Impact of mycotoxin on immune response and consequences for pig health

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THE MYCOTOXINS REGULATED IN PIG’S FEED

In animal feed, only aflatoxins (AF) are regulated in Europe. There are some recommendations (Table 1) for five other toxins, which occur regularly and which are known to be toxic to swine. There are the ochratoxin A (OTA), the deoxynivalenol (DON), the toxins T2 and HT2, the fumonisins (FB1, FB2) and the zearalenone (ZEN) (Bennett et Klich, 2003).
These molecules belongs to different families of mycotoxins, with various chemical structures, and so various toxic effects on pig. The dose, time of exposure, the specie, the age and the status of the animal (Bryden, 2007; Wild, 2007).
The Table 2 lists the major known effects of these mycotoxins on pig health.
AF are quickly absorbed and metabolized in the liver by the microsomal system which actives or modifies the metabolites (Riley, 1998; Haschek et al., 2002). AF alters the global immune response (innate and cellular) in pigs (Meissonnier et al., 2006).
OTA is mainly toxic to the liver and kidneys and causes kidney diseases in pigs. OTA affects the renal proximal tubule (Krogh, 1987; Marquardt and Frohlich, 1992). Moreover OTA acquires a genotoxic effect after its metabolization in the body (Ash et al., 2004; Pfohl-Leszkowicz and Manderville, 2007; Steyn et al., 2009). DON is the most common trichothecenes B. Pig is very sensitive to this mycotoxin, which can induce at low concentration feed refusal, and in higher concentrations vomiting (Haschek et al., 2002). Chronicle doses of DON (low concentrations on the long term), induce in pigs weight loss, anorexia, immunomodulation and a modification of the intestinal barrier function (Trenholm et al, 1984;. And Rotter al., 1996; Haschek et al., 2002; Pinton, Oswald, 2014). Toxins T2 and HT2 that belong to the family of trichothecenes A have similar effects but more pronounced than Trichothecenes B. They induce irritation to the gastrointestinal tract and skin, and they increase the sensitivity of the animal disease (Bryden, 2012).
Fumonisins are constituted of 12 compounds including fumonisin B1 (FB1), which is the most toxic and most studied. Fumonisins induce multiple toxic effects on animals with a known carcinogenic effect. In pigs, the FB1 affects the specific and the humoral responses by altering the balance of helper T cells, TH1 / TH2 (Taranu et al., 2005; Marin et al., 2006)
FB1 induced pulmonary edema in pigs (Haschek et al., 2002). Zearalenone (ZEN) has a significant effect on reproduction and fertility especially in swine. The α-zearalenol (α-ZEL) and β-zearalenol (β-ZEL), from the reduction of ketones by ZEN-reductase of the host, are non-steroidal estrogens that induce estrogenic activity in the animal (Fink-Gremmels and Malekinejad, 2007). ZEN and its derivatives cause redness and swelling of the vulva, vaginal prolapse and sometimes rectal prolapse in sows. In young sows, they can induce a significant swelling of the vulva (Gaumy et al., 2001).

« MODIFIED» MYCOTOXINS IN PIG FEED

Presentation

New analytical methods allowed putting in evidence new secondary metabolites and some molecules derivate from these mycotoxins. The term of “masked” mycotoxins” was introduced in 1990 by Gareis to describe a glucoside zearalenone not detected during routine analysis, but hydrolyzed during digestion (Gareis et al., 1990).
Indeed, different changes can occur in the structure of mycotoxin, which make them undetectable by conventional analytical techniques (Table 3). There are biological changes (did by a plant, fungus or animal body) or chemical ones such as the ones caused during thermal food processing methods.
The name of « masked mycotoxin » has often been an ambiguous use, and recently some authors have proposed a more precise terminology for the various forms of mycotoxins (Berthiller et al, 2013;. Rychlik et al, 2014).
These authors have redefined the terminology of « masked mycotoxin » strictly and introduced the concept of « modified mycotoxins. » Figure 1 shows for example, all the forms described for DON.
Mycotoxins called « native or free » correspond to the basic structures of mycotoxins formed by molds. Most likely to be found in the pig supply is DON, ZEN, fumonisin, aflatoxin and OTA.
Matrix-associated mycotoxins correspond to the « native » mycotoxins bound to a matrix, i.e. physically dissolved and / or trapped and / or forming a covalent bond with the matrix. Thus, Fumonisins are able to bind to polysaccharides or proteins by their two tricarballyliques acids chains, thus forming the hidden fumonisins (hidden F) or linked with starch (F related to starch) (Seefelder et al., 2003).
Excepted these binding phenomena in a matrix, « native » mycotoxins can undergo biological or purely chemical transformations. The term « modified mycotoxin » was proposed to describe any biological or chemical modification of the chemical structure of a « native » mycotoxin (Rychlik et al., 2014).
« Biologically modified » mycotoxins indicate compounds derived from biotransformation in an animal body, plant or a mold. Biotransformation are divided into two main types: Phase I reactions (oxidation, reduction or hydrolysis) and phase II reactions (conjugation).
Generally, biotransformation allows detoxification of toxics, for example in facilitating their excretion. However in some cases, it can lead to a more toxic molecule than the parent compound. This is for example the case of aflatoxin B1-epoxy which is derived from the oxidation of AFB1 by cytochromes P450 during the biotransformation reactions of stage I in animals. Glucuronide forms (DON3-GlcA, ZEN14-GlcA, T2-GlcA, HT2-3 / 4-GlcA) come from the Phase II biotransformation of the « native » mycotoxins corresponding by the animal, and represent examples of mycotoxins called « biologically modified – conjugated ». They correspond to the excretion of the native mycotoxins in animal body.
DON-3-β-D-glucopyranoside (D3G) and zearalenone-14-β-D-glucopyranoside (ZEN14G) are issued from DON or ZEN respectively after the phase II biotransformation of metabolization by plant. By convention, the terminology of « masked mycotoxin » was reserved only for the « biologically modified » mycotoxins from the conjugation reaction in a plant (Berthiller et al., 2013).
At present, the four major « hidden » mycotoxins in the strict sense are the ZEN14G, the D3G, T2 toxin-glucoside (T2-Glc) and HT2 toxin-glucoside (HT2-Glc) (Lattanzio et al. 2012). It is interesting to note the case of the acetylated derivative of 3ADON, an acetylated derivative of DON. This compound can be produced both by the fungus, in this case it is a « native » mycotoxin, and by transgenic varieties of rice, wheat and barley expressing the gene of the 3-O-acetyltransferase, and therefore considered as a « hidden »mycotoxin. Gene transfer of the 3-O-acetyltransferase to plants is a promising strategy to reduce the pathogenicity of Fusarium that affect some plant species. Indeed, it’s established that the conversion of DON into 3ADON by the plant can limit the aggressiveness of Fusarium (Karlovsky, 2011).
Other mycotoxins may be « biologically modified » by the action of a microorganism, and are grouped under the term of “mycotoxins differently modified”. The Deepoxy-DON (DOM-1) and 3-epi-DON, resulting from the transformation of DON by bacteria extracted from human microbiota or animal, belong to this group (Eriksen et al., 2002; Karlovsky, 2011; Gratz et al., 2013).
« Chemically modified » mycotoxins are the last group. The chemical modifications may or may not depend from the heat. « Chemically modified- thermally formed » mycotoxins appear during food processes such as baking, roasting, freezing or extrusion. These thermo-dependent changes are known for many mycotoxins, in particular fumonisins capable of entering into a Maillard reaction, due to the reduction of sugars with the production for example of fumonisin B1 N- (1-deoxy D-Fructos-1-yl) and fumonisin N- (carboxymethyl) (Hmph and Voss, 2004).
We can also mention the derivatives of DON (Nordon A-F and 9-hydroxymethyl DON lactone) as thermal degradation products; some of which can be found in commercial food samples (Bretz et al., 2005). « chemically modified – non-thermally formed » mycotoxins are formed by different processes, including hydrolysis carried out with fumonisins (HFBx), sulfation of DON leading to DON-sulfonate or the degradation products of ochratoxins by UV rays (Beyer et al, 2010;. Heydt-Schmidt et al, 2012.).

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Occurrence of “natives” and “modified” mycotoxins

Some « modified » mycotoxins, particularly the « hidden » forms but also the “matrix-associated” forms and some chemically modified forms can be found in pig feed. Table 4 shows data of occurrence of major mycotoxins and their « modified » forms in cereal samples (wheat, barley, corn, oats and rice) over the period from 2010 till 2014.
The « native » mycotoxins represent the major part in food contamination. However, other forms are also concomitantly found in foods. It is currently possible to detect many « modified » mycotoxins, but few quantitative data are available, particularly because of a lack of analytical standards and reference materials.
Table 5 provides more information on the proportion of certain « modified» mycotoxins for which few data are available, compared to their « native » form. For some mycotoxins, such as T2-HT2-Glc and Glc, the occurrence data are from only one study. Their presence was reported for the first time in 2012 in wheat and oats naturally contaminated (Lattanzio et al, 2012).
For the D3G, discovered earlier, more data are available on its occurrence and its ratio to DON. The proportion of this « masked » mycotoxin is stable in food and corresponds, to almost, 20% of the DON present (Berthiller et al, 2009). However, the ratios vary depending on the cereal, genotype concerned, the country and the year of harvest and can increase up to 46%. (Berthiller et al, 2009; De Boevre et al, 2012). Also the increasing use of Fusarium resistant plants, able to glucosylate DON in D3G, could increase the ratio D3G / DON. Some studies of these resistant plants have even found up to 2.7 times more D3G present in the plant than DON (Sasanya et al, 2008).
In terms of matrix- associated fumonisins (physically entrapped), their proportions compared to the ones of free fumonisins are more variable. Their presence has been shown after a hydrolysis step of raw materials (Dall’Asta et al., 2009). The proportion of physically trapped forms change according to the genotype of corn and according to the culture conditions (Dall’Asta et al., 2012).
In conclusion, more occurring data on different crops and in different countries are needed to properly assess the risk associated with the presence of these new mycotoxins.

Metabolization and toxicity of “modified” mycotoxins on pig

The occurrence of « modified » mycotoxins in feed and animal exposure to these new toxins raise a number of questions and the need to investigate the metabolism and toxicity of these compounds (EFSA, 2014). It is important to study the intrinsic toxicity of these toxins, but also to know their metabolism and in particular to determine if « modified » mycotoxins are converted into their « native » forms.
Some recent studies were interested in the effects of these « modified » mycotoxins on the pig, on in vitro or in vivo models. Most of these studies focus on the metabolism of these molecules and few about their toxicity.

Table of contents :

INTRODUCTION
I.Context general of study
II.Problem of DON contamination
III.Literature review
A. Mycotoxins in Feed: impact on pig intestinal health (Review n°1)
Introduction
1. Toxicity of the main mycotoxins in pig feed
2. Effects of mycotoxins on the pig intestine
3. Consequences of intestinal toxicity of mycotoxins for pig health
Conclusion
References
B. Impact of mycotoxin on immune response and consequences for pig health (Review n°2)
Introduction
1. Effect of major mycotoxins on the pig immune response
2. Consequences of mycotoxin induced immunomodulation for pig health
3. The problem of mycotoxins co-contamination
Conclusion
References
C. Masked mycotoxins: a risk in pig production? (Review n°3)
Original Version (French)
English Version
Introduction
1. The mycotoxins regulated in pig’s feed
2. ”Modified” mycotoxins in pig feed
3. Assessment of the exposure and characterization of the risk for the “modified” mycotoxins in
pig
Conclusion
References
AIMS OF THE THESIS
EXPERIMENTAL STUDIES
I. Toxicity assessment of derivatives of deoxynivalenol (DON)
A. Intestinal toxicity of the masked mycotoxin deoxynivalenol-3 -glucoside (Article n°1)
Introduction
Materials and Method
Results
Discussion
References
B. Microbial biotransformation of DON: molecular basis for reduced toxicity (Article n°2)
Introduction
Materials and Methods
Results
Discussion
References
II. In vivo toxicity of purified deepoxy-deoxynivalenol (DOM-1) in piglets (Article n°3)
Introduction
Materials and Method
Results
Discussion
References
GENERAL DISCUSSION & PERSPECTIVES
I. Discussion
A. Discussion on the analysis performed in the thesis
1. In silico analysis of the interaction between DON, DON derivatives and the ribosome
2. Pan genomic analysis of the effect of DON and its derivatives
3. The in vivo experimental protocol
B. Toxicity assessment of biological strategies to reduce toxic effects of DON
1. Efficiency of detoxifying strategies
a) Bacterial transformation
b) Plant transformation
II.Perspectives
CONCLUSION

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