dorninger et al. 2019;Novak et al. 2019). Among farm animals, pig is one of the most sensitive species to mycotoxins (Pinton et al. 2009;Pierron et al. 2016b). When exposed to DON, pig performances and immune response are altered. This lead the CONTAM Panel to determine the LOAEL of 1.3 mg DON/kg feed for pig using the reduction of feed intake as the most sensitive endpoint (Knutsen et al. 2017).

In addition to regulated mycotoxins, emerging mycotoxins also occur frequently and in high concentrations in cereals and cereal-derived products (Springler et al. 2016b;Malachová et al. 2014;[START_REF] Vaclavikova | Emerging' mycotoxins in cereals processing chains: Changes of enniatins during beer and bread making[END_REF]. Emerging mycotoxins are usually co-produced with regulated mycotoxins [START_REF] Hussein | Toxicity, metabolism, and impact of mycotoxins on humans and animals[END_REF] and the hazard of exposure to mycotoxin mixtures is gaining interest. The individual toxicity of mycotoxins is reported in different cell types [START_REF] Ivanova | Emodin -a secondary metabolite with multiple ecological functions in higher plants[END_REF][START_REF] Alshannaq | Occurrence, Toxicity, and Analysis of Major Mycotoxins in Food[END_REF][START_REF] Olleik | Comparative Structure-Activity Analysis of the Antimicrobial Activity, Cytotoxicity, and Mechanism of Action of the Fungal Cyclohexadepsipeptides Enniatins and Beauvericin[END_REF]Novak et al. 2019), whereas at the present very little is known about the health risk of mycotoxins mixture.

As we confirmed the co-contamination of finished pig feed by emerging mycotoxins and DON, we investigated the combined toxicity of DON and the 10 most prevalent and co-occurring with DON emerging mycotoxins. We focused on the effects on the intestine as a first barrier against food contaminants. When feed contaminated with mycotoxins is ingested, the gastro-intestinal tract is the first possible site of interaction (Fraeyman et al. 2018) and intestinal epithelial cells can be exposed to high concentrations of toxins. We tested the individual and combined effects of emerging based on their realistic concentrations on IPEC-1 cells. Indeed, the potential risk of chronic exposure to multiple mycotoxins bringing the same mechanism of action and/or targeting the same organs may lead to synergistic or at least additive effects [START_REF] Speijers | Combined toxic effects of mycotoxins[END_REF]. or more led to induce in the expression of certain genes. The total proteins contents were significantly decreased upon exposure to DON 10 µM in longer incubation time in accordance with its well-characterized inhibition of protein synthesis. At the cellular and subcellular level, DON binds to the ribosome, inhibits protein and nucleic acid synthesis and triggers ribotoxic stress, leading to the activation of mitogen-activated protein kinases (MAPKs) and their downstream signaling pathways (Pierron et al. 2016b). PCLS are considered as closely reflect the in vivo situation and are potentially useful for studying the toxicity of mycotoxins on liverresident cells. Application of the PCLS technique in the field of mycotoxicosis analysis however, has not yet been well developed. We confirm that PCLS is a promising model to assess hepatotoxicity of mycotoxins.

Perspectives

Toxic effect of emerging mycotoxins on PCLS

This study showed that most of the tested emerging mycotoxins were toxic, some of them being more toxic than DON. We analyzed the hepatic toxic effects of DON on several endpoints such as gene expression, ATP degradation and total proteins contents using PCLS. However, the toxic effect of emerging mycotoxins on above-mentioned markers through PCLS is still to be performed to obtain a picture of their potential toxicity on the liver.

Some of emerging mycotoxins such as ENNs and BEA have ionophoric properties and increase ions permeability (Na + K + and Ca 2+ ) in biological membrane. They increase oxidative stress by altering membrane ion permeability and uncoupling oxidative phosphorylation (EFSA 2014).

BEA can induce genes from different pathways. A toxicogenomic study of the effects of BEA on gene expression changes in Jurkat cells and human lymphoblastic T cells showed changes in the expression of genes involved in to apoptosis and programmed cell death (Escrivá et al. 2018). Furthermore, ENN altered the gene expression profile of primary rat hepatocytes [START_REF] Jonsson | Fusarium mycotoxin enniatin B: Cytotoxic effects and changes in gene expression profile[END_REF]. This demonstrates the need of analyzing the overall effect of emerging mycotoxins on gene expression using whole transcriptome approach.

Combined toxicity of DON and emerging mycotoxins on PCLS

Recently, due to the natural co-occurrence of mycotoxins, the hazard of exposure to mycotoxin mixtures is increasing day by day. The potential risk of chronic exposure to multiple mycotoxins, when the mechanism of action is similar, or if they target the same organs may lead to a synergistic or at least an additive effect when combined together [START_REF] Speijers | Combined toxic effects of mycotoxins[END_REF]. However, in our study performed on intestinal cells, we reported that emerging mycotoxins did not exacerbated the toxicity of DON in mixture. In order to improve the knowledge on the combined toxicity of emerging mycotoxins and DON using the ex vivo model of PCLS is the continuation of the present work.

Slices from other organs

The Krumdieck tissue slicer, which underwent regular improvements, is currently one of the most widely used instruments and can produce hundreds of slices in a semi-automatic procedure within 1 hour. Precision-cut tissue slices (PCTS) are widely used by many researchers as an ex vivo model of different animals including rat, mouse, hamster, guinea pig, cow, pig, deer, dog, monkey, trout and humans. Slices can be harvested from different tissues (liver, intestine, lung, kidney, brain, spleen, heart, thyroid gland, prostate, lymph node and several types of tumours) (Groothuis et al. 2014). If the effects of mycotoxins have already been investigated ex vivo using intestinal explants (Pinton et al. 2009;Alassane-Kpembi et al. 2017a) their toxicity on explants from other organs is poorly investigated. Thus, PCTS of other organs, such as kidney, spleen or reproductive tissue could provide original and useful results.

Investigation of toxic effect of emerging mycotoxins in vivo

It is important, ultimately to confirm the results obtained with in vitro or ex vivo approaches by performing in vivo trials. Contaminants that show certain effects in vitro may therefore have different effects in vivo. In contrast to in vitro studies, in vivo studies are needed to evaluate how the body as a whole, will respond to a particular substance [START_REF] Goodwin | In vitro assays of angiogenesis for assessment of angiogenic and anti-angiogenic agents[END_REF]. The toxic effects of DON have been already addressed via several endpoints in vivo, whereas the toxicity of emerging mycotoxins in vivo are fragmentary. We reported the occurrence and cocontamination of emerging and regulated mycotoxins in pig feed. Perform in vivo experiments with pigs exposed to feed contaminated at realistic level and, of course, upon regulations and ethics in animal studies, is of high interest for obtaining useful data for risk assessment.

  % des échantillons étaient co-contaminés avec du DON et d'autres mycotoxines réglementées et émergentes. La toxicité du DON et des 10 mycotoxines émergentes les plus répandues a été évaluée en mesurant la viabilité de cellules épithéliales intestinales porcines (IPEC-1) après 48 h d'exposition. Trois mycotoxines émergentes (brevianamide F, cyclo-(L-Pro-L-Tyr) et tryptophol) n'ont pas eu d'impact sur la viabilité cellulaire. Les autres toxines ont été classées dans l'ordre de toxicité suivant: apicidine> enniatine A1> DON> beauvéricine> enniatine B> enniatine B1> émodine> aurofusarine. La toxicité combinée du DON et des 10 mycotoxines émergentes a été évaluée en fonction de leurs concentrations réelles dans les aliments analysés. Nous avons observé que malgré la très forte fréquence des co-contaminations, la corrélation entre les concentrations de DON et des mycotoxines émergentes étudiées était faible. Nous avons donc évalué les effets toxiques de trois mélanges correspondant à des situations auxquelles les animaux peuvent être exposés. Le ratio n°1 a été calculé en utilisant la concentration P25 (1 er quartile) de la mycotoxine émergente et la concentration P75 (3 ème quartile) du DON. Le ratio n°3 correspondait au scénario inverse du ratio n°1. Le ratio n°2 a été calculé en utilisant la concentration médiane (2 ème quartile) du DON et de chaque mycotoxine émergente.

  Figure Number Details Page Number 1 General schematization of physical, chemical and microbial strategies used for mycotoxin reduction in food and feed chains 2 Layers of the Alimentary Canal. The mucosa, submucosa, muscularis, and serosa 3 Anatomy of different lob of pig liver 4 Four steps (an overview) of PCLS preparation 5 Preparation and incubation of liver slices 6 Total protein in PCLS 7 Total ATP contents in PCLS 8 RIN obtained from an individual control PCLS in different time of incubation 9 RIN curve of three controls animals over 20 h of incubation 10 PCLS treated with DON 10 µM. Inflammatory cytokines, gene modulation 11 PCLS treated with DON 10 µM. Lipids metabolisms, gene modulation 12 PCLS treated with DON 10 µM. Oxidative Stress, gene modulation 13 PCLS treated with DON 10 µM. Protein junction, gene modulation 14 PCLS treated with DON 3 µM and 10 µM at 20 h incubation 15 Experimental models

Figure 1 :

 1 Figure 1: General schematization of physical, chemical and microbial strategies used for mycotoxin reduction in food and feed chains (Luz et al. 2017).

Figure 2 :

 2 Figure 2: Layers of the alimentary canal. The wall of the alimentary canal has four basic tissue layers The mucosa, submucosa, muscularis, and serosa (Betts et al. 2017):

Figure 3 :

 3 Figure 3: Anatomy and different lob of pig liver (Kim and Lee 2013).

  mycotoxins and represents a target organ for food contaminants. Liver detoxifies mycotoxins via biotransformation before reaching any tissue through the general bloodstream. The biotransformation process comprises a series of biochemical reactions leading to changes in the structure of contaminants facilitating their excretion from the body. A typical xenobiotic metabolism pathway contains a variety of enzymes that are involved in both phase I and phase II xenobiotics metabolism. The examples of phase I reactions are oxidation, reduction, dehalogenation, or hydrolysis and are catalyzed by several enzymes including cytochrome P450

2 .

 2 Objectives of the studyMycotoxins are common natural contaminants present in food and feed samples(FAO 2004), and they occur throughout the world. More than 50% of the world's cereal grains are contaminated with mycotoxins. Despite of many years of research and the introduction of good agricultural and manufacturing practices, mycotoxins occurrence remains a global problem.

Figure 1 .

 1 Figure 1. (A) Worldwide contamination of deoxynivalenol (DON). Concentration of DON is highlighted by colors, where yellow indicates > 100 µg/kg, green > 200 µg/kg, dark green > 300 and dark blue indicates > 400 µg/kg. (B) Abundance of DON in pig finished feed (P25, median, mean, P75). X axis represents the distribution of the concentration, Y axis describes the number of contaminated samples.

Figure 2 .

 2 Figure 2. Dose effect curve of individual toxicity of apicidin (API). Data are mean ± SEM of three biological replicates. Bonferroni multiple comparison test. Significant difference between control and different doses of API *** p < 0.001.

Figure 3 .

 3 Figure 3. Dose-effect curves of deoxynivalenol (DON) (blue lines and symbols), emerging mycotoxins (brevianamide-F (BRV-F), cyclo-(L-Pro-L-Tyr) (Cyclo) and tryptophol (TRPT)) alone (red lines and symbols), or in combination with DON (black lines and symbols) at different ratios: ratio 1 was calculated from the P25 concentration of emerging mycotoxin and P75 concentration of DON; ratio 2 was calculated from the median concentration of emerging mycotoxin and DON; ratio 3 was calculated from the P75 concentration of emerging mycotoxin and the P25 concentration of DON. Six serial dilutions of each ratio were tested (Emerging mycotoxin alone, DON alone, mixture). Data are mean ± SEM of three biological replicates. Bonferroni multiple comparison test. Significant difference between emerging mycotoxins alone and the mixtures *** p < 0.001. Significant difference between DON alone and the mixtures # # # p < 0.001.

Figure 4 .

 4 Figure 4. Dose-effect curves of DON (blue lines and symbols), emerging mycotoxins (AFN, EMO and ENN-B1) alone (red lines and symbols) or in combination with DON (black lines and symbols) at different ratios: ratio 1 was calculated from the P25 concentration of emerging mycotoxin and P75 concentration of DON; ratio 2 was calculated from the median concentration of emerging mycotoxin and DON; ratio 3 was calculated from the P75 concentration of emerging mycotoxin and P25 concentration of DON. Six serial dilutions of each ratio were tested (Emerging mycotoxin alone, DON alone, mixture). Data are mean ± SEM of three biological replicates. Bonferroni multiple comparison test. Significant difference between emerging mycotoxins alone and the mixtures *** p < 0.001. Significant difference between DON alone and the mixtures # # # p < 0.001.

  Toxicity of DON and The Highly Toxic Secondary Metabolites (ENN-B, BEA, ENN-A1and API)

Figure 5 .

 5 Figure 5. Dose-effect curves of DON (blue lines and symbols), emerging mycotoxins (ENN-B, BEA, ENN-A1 and API) alone (red lines and symbols) or in combination with DON (black lines and symbols) at different ratios: ratio 1 was calculated from the P25 concentration of the emerging mycotoxin and the P75 concentration of DON; ratio 2 was calculated from the median concentration of emerging mycotoxin and DON; ratio 3 was calculated from the P75 concentration of emerging mycotoxin and the P25 concentration of DON. Six serial dilutions of each ratio were tested (Emerging mycotoxin alone, DON alone, mixture). Data are mean ± SEM of three biological replicates. Bonferroni multiple comparison test. Significant difference between emerging mycotoxins alone and the mixtures *** p < 0.001. Significant difference between DON alone and the mixtures # # # p < 0.001.For ENN-B and ENN-A1, the toxicity of the mixture was similar to the toxicity of DON alone, except at the highest concentrations of ENN-B (3.6 µM) and ENN-A1 (1.7 µM). The combined toxicity

Figure S5a .

 S5a Figure S5a. LC-ESI(+)-MRM chromatogram (overlay of all acquired MRMs) of AT5-4153-009 (only compounds for toxicity tests are annotated).

Figure S5b .

 S5b Figure S5b. LC-ESI(-)-MRM chromatogram (overlay of all acquired MRMs) of AT5-4153-009 (only compounds for toxicity tests are annotated).

Figure S6 .

 S6 Figure S6. Overlay of the Extracted Ion Chromatograms of both quantifier (pink) as well as qualifier (blue; filled) of the 11 investigated compounds in sample AT5-4152-9; the horizontal lines denote the target height of the qualifier (± 30% tolerance) calculated from the height of the quantifier and the target ion ratio calculated from the standards.

Figure 4 :

 4 Figure 4: Four steps (an overview) of PCLS preparation (Olinga and Schuppan 2013).

Figure 5 :

 5 Figure 5: Preparation and incubation of liver slices. Liver cores are prepared using a drill and a tissue coring tool and transferred to the cylindrical core holder of the Krumdieck slicer (Graaf et al. 2010).

Figure 6 ,Figure 6 :

 66 Figure 6, incubation time did not affect the total protein content. By contrast, 20 hours of exposure to DON 10 µM decreased by 50% the total proteins content, whereas 2 hours exposure had no effect.

Figure 7 :

 7 Figure 7: Total ATP content in a PCLS. Samples were treated by DON 10 µM during 2 h and 20 h of incubation treatments. Data are mean ± SEM of three biological replicates. Bonferroni multiple comparison test. Significant difference between control and treated groups * P < 0

5. 5 . 1 . 2 .

 512 Reverse transcription and PCRReverse transcription (RT) was performed with the Kit High Capacity cDNA-RT(Fisher Scientific, Illkirch, France). Each reaction contained 10 µL RNA (2 µg RNA) and 10 µL of the mixture (2 µL of 10X RT buffer; 0.8 µL of 25X dNTP MIX 100 µM; 2 µL of 10X RT random primer; 1 µL of RNase inhibitor, 1 µL of multiSribe reverse Transcriptase and 3.2 µL Nucleasefree H2O) (Thermo Fisher, Life Sciences). The conditions of RT reaction were 25℃ for 10 min, 37℃ for 2 h, 85℃ for 5 min, maintain at 4℃. Ultimately, 5 µL of cDNA were diluted in 95 µL of sterile water. Diluted cDNA (5 ng/µL) was stored at -20 ⁰C for the next steps of qPCR (quantitative polymerize chain reaction).

Figure 8 :

 8 Figure 8: RIN obtained from an individual control PCLS in different time of incubation: Each letter (D, E, F) represent a biological replicate (animal).

Figure 9 :

 9 Figure 9: RIN curve of three controls animals over 20 h of incubation. Data are mean ± SEM of three biological replicates.

Figure 10 :Figure 11 :Figure 12 :

 101112 Figure 10: PCLS treated with DON 10 µM. Inflammatory cytokines, gene modulation: Data are mean ± SEM of three biological replicates. Bonferroni multiple comparison test. Significant difference between treated and corresponding control at each time point * P < 0.05, ** P < 0.01, *** P < 0.001

Figure 15 :models 5 . 7 . 1 .

 15571 Figure 15: Experimental models



  many problems in predictive toxicology and furthering knowledge and reducing the need to resort to animal testing. Predictions are done based on the chemical structure and selected properties of a compound. So, modelling and simulation via in silico contribute to reduce, refine and replace animal experimentation. Mainly modelling and simulation are based on experimental data, quantitative structure activity relationship and scientific data (Cronin and Madden 2010). Following are briefly listed pros and cons of this model: Predictions for a large set of compounds in a high-throughput mode  Simulation based on the structure of a compound Performing without animals  Less cost  Difficult to extrapolate result to in vivo/ex vivo  Need special software and expert people

 5 . 7 . 3 .

 573 Rapid and easy to perform  Allow to analyze the mode of action  Adapted to assess interaction  Effect on a single cells does not represent effect on the whole body  Difficult to assess pharmacokinetics/ absorption/excretion  Difficult to extrapolate results to in vivo Ex vivo models Ex vivo models represent a tissue or slices culture of an organ, where the natural original threedimensional architecture and original cell composition and intercellular connections areretained. An ex vivo model is ethically advantageous, requires no postsurgical animal care, enables more reproducibility, and provides a tightly controlled artificial environment for toxicological studies[START_REF] Dhawan | Vitro Toxicology[END_REF]. Although, in vitro and ex vivo appear somehow similar, as both testing methods involve experiments on biological matter, conducted outside of a living organism in an artificial environment. For instance, ex vivo models are much more complex than in vitro in terms of cell diversity, and therefore closer to in vivo conditions.

mechanism

  Study animal behaviour against toxins  Assessment of zootechnical parameters  Pharmacokinetics assessment  Sampling of different organs  Authorization of protocol from Ethical Committee  Expertise required  Large number of animals for statistical analysis  Costly The two forms of in vivo experiments are animal studies and clinical trials in toxicological and pharmacological studies. Animal models are classical toxicological models used as a mandatory element in preclinical (toxicological and pharmacological) studies of new compounds (Parvova



  Need validation via in vivoIn vitro presents a rapid, easy and economic model that is widely used to investigate toxicity of mycotoxins. For instance Novak et al. assessed the individual toxicity of emerging and regulated mycotoxins in porcine intestinal cells (IPEC-J2) using an in vitro model(Novak et al.



  Studies on human liver possible  Studies on several compounds at different concentrations  Viability: 6 h to 2 days  No collection of bile possible  Not all the cells preserved similarly (inter-assay variability)

  Their individual effects lead to identify 3 classes of toxicity: Class #1 (nontoxic) included BRV-F, Cyclo and TRPT, Class #2 (moderately toxic) included AFN, EMO and ENN-B1 and Class #3 (highly toxic) included ENN-A1, B, BEA, and API. Despite of the high percentage of co-occurrence with DON, there was very low correlation between the concentration of emerging mycotoxins and the one of DON. Thus, we tested the toxicity of the mixtures based on 3 scenarios representing plausible to situations of exposure for animals. First scenario was low concentration of emerging mycotoxins + high concentration of DON (P25/P75), second scenario was median concentration of emerging mycotoxins + median of DON, whereas the 3 rd scenario was the reverse case of scenario #1 (P75/P25). In most of the cases, the combined effect was similar to the individual effect of DON, or lesser than DON.In addition to intestine, liver is the main site of detoxification for xenobiotics, including mycotoxins and represents a target organ for food contaminants. So, we developed an ex vivo model to study the effects of mycotoxins on liver, the Precision Cut Liver Slices (PCLs). The first steps of developing this tool aimed to verify the maintenance of the quality of the tissue during time. We confirmed through analyzing liver damage makers (AST, ALP, AST, LDH) and as well via RIN that quality of tissue maintained stable over time of incubation. This tool was then used to assess the toxicity of DON (3 and 10 µM) at different incubation times (0 to 20 h), by studying gene expression, ATP and total proteins content. Exposure to DON for 4 h

Table 1 :

 1 EU recommendation of mycotoxins in pig feed

			Guidance value in mg/kg
	Mycotoxins	Feedstuff	(ppm) relative to pig
			feedingstuff with a
			moisture content of 12 %
	DON	Complementary and complete feedingstuffs for pigs	0.9
		Complementary and complete feedingstuffs for piglets and gilts (young sows)	0.1
	ZEN
		Sows and fattening pigs	0.25
	The sum of
	FUMs (B1 +	Complementary and complete feedingstuffs for pig	5
	B2)
	The sum of T2 & HT2	Compound feed for pig	0.25
	Ochratoxin A	Complementary and complete feedingstuffs for pigs	0.05
	(European-Commission 2016)

Table 2 :

 2 Characteristics of DON

	Property	Information
	Name	Deoxynivalenol (DON) vomitoxin
	Chemical structure
	IUPAC name	12,13-epoxy-3α,7α,15-trihydroxytrichothec-9-en-8on
	Molecular formula	H15O20O6
	Molar mass	296.32 g/mol
	CAS number	51481-10-8
	Physical state	Colourless fine needles
	Soluble in:

Polar organic solvents (e.g., aqueous methanol, ethanol, chloroform, acetonitrile, and ethyl acetate) and water

Table 3 :

 3 EU regulation for DON in foodstuff

		Foodstuff	Maximum recommendation level (mg/kg)
	1	Unprocessed cereals other than durum wheat, oats and maize	1.25
	2	Unprocessed durum wheat and oats	1.75
	3	Unprocessed maize with the exception of unprocessed maize intended to be processed by wet milling	1.75
		Cereals intended for direct human consumption, cereal flour, bran
	4	and germ as end product marketed for direct human consumption,	0.75
		with the exception of foodstuffs
	5	Pasta (dry)	0.75
	6	Bread (including small bakery wares), pastries, biscuits, cereal snacks and breakfast cereals	0.5
	7	Processed cereal-based foods and baby foods for infants and young children	0.2
		Milling fractions of maize with particle size > 500 micron falling
	8	within and other maize milling products with particle size > 500	0.75
		micron not used for direct human consumption
		Milling fractions of maize with particle size ≤ 500 and other maize
	9	milling products with particle size ≤ 500 micron not used for direct	1.25
		human consumption
	(European-Commission 2007)

Table 4 : EU regulation for DON in feedstuff Feedstuff Guidance value in mg/kg (ppm) relative to a feedingstuff with a moisture content of 12 %

 4

	1	Feed Materials :
		 Cereals and cereal products with the exception of maize by-	8
		products
		 Maize by-products	12
	2	Complementary and complete feedingstuffs with the exception of:	5
		 complementary and complete feedingstuffs for pig	0.9
		 complementary and complete feedingstuffs for calves (< 4	2
		months), lambs and kids
	(European-Commission 2006)

Table 5 :

 5 Characteristics of ENNs

	Property	Information
	Chemical structures
	Molar mass g/mol	ENN-A 681.9	ENN-A1 667.9	ENN-B 639.8	ENN-B1 653.9
	CAS number	2503-13-1	4530-21-6	917-13-5	19914-20-6
	Physical state	Colourless fine needles
	Soluble in:

Table 6 :

 6 Cytotoxicity of ENNs in different cell lines

	Organ	Cell Type	Toxicity	Range of IC50
	Human intestinal cells	Caco-2		1.1 µM to 4.6 µM
	Human lung epithelial cells Human epidermal keratinocytes	BEAS-2B HEK	ENN-A > ENN-A1 > ENN-B1 >ENN-B	5.7 µM to 43.7 µM 2 µM to 54 µM
	Human endothelial cells	HUVEC		2.8 µM to 17.3 µM
	Human liver cells	HepG2	ENN-A > ENN-B > ENN-A1 ≈ENN-B1 3 µM to 5.6 µM
	Human gastric carcinoma cell line	N87	ENN-A1 > ENN-B1 > ENN-A >ENN-B 0.003 µM to 1.7 µM

Table 7 :

 7 Characteristics of BEA

	Property	Information
	Chemical structures
		C45H57N3O9
	Molar mass	784.0 g/mol
	Physical state	Colourless fine needles
	CAS number	26048-05-5

Table 8 :

 8 Characteristics of AFN

	Property	Information
	Chemical structures
		C30H18O12
	Molar mass	570.4 g/mol
	Physical state	Black and brown fine needles
	CAS number	88360-87-6

Table 9 :

 9 Characteristics of API

	Property	Information
	Chemical structures
		C34H49N5O6
	Molar mass	623.8 g/mol
	CAS number	183506-66-3
	Physical state	Colourless fine needles
	Soluble in:

Table 10 :

 10 Characteristics of EMO

	Property	Information
	Chemical structures
		C15H10O5
	Molar mass	270.24 g/mol
	CAS number	518-82-1

Physical state

The orange-red, crystalline Soluble in: Polar organic solvents DMP, DMSO and in water

Table 11 :

 11 Characteristics of BRV-F

	Property	Information
	Chemical structures
		C16H17N3O2
	Molar mass	283.3 g/mol
	CAS number	38136-70-8
	Physical state	White solid fine needless
	Soluble in:

Table 12 :

 12 Characteristics of Cyclo-Pro-L-Tyr

	Property	Information
	Chemical structures
		C14H16N2O3
	Molar mass	260.3 g/mol
	CAS number	357-67-5
	Physical state	White solid fine needless
	Soluble in:

Soluble in ethanol, methanol, DMF or DMSO. Limited water solubility.

Table 13 :

 13 Characteristics of TRPT

	Property	Information
	Chemical structures
		C10H11NO
	Molar mass	161.2 g/mol
	CAS number	526-55-6
	Physical state	White solid fine needless
	Soluble in:

Soluble in ethanol, methanol, DMF or DMSO. Limited water solubility.

Chemical structure and characteristics of TRPT

[START_REF] Kosalec | Genotoxicity of Tryptophol in a Battery of Short-Term Assays on Human White Blood Cells in vitro[END_REF]

Table 14 :

 14 The roles of the cells in the small intestinal mucosa[START_REF] Betts | Anatomy & Physiology[END_REF].

	Cell type	Location in the mucosa	Function
	Absorptive	Epithelium/intestinal glands	Digestion and absorption of nutrients in chime
	Goblet	Epithelium/intestinal glands	Secretion of mucus
	Paneth cells Intestinal glands	Secretion of the bactericidal enzyme lysozyme;
			phagocytosis
	G cells	Intestinal glands of	Secretion of the hormone intestinal gastrin
		duodenum
	I cells	Intestinal glands of
		duodenum

Secretion of the hormone glucoses-dependent insulinotropic peptide, which stimulate the release of insulin

K cells

Intestinal glands Secretion of the hormone glucose-dependent insulinotropic peptide, which stimulates the release of insulin

M cells

Intestinal glands of duodenum and jejunum Secretion of the hormone motilin, which accelerates gastric emptying, stimulates intestinal peristalsis, and stimulates the production of pepsin

S cells

Intestinal glands Secretion of the hormone secretin

  in vivo, in vitro and ex vivo model are generally used so far. In vitro models of intestinal mucosa have been developed for studying enteritic diseases. Ex vivo tissue cultures represent a model between in vitro and in vivo, where whole tissue slices are cultured, such as organotypical slice cultures. In the ex vivo models, the cytoarchitecture is retained as well as many of the intercellular connections and interplays. Thus, metabolic processes more closely represent the in vivo situation. In the context of reducing the number of experimental animals, intestinal explants represent a powerful model[START_REF] Kolf-Clauw | Development of a pig jejunal explant culture for studying the gastrointestinal toxicity of the mycotoxin deoxynivalenol: Histopathological analysis[END_REF][START_REF] Fadeel | Crop Prospects and Food Situation | GIEWS -Global Information and Early Warning System | Food and Agriculture Organization of the United Nations[END_REF][START_REF] Pearce | Intestinal in vitro and ex vivo Models to Study Host-Microbiome Interactions and Acute Stressors[END_REF]

2018

). Although, the in vitro studies can be criticized for being very different from the natural environment they have several advantages. First, they do not demand the technical skills in animal handling and have typically lower costs. Secondly, in vitro assays are more rapid and can be more easily performed. Furthermore, in vitro assays allow identification of direct effects on target cells. Finally, in vitro studies have the advantage of allowing genetic manipulation of cells as well as the utilization of cells and tissues from transgenic species

Table 15 :

 15 Effect of DON on the intestine in vitro

	Cells Type	Dose and time of exposure	Range or toxicity time point of	Endpoint	References
				Decrease in claudin-3 & 4
				tight junction protein
		10-50 µM (48 h)	Toxicity >5 µM	Increase in 4 kDa dextran permeability	(Pinton et al. 2009)
		TEER 2-16 days	TEER >2 days	Increase in E coli 28C translation
	IPEC-1			Decrease in TEER Decrease in TEER
		10-30 µM		Decrease in claudin-3 & 4
			>10 µM	tight junction protein	(Pinton et al. 2012)
		(24 -48 h)
				Increase in 4 Dka dextran
				permeability
		0. -13.5 µM (72 h)	>3 µM	Decrease in protein expression of ZO-1	(Diesing et al. 2011b)
		0. -13.5 µM (72 h)	>6 µM	Affect the distribution pattern of ZO-1	(Diesing et al. 2011b)
				Decrease in TEER values
		6.74 µM (1 -48 h)	>1 h	ZO-1 Affect the distribution Decrease in protein expression of claudin-3, occludin and	(Gu et al. 2014)
				pattern of ZO-1
	IPEC-J2	2.5-10 µM	>2.5 µM	Inhibit proliferation Increase LDH release	(Awad et al. 2012)
		(24 -48 h)	24 -48 h
				Decrease ATP contents
		20 µM
			-	Decreased proliferation	(Novak et al. 2019)
		(48 h)
		20 µM	>1 h	Decrease in TEER
		(0 -72 h)	TEER >7	Decrease in protein	(Springler et al. 2016a)
		TEER (30 days)	days	expression, claudin-3 & 4

Table 16 :

 16 Effect of emerging mycotoxins on the intestine in vitro

	Cells Type	Toxins	Dose and exposure time of	Range or time toxicity point of	Endpoint	References

Table 17 :

 17 Cellular microanatomy and their function

	Cells type		Function
			Hepatic regeneration
	Hepatocytes	Protein and lipids synthesis and metabolism
			Xenobiotic metabolisms
	Stellate/Ito cells	Vitamin A and fat storage Scar tissue and wound healing
	Liver Sinusoidal	Regulate vascular resistance
	Endothelial cells
	Kupffer cells	Innate immunity Ischemia reperfusion injury
	Dendritic cells	Innate immunity
	Cholangiocyte	Bile duct cells
		NK	Nonspecific targeting of tumor and viruses
	Lymphocytes	NKT T cells	Innate immunity Target lipid antigens Innate and adaptive immunity
			Cell mediated, adaptive immunity
		B cells	Humoral mediated, adaptive immunity

Table 18 :

 18 Effect of DON on liver cells

In vitro Cells Type Dose and time of exposure Range or time point of toxicity Endpoint References

				Decrease mitochondrial
				function
	Rat's MH1C1		>0.1 μg/mL
	Rat's NBL CL2	0 -25 μg/mL (24 h)	>0.1 μg/mL	Oxidative stress mitochondrial function Decrease in the	(Sahu et al. 2010)
	Rat's WRL68		>0.1 μg/mL
		0 -25 μg/mL		Oxidative stress
	HepG2	(24 h)	>0.1 μg/mL	mitochondrial function Decrease in the	(Sahu et al. 2010)
		1-10 μM (48 h)	>1 μM	Alteration of ATF3 expression	(Nielsen et al. 2009)
				Obvious cytotoxicity
	Human	0.1-100 μM
	primary		>1 μM	LDH release	(Königs et al. 2008)
	hepatocytes	(8 -48 h)
				Activation of caspase-3
		0.01 -100		Cytotoxicity
	Rat's primary	μg/mL (3 h)	>10 μg/mL	frequencies Increases of the micronucleus (MN)	(Knasmüller et al. 1997)
	hepatocytes	0.1-100
		μg/mL	>5 μg/mL	An increased percentage of large-sized nuclei	(Bradlaw et al. 1985)
		(24 h)
		0.1 -25
		μg/mL		Oxidative stress
		(24 h)	>0.1 μg/mL	Decrease in the	(Sahu et al. 2010)
				mitochondrial function
	Rat's Clone9
		0 -100		Cytotoxicity
		μg/mL
		(48 h)	>10 μg/mL	Double-stranded DNA (ds-DNA)content	(Sahu et al. 2008)
				Oxidative stress

Table 19 :

 19 Effect of DON on the liver

	In vivo

Table 20 :

 20 Effect of emerging mycotoxins on liver cells

	Cells Type	Toxins	Dose and time of exposure	Range or time point of toxicity	Endpoint	References

Table 1 .

 1 Top 60 emerging and regulated mycotoxins co-occurring with DON in finished pig feed.

		Metabolites	Occurrence n (%)	Co-occurrence with DON n	Contamination Level (µg/kg Feed)	Correlation (DON and Other Mycotoxins)
				(%)	P25	P50	P75	Coefficient p-Value
	1	Deoxynivalenol	463 (88%)	463 (100%)	111	206	389	1.00	NA
	2	Culmorin	492 (94%)	458 (99%)	38	107	247	0.50	0.00
	3	Zearalenone	502 (96%)	449 (97%)	9	18	46	0.64	0.00
	4	Brevianamide F	500 (95%)	446 (96%)	17	28	45	0.17	0.00
	5	Cyclo-(L-Pro-L-Tyr)	494 (94%)	434 (94%)	117	196	371	0.14	0.00
	6	Enniatin B	479 (91%)	430 (93%)	9	32	83	0.02	0.61
	7	Enniatin B1	481 (92%)	430 (93%)	10	37	87	0.04	0.39
	8	Asperglaucide	470 (90%)	419 (90%)	18	38	94	-0.11	0.02
	9	Emodin	472 (90%)	418 (90%)	3	5	10	-0.01	0.91
	10	Aurofusarin	464 (89%)	417 (90%)	87	211	548	0.59	0.00
	11	Moniliformin	469 (90%)	416 (90%)	7	17	45	0.11	0.03
	12	Beauvericin	464 (89%)	411 (89%)	4	7	13	0.32	0.00
	13	Enniatin A1	459 (88%)	411 (89%)	5	16	33	0.09	0.08
	14	3-Nitropropion acid	455 (87%)	407 (88%)	3	6	10	-0.03	0.57
	15	Tryptophol	454 (87%)	407 (88%)	119	197	319	0.10	0.04
	16	15-Hydroxyculmorin	429 (82%)	391 (84%)	76	142	277	0.79	0.00
	17	Equisetin	424 (81%)	386 (83%)	5	10	23	0.01	0.80
	18	Infectopyron	409 (78%)	366 (79%)	108	263	449	-0.16	0.00
	19	DON-3 Glucoside	380 (73%)	362 (78%)	10	21	47	0.79	0.00
	20	Neoechinulin A	407 (78%)	357 (77%)	10	19	42	0.06	0.22
	21	Tenuazonic-acid	384 (73%)	347 (75%)	53	90	182	0.04	0.49
	22	Alternariol	366 (70%)	333 (72%)	2	4	9	0.01	0.79
	23	Rugulusovin	373 (71%)	332 (72%)	4	7	14	0.15	0.01
	24	Tentoxin	342 (65%)	319 (69%)	2	3	6	-0.03	0.56
	25	Apicidin	341 (65%)	310 (67%)	3	7	11	-0.13	0.02
	26	Fumonisin B1	332 (63%)	304 (66%)	26	70	254	0.14	0.02
	27	Nivalenol	315 (60%)	296 (64%)	10	24	57	0.14	0.02
	28	Cyclo-(L-Pro-L-Val)	337 (64%)	286 (62%)	85	137	246	0.15	0.01
	29	Epiequisetin	307 (59%)	285 (62%)	2	4	7	0.05	0.42
	30	Citreorosein	317 (60%)	283 (61%)	3	5	8	0.12	0.05
	31	Enniatin A	306 (58%)	282 (61%)	2	3	5	-0.01	0.81
	32	Alternariolmethylether	307 (59%)	275 (59%)	2	3	5	0.09	0.14
	33	Altersetin	301 (57%)	275 (59%)	13	29	76	0.18	0.00
	34	Asperphenamate	311 (59%)	269 (58%)	5	11	27	-0.02	0.75
	35	Lotaustralin	288 (55%)	257 (56%)	15	30	85	-0.10	0.13
	36	Butenolid	253 (48%)	242 (52%)	22	37	70	0.32	0.00
	37	Kojic acid	262 (50%)	241 (52%)	43	74	148	-0.06	0.39
	38	Enniatin B2	258 (49%)	238 (51%)	2	3	5	-0.01	0.84
	39	Fumonisin B2	258 (49%)	237 (51%)	19	50	143	0.16	0.01
	40	Zearalenone Sulfate	236 (45%)	222 (48%)	10	25	53	0.25	0.00
	41	Antibiotic Y	233 (44%)	215 (46%)	40	111	402	-0.02	0.75
	42	T2 Toxin	235 (45%)	209 (45%)	2	4	9	0.12	0.07
	43	Macrosporin	219 (42%)	202 (44%)	2	3	8	0.02	0.76
	44	N-Benzoyl-Phenylalanine	220 (42%)	191 (41%)	3	5	11	-0.02	0.82
	45	Flavoglaucin	206 (39%)	175 (38%)	7	16	34	0.05	0.51
	46	Curvularin	196 (37%)	171 (37%)	2	4	8	-0.09	0.27
	47	Questiomycin A	178 (34%)	162 (35%)	4	10	20	0.22	0.01
	48	Rubellin D	179 (34%)	161 (35%)	4	8	18	0.10	0.21
	50	Bikaverin	171 (33%)	153 (33%)	10	25	56	0.27	0.00
	50	Fusarinolic-acid	157 (30%)	153 (33%)	47	130	320	0.3	0.00
	51	Fumonisin B4	165 (31%)	149 (32%)	11	23	68	0.2	0.03
	52	Cytochalasin J	170 (32%)	146 (32%)	13	29	63	0.1	0.46
	53	Ergometrine	152 (29%)	145 (31%)	6	11	24	0.0	0.57
	54	Ergocristine	151 (29%)	143 (31%)	2	5	13	0.2	0.02
	55	Fumonisin B3	154 (29%)	136 (29%)	24	48	103	0.1	0.15
	56	HT2-toxin	149 (28%)	134 (29%)	13	20	30	0.2	0.01
	57	Monocerin	144 (27%)	133 (29%)	1	2	3	0.2	0.02
	58	Chrysogin	136 (26%)	126 (27%)	7	12	17	0.4	0.00
	59	Ergosin	128 (24%)	123 (27%)	3	6	13	-0.1	0.39
	60	5-Hydroxyculmorin	121 (23%)	117 (25%)	107	170	304	0.7	0.00

Table 2 .

 2 IC 50 values of the selected emerging mycotoxins on IPEC-1 cells.

	Metabolites	Abbreviation	IC 50 (µM)	Toxicity
	Brevianamide F	BRV-F	Non-Toxic
	Cyclo-(L-Pro-L-Tyr)	Cyclo	Non-Toxic	Non-toxic
	Tryptophol	TRPT	Non-Toxic
	Aurofusarin	AFN	19.1 ± 3.4
	Emodin	EMO	19.0 ± 0.7	Moderately toxic
	Enniatin B1	ENN-B1	13.5 ± 2.5
	Enniatin B	ENN-B	4.4 ± 0.9
	Beauvericin	BEA	4.3 ± 1.8
	Deoxynivalenol	DON	3.2 ± 0.7	Highly toxic
	Enniatin A1	ENN-A1	1.6 ± 0.3
	Apicidin	API	1.5 ± 0.5

Table 21 :

 21 PCLS damage markers (ALP, AST, ALT, total protein, LDH) level in the incubation medium

				Controls				DON 10 µM
	Markers	2 h	4 h	6 h	8 h	20 h	2 h	4 h	6 h	8 h	20 h
	ALP (U/L)	2.9±0.5	4.4±1. 8	4.8±2 .3	3.9±1 .3	4.1±2. 5	4.6±1	4.7±2. 2	5.1±1. 8	4.3±2. 6	4.5±1. 2
	ALT (U/L)	4.7±0.6 5.0±1	6.3±1 .5	6.3±0 .6	5.3±2. 3	5.3±0.6	6.3±0. 6	6.0±0	5.3±0. 6	6.7±0. 6
	AST (U/L)	77±40	103±2 7	112± 57	147± 34	64±20 98±23.6	117±1 5	115±3 8	152±4 0	84±38
	LDH (U/L)	43±4.4 73±31	85±4 3	97±3 3	32±5	67±28	87±24 83±44	117±3 1	50±17
	Total proteins (g/L)	5.2±0.2	5.2±0. 3	5.4±0 .4	5.1±0 .1	5.3±0. 5	5.2±0.2	5.2±0. 2	5.3±0. 4	5.0±0. 2	5.7±0. 3
	Samples were treated to DON 10 µM over 20 h. Treated samples were compared to corresponding
				control at eac incubation time.

Table 22 2

 22 : List of selected genes assessed in PCLS in response ofDON 10 µM

	Inflammatory	Lipids	Oxidative	Junctional	Transcriptional	Protease	Housekeeping
	cytokines	Metabolism	Stress	Proteins	Factor	Inhibitors	Genes
	CCL20	SCD1	SOD1	E-Cadherin	JUN	Serpin	RPL32
	IL-6	SCARB-2	CAT	OCLN	FOS		B2-microglobulin
	IL-1b	CYP7A1	CCS	CLDN-3			TBP
	IL-10	LDLR	DUOX
	IL-22	LXR	NFKB
	IL-8	ABCG-8
	TNF-alpha	NR1H3

  that are involved in cellular function of lipid metabolism may cause an autosomal dominant disease. Mainly, this disease in liver is associated due to the deficiency of the low density lipoprotein.

	2 Definition of abbreviations:
	CCl-20 (C-C Motif Chemokine Ligand), IL (Interlukin), TNF-a (Tumor Necrossis Factor Alpha), SCD1 (Stearoyl-

CoA Desaturase), SCARB-2 (Scavenger Receptor Class B), CYP7A1 (Cytochrome P450 Family), LDLR (Low Density Lipoprotein Receptor), LXR (Liver X Receptor), ABCG-8 (ATP Binding Cassette), NR1H3 (Nuclear Receptor Subfamily 1), SOD1 (Superoxide Dismutase), CAT (Catalase), CCS (Copper Chaperone), DUOX (Dual Oxidase), NFKB (Nuclear Factor kappa-light-chain-enhancer of activated B cells), OCLN (Occludin), CLDN-3 (Claudin-3) JUN (Jun Proto-Oncogene), FOS (Fos Proto-Oncogene), RPL32 (Ribosomal Protein L32), B2 (Beta-2)-Microglobulin, TBP (TATA-Box Binding Protein) modulation

  However, it is reported that inflammatory cytokines are regulated due to DON in the intestinal cells[START_REF] Stadnyk | Intestinal Epithelial Cells as a Source of Inflammatory Cytokines and Chemokines[END_REF][START_REF] Wan | Individual and combined cytotoxic effects of Fusarium toxins (deoxynivalenol, nivalenol, zearalenone and fumonisins B1) on swine jejunal epithelial cells[END_REF]), but we found that DON can induce other genes in hepatic cells in addition to inflammatory cytokines. Other studies have also shown that in addition to inflammatory cytokines, DON can induce genes from other pathways. DON modulated

	significantly inflammatory cytokines IL-1β, IL-1α, IL-8, and TNF-α as well genes associated
	with oxidative stress MT1A, MT2A, when intestinal jejunal explants were treated with DON
	0.75-24 µM for 4 h (Luo et al. 2017). Furthermore, modulation of inflammatory cytokines (IL-
	1β, IL-1α, IL-8, TNF-α) were observed following DON exposure in vitro and in vivo (Chung
	2.25					3.0				CCS
	1.80					2.4					***
						1.8						*
						1.2
						0.6
						0.0
							C t r l	2 h	4 h	6 h	8 h	2 0 h
						5
	3.0
									Claudin3
			E-Cadherin
						**
						4
	2.4
	1.8					3
	1.2					2
	0.6					1
	0.0					0
	C t r l	2 h	4 h	6 h	8 h	2 0 h	C t r l	2 h	4 h	6 h	8 h	2 0 h
	2.0
			Occludin
	1.6
	1.2
	0.8
	0.4
	0.0
	C t r l	2 h	4 h	6 h	8 h	2 0 h

Micronucleus

Toxins 2019, 11, 727; doi:10.3390/toxins11120727 www.mdpi.com/journal/toxins

Toxins 2019,11, 727

Acknowledgements

Acknowledgments: We would like to thank Terciolo for her critical review of the paper and Daphne Goodfellow for English editing.

Funding: This research was supported by the ANR grants "EmergingMyco" (ANR-18-CE34-0014) and "Newmyco" (ANR-15-CE21-0010) and a grant from BIOMIN. A.K.K was partially supported by the regional contract 15066653.

Conflicts of Interest: B.N., T.J. and G.S. are employed by BIOMIN. However, this circumstance did not influence the design of the experimental studies or bias the presentation and interpretation of results. The other authors, A.K.K., P.G.P.P., M.N., P.P. and I.P.O. declare no conflict of interest. Then the effect of the doses of DON 3 µM and 10 µM was assessed on gene regulation at one incubation time 20 h (Figure 14). Genes were selected involved in different cellular function or process such as (inflammatory cytokines, lipids metabolism, oxidative stress, junctional proteins, transcriptional factor and protease inhibitor) (Table 22). All the genes were regulated dose dependently except CCS and SOD1 genes involved in cellular function of oxidative stress and Serpin which is the largest and most broadly distributed superfamily of protease inhibitors.

Supplementary Data of Article

Expression of some studied genes was modulated when PCLS were treated with DON 10 µM. However, expression of some genes seems to be modulated with DON 3 µM but not significantly. Only one gene of inflammatory cytokine (CCL20) was significantly regulated when PCLS were treated with DON 3 µM (Figure 14).

Overall, genes from different pathways were up regulated at 20 h of exposure in response to DON 10 µM. Most of the genes were up-regulated in longer exposure time, whereas some of the genes were down-regulated. Likewise, most of the genes were regulated dose dependently. PCLS represent the closest in vitro model of the in vivo situation. The major advantages are that the three-dimensional architecture is preserved. PCLS have been applied by many researchers [START_REF] Graaf Ia De | Precision-cut tissue slices as a tool to predict metabolism of novel drugs[END_REF]. One of the great advantages of the PCLS model is that numerous slices can be prepared from a single liver. On the other hands, PCLS can be employed for studies of xenobiotic metabolism, xenobiotic-induced cytotoxicity and genotoxicity, and to evaluate the effects of xenobiotics on hepatic enzyme activities [START_REF] Price | Use of precision-cut rat liver slices for studies of xenobiotic metabolism and toxicity: comparison of the Krumdieck and Brendel tissue slicers[END_REF].

Level of expression

Moreover, slices can be prepared from a range of tissues (including the heart, kidney, lung, intestine and spleen) from both experimental animals and human organs depending on the purpose of the study. For example, the effects of 4 h exposure of ENN-B1 alone and mixed with the most toxic trichothecene the T-2 toxin were assessed on the histopathology of piglets intestinal explants (Kolf-Clauw et al. 2013). Intestinal explants mounted in Ussing chambers were used to investigate the effect of mycotoxins on paracellular passage of FITC-dextran (Pinton et al. 2009). Mouse precision-cut liver slices (mPCLS) were incubated for 24 h to assess drug-induced liver injury [START_REF] Hadi | Mouse Precision-Cut Liver Slices as an ex Vivo Model To Study Idiosyncratic Drug-Induced Liver Injury[END_REF]. Furthermore, splenic explants were used to characterize the clinical, immunological, and parasitological features of the hamster spleen infected with the parasite Leishmania donovani [START_REF] Osorio | Identification of Small Molecule Lead Compounds for Visceral Leishmaniasis Using a Novel Ex Vivo Splenic Explant Model System[END_REF].

We investigated the toxicity of DON in the PCLS model. To the best of our knowledge this is the first study, investigating toxicity of mycotoxins using PCLS. Our aim in developing PCLS model was to introduce an appropriate alternative to the in vivo model contributing to the reduction of animal number and adapted to the simultaneous study of several toxins.

In conclusion, all above mentioned models are widely used whereas none of them can be considered as ideal model. Each model has its own advantages and limitations, but all those models give us a wide and global perspectives to use and implement them in the research. The purpose of this discussion was to explore the historical evolution of different culture models and the reasons why the continues development of different models exists.

.