The mycotoxins deoxynivalenol and nivalenol show in vivo synergism on jejunum enterocytes apoptosis
Graphical abstract
Introduction
The worldwide contamination of agricultural grain commodities by mycotoxins raise a high concern for food and feed safety (Streit et al., 2013). Mycotoxins are low-molecular-weight secondary metabolites produced by toxigenic fungi (Bouhet and Oswald, 2005).
The Fusarium fungi are commonly found on cereals grown worldwide in the temperate regions. They produce mycotoxins, including deoxynivalenol (DON) and nivalenol (NIV), often associated. F. graminearum and F. culmorum on wheat are both co-producers of DON and NIV (Logrieco et al., 2002, Bottalico and Perrone, 2002). These toxins cause a variety of toxic effects in both animals and human (Creppy, 2002). DON may have adverse health effects after acute or chronic exposure. Acute administration of DON at high dose decreases feed consumption (up to anorexia), and induces emesis of neurogenic origin (Gaigé et al., 2013). Repeated ingestion of low dose DON in pig induced intestinal changes and might predispose animals to infections by enteric pathogens (Pinton et al., 2009, Bracarense et al., 2012). DON is capable to disrupt proliferation, to induce programmed cell death and to alter genes expression (Pestka, 2010). Pig is the most sensitive species to DON and to NIV toxicity (EC, 2000, Pestka and Smolinski, 2005). Because of a digestive physiology very similar to that of human (Kararli, 1995), pig can be regarded as the most relevant animal model for extrapolating to human (Rotter et al., 1996).
NIV is considered to be one of the mycotoxins needing regulation (SCOOP European Union, 2003, EFSA, 2013). However, the occurrence of NIV contamination is limited to some parts of Europe and Asia. Consequently, NIV has been poorly studied, and the health risks have not been evaluated (EFSA, 2013). In vitro, NIV inhibited proliferation of human lymphocytes (Thuvander et al., 1999). In young male pigs fed with 2.5 or 5 mg purified NIV/kg feed for 3 weeks, there was no sign of altered feed intake or body weight and no vomiting or clinical sign (Hedman et al., 1997).
Globally, mycotoxin co-occurrence is common (Schatzmayr and Streit, 2013), particularly in finished feed: for example 58% of 4548 samples contained two or more mycotoxins (Streit et al., 2013). DON and NIV usually co-occur in grains and grain products, and the DON concentration is generally higher than that of NIV (Schothorst and Van Egmond, 2004, Yazar and Omurtag, 2008). DON and NIV were detected in 57% of 11,022 samples, and in 16% of 4166 food samples, respectively, from European Union (Schothorst and van Egmond, 2004). The combination of NIV with DON resulted in an additive effect in vitro on human lymphocytes (Thuvander et al., 1999). Interaction between DON and NIV were synergistic in vitro on Caco-2 cells (Alassane-Kpembi et al., 2013). Less additive effect was observed in ex vivo explants (Kolf-Clauw, data not published).
Research has been recently focused on gastrointestinal tract toxicity because it is the first organ exposed to food/feed contaminants (Haschek et al., 2010) playing multi-function roles in regulation, storage, propulsion, digestion, absorption, secretion, barrier activity and elimination (Haschek et al., 2010, Gelberg, 2012, Pinton and Oswald, 2014). In the approach of “3 Rs”-Replacement, Reduction and Refinement (Russel and Burch, 1959), alternatives to animals experiments are needed for this research. Many biological models have been used in toxicity study such as in vitro (cell lines: Pinton et al., 2009) and ex vivo (explants: Kolf-Clauw et al., 2009), in parallel to conventional animal experiments (Hedman et al., 1997). The intestinal loops, an in vivo model, have been developed previously in parasitology or in bacteriology studies (Pernthaner et al., 1996, Gerdts et al., 2001, Vandenbroucke et al., 2011). Jejunal loops were previously shown to allow to analyze the in situ effects of toxins on intestinal mucosa (Cheat et al., 2015).
In our previous study, the digestive effects of DON and NIV were investigated in explants and in loops after 4-h exposure, and we identified villus apoptosis and proliferation as sensitive endpoints (Cheat et al., 2015). In the present study, we investigated these endpoints to compare the digestive effects of DON and NIV alone or associated, after a single 24-h exposure in loops, or after 28-day repeated exposure of pigs.
Section snippets
Purified toxins
DON was acquired from Sigma (St Quentin Fallavier, France) and NIV from Waco Pure Chemical Industries LTD (Osaka, Japan). Stock solutions of these mycotoxins were dissolved in dimethyl sulfoxide (DMSO) at 30 mM DON and NIV for the loops experiments. These stock solutions were stored at −20 °C. Working dilutions were prepared in physiological saline solution. The concentrations at 0 (Ctrl), 1, 3 and 10 μM were used for the dose–response of individual or combined mycotoxins.
Loops
For the loops
Loops model
The loop results were obtained from 2 pigs after 24 h in situ incubation.
Models
A conventional experimental animal model (animal experiment) was used to confirm endpoints described with loops model. The animal experiment allows to expose the animals to natural contaminated feed repeatedly (28-day study). Loops model is the alternative model that enables to reduce the number of animals in toxicology study. The interest of the loops model is that it has tremendous advantages compared with the models using individual pigs because one pig can provide approximately 30
Conclusion
Enterocytes apoptosis and total-cell proliferation at the villus tips were affected by DON and DON + NIV in both models. The loops allowed to show the synergism between DON and NIV on enterocytes apoptosis. Taking together from the previous study (Cheat et al., 2015), our data indicate that intestinal loops model, in the context of 3 Rs, represents a relevant and sensitive model to investigate the digestive effects of food contaminants. Limitations to unique exposure and to non-natural
Acknowledgment
This study was supported by the DON & Co project from ANR-10-CESA. S Cheat was supported by doctoral fellowships from TECHNO I Scholar Program, Erasmus Mundus. The authors are grateful to the CIRE team for the animals care and handling, to the E5-team for helping in animal experiment sampling, to C Bleuart and I Pardo for technical assistance for immunohistochemistry, and to F Lyazhri for statistical advice. The loops surgery was conducted at INRA Centre de recherche Val de Loire (Plate-forme
References (63)
- et al.
New insights into mycotoxin mixtures: the toxicity of low doses of type B trichothecenes on intestinal epithelial cells is synergistic
Toxicol. Appl. Pharmacol.
(2013) - et al.
The effects of mycotoxins, fungal food contaminants, on the intestinal epithelial cell-derived innate immune response
Vet. Immunol. Immunopathol.
(2005) - et al.
Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors
Adv. Enzyme Regul.
(1984) Update of survey, regulation and toxic effects of mycotoxins in Europe
Toxicol. Lett.
(2002)- et al.
Systemic and local effects of the Fusarium toxin deoxynivalenol (DON) are not alleviated by dietary supplementation of humic substances (HS)
Food Chem. Toxicol.
(2012) - et al.
C-Fos immunoreactivity in the pig brain following deoxynivalenol intoxication: focus on NUCB2/nesfatin-1 expressing neurons
Neurotoxicology
(2013) - et al.
Multiple intestinal “loops” provide an in vivo model to analyse multiple mucosal immune responses
J. Immunol. Methods
(2001) - et al.
Deoxynivalenol alone or in combination with nivalenol and zearalenone induce systemic histological changes in pigs
Exp. Toxicol. Pathol.
(2015) - et al.
Enterocyte apoptosis and proliferation are increased in microvillous inclusion disease (familial microvillous atrophy)
Hum. Pathol.
(2000) - et al.
Development of a pig jejunal explant culture for studying the gastrointestinal toxicity of the mycotoxin deoxynivalenol: histopathological analysis
Toxicol. Vitro
(2009)
Lutein protects HT-29 cells against Deoxynivalenol-induced oxidative stress and apoptosis: prevention of NF-κB nuclear localization and down regulation of NF-κB and cyclo-oxygenase - 2 expression
Free Radic. Biol. Med.
Impact of deoxynivalenol (DON) contaminated feed on intestinal integrity and immune response in swine
Food Chem. Toxicol.
Pro-apoptotic effects of nivalenol and deoxynivalenol trichothecenes in J774A.1 murine macrophages
Toxicol. Lett.
Role of oxidative stress in Deoxynivalenol induced toxicity
Food Chem. Toxicol.
The immune response of sheep surgically modified with intestinal loops to challenge with Trichostrongylus colubriformis
Int. J. Parasitol.
Deoxynivalenol: toxicity, mechanisms and animal health risks
Anim. Feed Sci. Technol.
The food contaminant deoxynivalenol, decreases intestinal barrier permeability and reduces claudin expression
Toxicol. Appl. Pharmacol.
Report from SCOOP task 3.2.10 “collection of occurrence data of Fusarium toxins in food and assessment of dietary intake by the population of EU member states” Subtask: Trichothecenes
Toxicol. Lett.
Deoxynivalenol transport across human intestinal Caco-2 cells and its effects on cellular metabolism at realistic intestinal concentrations
Toxicol. Lett.
Mycotoxins nivalenol and deoxynivalenol differentially modulate cytokine mRNA expression in Jurkat T cells
Cytokine.
In vitro exposure of human lymphocytes to Trichothecenes: individual variation in sensitivity and effects of combined exposure on lymphocyte function
Food Chem. Toxicol.
Individual and combined cytotoxic effects of Fusarium toxins (deoxynivalenol, nivalenol, zearalenone and fumonisins B1) on swine jejunal epithelial cells
Food Chem. Toxicol.
The AF-1 activation function of estrogen receptor α is necessary and sufficient for uterine epithelial cell proliferation in vivo
Endocrinology
Deoxynivalenol: a trigger for intestinal integrity breakdown
FASEB J.
Deoxynivalenol impairs weight gain and affects markers of gut health after low-dose, short-term exposure of growing pigs
Toxins (Basel)
The Design and Statistical Analysis of Animal Experiment
Nivalenol and deoxynivalenol affect rat intestinal epithelial cells: a concentration related study
PLoS One
Interaction of Salmonella choleraesuis, Salmonella dublin and Salmonella typhimurium with porcine and bovine terminal ileum in vivo
Microbiology
Toxigenic Fusarium species and mycotoxins associated with head blight in small-grain cereals in Europe
Eur. J. Plant Pathol.
Porcine in vitro and in vivo models to assess the virulence of Salmonella enterica serovar Typhimurium for pigs
Lab. Anim.
Chronic ingestion of deoxynivalenol and fumonisin, alone or in interaction, induces morphological and immunological changes in the intestine of piglets
Br. J. Nutr.
Cited by (27)
Deoxynivalenol hijacks the pathway of Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT-3) to drive caspase-3-mediated apoptosis in intestinal porcine epithelial cells
2023, Science of the Total EnvironmentCitation Excerpt :According to the kinetics of DON in the small intestine of the pigs, the in vitro concentrations used in this study (10 μmol/L) corresponded to 10.5 mg/kg food contamination (Danicke et al., 2004). DON-induced IECs apoptosis has been demonstrated in previous studies, and the underlying mechanisms have primarily focused on the MAPKs pathways (Cheat et al., 2016; Wang et al., 2014; Xu et al., 2020a; You et al., 2021). The STAT-3 gene is a known susceptibility loci for inflammatory bowel disease, and substantial evidence supports its role in the regulation of apoptosis (Lin et al., 2005).
Nivalenol affects spindle formation and organelle functions during mouse oocyte maturation
2022, Toxicology and Applied PharmacologyImpact of deoxynivalenol on intestinal explants of broiler chickens: An ex vivo model to assess antimycotoxins additives
2021, ToxiconCitation Excerpt :This analysis demonstrates the damaging effect of DON on the intestinal epithelium and the presence of a significantly greater amount of apoptotic cells in the toxin-exposed group compared to control (Fig. 5). A similar pattern of results was obtained by Cheat et al. (2016) in an assessment with pig intestinal segments. In an in vivo experiment with aflatoxin B1 (AFB1), Peng et al. (2014) showed the same toxin-induced effect on the intestinal epithelium of broilers.
Mechanism of deoxynivalenol mediated gastrointestinal toxicity: Insights from mitochondrial dysfunction
2021, Food and Chemical ToxicologyMycotoxins in human food: A challenge for research
2021, Cahiers de Nutrition et de Dietetique