Proiectul PN 2 - IDEI 101

Project PN 2

Project title: Studying the toxicity of zearalenone, natural contaminant of feed, using new tools of nanobiotehnology

Financial support: UEFISCDI

Project code: PN-II-ID-PCE-2011-3-0339

Contract no.: 101

Project abstract:

Zearalenone (ZEA), a mycotoxin produced by several fungi of Fusarium genra could contaminate food and feed being a risk factor for both a animal and human health and agronomic perspective. Most studies revealed the specific toxic effect of this toxin on the reproductive apparatus in laboratory and farm animals. In addition, ZEA has been shown to be immunotoxic in low concentrations. The pig, a great cereal consumer, is considered by EFSA the most sensitive species to ZEA action (estrogenic and other effects). The economic losses caused by feed contamination with ZEA can be important for this species, particularly during the weaning period, which is much more difficult for the pigs than for other animal species. Although the studies on ZEA intensified lately, the mechanisms of its toxicity are yet to be ascertained. Using new tools of genomics and proteomics (PCR microarrays, real time PCR, western blot, ELISA), the aim of the present project is to obtain a better insight in the mechanisms of action of ZEA and the changes produced by this toxin in gene expression of pig immune response and signalling pathway driving activation of genes responsible for the immune competence at local and systemic level. The project will develop a microarray study (not been performed until now) in order to investigate the possible genotoxic effect of ZEA in pig. Using data generated by the in vitro and in vivo studies the project will contribute to the determination of the maximal tolerance levels (regulation) for ZEA in the growing pigs.

Objective:

Using new tools of genomics and proteomics (PCR microarrays, real time PCR, western blot), the aim of the present project is to obtain a deep insight in the mechanisms of action of ZEA at the cellular and molecular level and the changes produced by this toxin in genes involved the pig immune response and cellular signalling pathway responsible for the defence processes. . Using data generated by the in vitro and in vivo studies the project will contribute to the determination of the maximal tolerance levels (regulation) for ZEA in the growing pigs.

The European Food Safety Authority (EFSA) have recommended that the EU regulations concerning the tolerance levels for food and feed contaminants have to take into consideration the concentrations with no effect both for animal performance and also for other important parameters reflecting the animal health, such as the immune defence. More recently, EFSA (EFSA Journal, 2010) recommends conducting in-depth studies to document that the doses accepted as maximal limits have no genotoxic effects. For many contaminants of the agro-food chain there are no norms of tolerance issued by the European Commission, or by the national Ministries of Agriculture and the National Sanitary Veterinary Agencies. For zearalenone and other contaminants (ochratoxin, deoxynivalenol, fumonisin B1+B2) the tolerance limits are regulated by the European Union only through recommendation CE/576/2006 which means that new experimental evidence (in-depth investigation at the cellular level) are required in order to confirm the proposed/recommended limit and record it as limit. The elaboration of a regulation and its enactment is imperiously necessary.

Method and approach:

The research methodology used in this project is adapted to each specific objective and promotes the use of new technologies in cell toxicology, functional, proteomic and bioinformatics genomics. These technologies supply the required technical-scientific data in order to estimate the tolerance level for zearalenone in growing pigs.

The project involves two complementary research approaches:

1) In vitro approach, including studies on intestinal cell cultures treated with different concentrations of mycotoxin and analysed by DNA microarray and qPCR in order to assess the effect of zearalenone on the genes responsible for the local intestinal and systemic defence response; the in vitro studies have been used two type of intestinal epithelial cells (pig IPEC-1 and human Caco-2).

2) Integrated in vivo approach including feeding trials in growing pigs in order to assess the effect of ZEA on different pig responses.

Project team:

• Taranu Ionelia-coordinator
• Marin Daniela-team member
• Pistol Gina-team member
• Stancu Mariana-team member
• Calin Loredana-team member
• Gras Mihai-team member
• Ghita Elena-team member
• Pelmus Rodca-team member
• Lazar Cristina-team member

Project Workplan:

Results of in vitro studies (phase 2 and 3)

Using a DNA microarray, the genome wide response specifically expressed in response to low concentration of ZEA and the various pathways that control intestinal barrier function was evaluated on porcine (IPEC-1) and human (Caco-2) intestinal epithelial cells. After exposing intestinal porcine epithelial cells (IPEC-1) to 10 M of ZEA for 24h, genes expression profiles were analysed by microarray and qPCR. The results obtained in in vitro studies with porcine IPEC-1 suggest that zearalenone at a low, non-cytotoxic concentration (10 M) induced significant changes in gene expressions of porcine intestinal cells that were clearly distinct from the control. The microarray analyses identified 1954 genes with an altered profile compared to the control group, 1164 being down-regulated and 792 up-regulated. 190 genes were significantly (P<0.05) differentially expressed, of which 70% were up-regulated, most of them being involved in signalling pathways (35 genes) and transcriptional regulation (16 genes). These alterations concern among other the intestine immune response to pathogens by over expression of ITGB5 gene, which plays a key role against the attachment and adhesion of ETEC to porcine jejunal cells, TFF2 implicated in mucosal protection, as well as the oxidative stress by up-regulation of glutathione peroxidase enzymes (GPX 6, GPX2, GPX1). The deeper insight obtained by microarray analyses revealed the effect of ZEA on transcription. Upon ZEA challenge GTF3C4 (general transcription factor IIIC, polypeptide 4) gene responsible for the recruitment of polymerase III and initiating of tRNA transcription in eukaryotes and STAT5B (signal transducers and activators of transcription 5b) were significantly higher induced. Further in vivo studies are needed to confirm these results (Taranu et al., Toxicol Letter, 2014).

In vitro ZEA is a genotoxic mycotoxin able to modify the expression of genes implicated in various biological and molecular processes and the cellular components also in human intestinal epithelium. After exposing intestinal human cells (Caco-2) to 10�M of ZEA for 24h, our microarray analyses results revealed that the up-regulation of gene expression was the most common effect produced by ZEA in Caco-2 cells. Microarray analyses identified 377 genes differentially regulated in comparison to the control, of which 11 genes were down-regulated and 319 up-regulated with a fold change = 2. 102 of up-regulated genes had a fold change between 3 and 6. The highest expressed gene (19.26 fold change) was STRN3 gene enconding for calmodulin protein which mediate many crucial processes such as inflammation, metabolism, apoptosis etc (Taranu et al., Bulletin UASVM Animal Science and Biotechnologies, 2014).

Microarray data also reveal that pig is more sensitive to mycotoxins effect that human.

Results of in vivo studies (phase 3 and 4)

The toxicity of ZEA (250 and 100ppb) was evaluated in growing pigs in in vivo two feeding trials (250ppb ZEA-trail 1 and 100ppb ZEA-trial 2). Two groups of weaned piglets with an initial average body weight of 9.5 0.6 Kg were fed the control or ZEA-contaminated diet and studied for 30 days. Our results showed that ZEA neither at higher (250ppb) or lower (100ppb) did not influence the performance (average daily gain, daily feed intake or feed: gain ratio) of pigs fed contaminated diet.

Plasma biochemistry constituents as well as the concentration of non-specific immunoglobulin subsets (IgM, IgA, IgG) were not affected by the two mycotoxin levels, the observed differences being insignificant in comparison with the control group. However, a trend towards a decrease in glucose, iron and Gama GT concentration, not statistically significant for the duration of the experiment, was identified in the plasma of pigs receiving the diet with higher concentration of ZEA.

The in vivo results suggest that at higher level ZEA is a hepato- immunotoxic Fusarium toxin in pigs. By inhibiting the gene expressions and protein concentrations of MAPK (TAK1/JNK/p38) and NF-kB, the key effectors of signal transduction pathways, this mycotoxin caused severe hepatic immunosuppression (significant reduction in the expressions and protein concentrations of several markers of pro- and anti-inflammatory processes), which might have important consequences during an infection process. The toxic action of ZEA begins in the upstream of the signal transduction pathway of MAPK by the inhibition of the expression of TAK1 gene, an activator of MAPK and NF-kB (Pistol et al., British J. Nutr., 2014).

The in vivo results suggest also that ZEA has a toxic effect at spleen level. Indeed, our results indicate that ZEA increased in spleen the expression and the synthesis of pro-inflammatory cytokines (TNF-a, IL-8, IL-6, IL-1). The pattern of spleen inflammatory cytokines observed in this experiment is in contrast with the results obtained in liver which showed a dramatically decrease for all pro-and anti-inflammatory cytokines and signaling molecules p-38MAPK and NF-kB under the effect of ZEA dietary contamination.

Publications:

Peer review articles (ISI):

1. Streit E., Schatzmayr G., Tassis P., Tzika E., Marin D., Taranu I., Tabuc C., Nicolau A., Aprodu I., Puel O. and Isabelle P. Oswald. 2012. Current Situation of Mycotoxin Contamination and Co-occurrence in Animal Feed Focus on Europe. Toxins, 4: 788-809.

2. Daniela E. Marin, Gina C. Pistol, Ionela V. Neagoe, Loredana Calin, Ionelia Taranu. Effects of zearalenone on oxidative stress and inflammation in weanling piglets. Food and Chemical Toxicology, 58: 408-415.

3. Gina Cecilia PISTOL, Mihail Alexandru GRAS, Daniela Eliza MARIN, Florentina ISRAEL-ROMING, Mariana STANCU and Ionelia TARANU. 2014. Natural feed contaminant zearalenone decreases the expression of important pro-and anti-inflammatory related mediators and MAPKs/NFkB signaling molecules in pig. British J. Nutr., 111: 452-464.

4. Ionelia TARANU, Cornelia BRAICU, Gina Cecilia PISTOL, Daniela Eliza MARIN, Loredana BALACESCU, Ioana Beridan-NEAGOE, Radu BURLACU. 2014. Exposure to zearalenone mycotoxin alters in vitro porcine intestinal epithelial cells by differential gene expression. Toxicology Letters, 232: 310-325.

5. Ionelia TARANU, Aurora ARGHIR, Gina Cecilia PISTOL, Daniela Eliza MARIN, Ana-Maria NICULESCU. 2014. Effect of Fusarium mycotoxin Zearalenone on gut epithelium. Bulletin UASVM Animal Sciences and Biotechnologies, 72, 315-316.

Scientific communications:

Results of in vivo studies (phase 5 and 6)

Bioinformatic and biostatistic analyse of in vivo data; correlation of in vitro and in vivo results (phase 5 and 6)

During the 2015 and 2016 phases the biochemical, bioinformatic and statistical analyses of the parameters produced by the in vivo experiments were further conducted:

-The effect of zearalenone (100ppb) on the genes expression involved in the immune response measured by real time PCR in organ tissues collected in the second in vivo experiment (liver, spleen, duodenum and colon) showed that 100ppb of ZEA is able to modulate the expression of genes enconding for pro-inflammatory cytokines and antoxidative enzymes, but only for some of them the difference against the control is statistic significant; the different modulation (over-or under expression) is organ specific: e. g. activation in spleen (IL-1, IL-8) and kidney (IL-6) and inhibition in duodenum (IL-8) and liver (CAT, SOD). ZEA 100 ppb modulated differently the gene expression of enzymes involved in oxidative stress and the effect is also organ specific: e. g. activation in kidney (SOD, CAT, GPx) and inhibition in liver (SOD, CAT) and duodenum (CAT, SOD). The difference is significant only of kidney. No differences in their activity was found.

- the microarray analysis on intestine and spleen samples collected in the second in vivo experiment of the project was refined and, using INGENUITY software, it was extrapolated to the human genome. A prediction of the effect of zearalenone in human was thus obtained. The bioinformation analysis revealed 48 genes whose expression was under-expressed and 55 whose expression was over-expressed under the effect of zearalenone, with fold change higher than 2.

- ZEA affected the gene encoding for transporting proteins (e.g. CLCA4), transmembrane proteins (TMEM233), proteases (SERPINA 3), phospholipases (PNPLA4), olfactory receptors (OR2T2), membrane receptors (STRA6) etc in spleen. ZEA suppressed the gene encoding for MYD88, important for viral infections in intestine and modulated also in intestine the Toll like receptors genes (suppression of TLR4, responsible for innate immune response to E. coli and activation of TLR1, mediator of cytokine synthesis.

Statistical correlations to compare the results obtained in vitro on intestinal cells and in vivo on intestinal-duodenum tissue regarding the inflammatory response elicited by zearalenone were further realized during the 5 and 6^th phases. A negative correlation regarding the effect of ZEA on the genes expression encoding the synthesis of several pro-inflammatory markers (TNF-alpha, IL-1beta and IL-8) was found; these genes increased in vitro at the cellular level and decreased in vivo at tissue level. By contrast, a positive correlation for others (IL-6 and IFN-gamma) and the anti-inflammatory genes (IL-4 and IL-10) was noticed.

Positive correlation were identified either for in vitro or in vivo between gene expression and protein level for both pro- and anti-inflammatory markers. Positive correlation between vitro and vivo were found for blood cell proliferation which decreased under the ZEA action in both type of investigations.

Publications:

Peer review articles (ISI):

1. Daniela Eliza Marin, Monica Motiu and Ionelia Taranu. 2015. Food Contaminant Zearalenone and Its Metabolites Affect Cytokine Synthesis and Intestinal Epithelial Integrity of Porcine Cells. Toxins, 1979-1988.

Proceeding articles:
2. Taranu Ionelia, Gras Mihai Alexandru, Pistol Gina, Motiu Monica, Marin Daniela, Stancu Mariana. 2015. Investigation of zearalenone tolerance limit in the feedstuffs for weaned pigs. Lucrari stiintifice seria zootehnie, proceeding of USAMV-Iasi Symposium (Modern Animal Husbadry-Food Safety and Durable Development), Iasi, 22-24 oct. 2015.

Disemination articles

Ionelia TARANU, Daniela Eliza MARIN, Gina Cecilia PISTOL, Mihail Alexandru GRAS,Catalin ROTAR, Mihai Lauren?iu PALADE, Mariana STANCU, Loredana Georgeta CALIN. 2016. Mycotoxins and recomandation for tolerance limits in feed for pigs.

Scientific communications: