Emerging mycotoxins: a new risk for Human and animals? – EmergingMyco

The consortium includes an industrial partner specialized in crop protection and in relation with the actors of agriculture in France. It has the capacity to collect numerous samples in fields to analyses the mycotoxin content and to make links to agricultural practices. INRA and CNRS research teams have varied and complementary expertise in mycotoxin toxicology, metabolomics, transcriptomics and the study of the intestinal microbiota. The toxicity of emerging mycotoxins the most present in cereals will be assessed using in vitro (cell cultures), ex vivo (explants) and in vivo (experiments on pigs and rodents) approaches, focusing on two target organs - the intestine and the liver. Omics techniques will allow a global evaluation without a priori of the effect of these mycotoxins on the expression profile of genes and metabolites. Intestinal and hepatic mycotoxin biotransformation products will be studied using chromatographic and mass spectrometric techniques. The study of the intestinal microbiota by sequencing bacterial ribosomal RNA will allow the evaluation of how emerging mycotoxins affect its composition and how they may alter microbial equilibrium. Using another metatranscriptomic approach, it will be possible to study how genes and bacterial functions are affected by the presence of different mycotoxins.

Eighteen months after starting the project, the first results of field analyses have identified emerging metabolites produced by molds, present both in wheat in France and in pig feed. More than 200 metabolites were identified in the samples analyzed, of which nearly 90% were also co-contaminated by deoxynivalenol. Among these metabolites, those presented below, which are among the most abundant, could be classified according to their decreasing toxicity to pig intestinal cells: apicidin > enniatine-A1> deoxynivalenol > beauvericin>ENN enniatine-B> enniatine-B1>emodine > aurofusarin>> brevianamide F= cyclo-(L-Pro-L-Tyr)= tryptophol. The toxicity of these metabolites in mixture with deoxynivalenol was tested by reproducing realistic animal exposure situations to mycotoxins. In most cases, the toxicity of the mixture was similar to or sometimes lower than that of deoxynivalenol alone, underlining the fact that its toxicity under these experimental conditions is not exacerbated by the mixture. Research on the antimicrobial and cytotoxic properties and mode of action of enniatins and beauvericin have shown that they are active against Gram + bacteria, mycobacteria and various human cell lines (pulmonary, intestinal, and hepatic in particular) and that enniatins A and A1 are more toxic than enniatins B, B1 and beauvericin. The study of their mode of action has shown that they interact with bacterial lipids, depolarize the membrane and inhibit the synthesis of bacterial macromolecules at low doses.

This project will enable the collection of missing data on the presence of emerging mycotoxins in French cereals, their co-occurrence with other regulated mycotoxins and the link with agricultural practices to identify those that limit contamination. In addition, this project will provide toxicological and metabolism data on emerging mycotoxins in humans and animals in order to complete the existing data and produce original data. The evaluation of toxic effects by targeted (classical physiological parameters) and non-targeted approaches (transcriptomic, proteomic, metabolomic approaches and study of the intestinal microbiome) will make it possible to identify the metabolic pathways that have been disrupted. Tests in pigs will make it possible to determine a tolerable daily intake (TDI) for this species. Finally, the comparison of the toxicity of emerging mycotoxins between humans and pigs will confirm that pigs are a relevant model species for mycotoxin toxicology studies in humans.

The results of the first 2 studies were communicated in conferences and published in scientific articles. The first study produced original results on the toxicity on intestinal epithelial cells of several emerging mycotoxins which were also tested in mixture with a regulated mycotoxin. In these mixtures reproducing realistic situations, they do not alter the toxicity of deoxynivalenol.
The second study examined the antimicrobial and cytotoxic properties and mode of action of enniatins and beauvericin. After showing that these mycotoxins are active against Gram + bacteria, mycobacteria and various human cell lines, the work showed that they interact with bacterial lipids, depolarize the membrane and inhibit the synthesis of bacterial macromolecules at low doses.

Mycotoxins are frequent contaminants of cereals, found in more than 25 % of samples worldwide. They are of high concern due to their potential health risks for humans and/or livestock. When ingested at low to moderate doses, their impact on human health is likely to be significant as they can induce stunting, immune dysfunction and contribute to cancers. In livestock, they can induce acute poisoning, but also various metabolic disturbances resulting in poor animal productivity following a chronic exposure. In France as well as in Europe, the climate conditions favor the contamination by Fusarium fungi. The main Fusarium toxins, ie deoxynivalenol (DON), fumonisins (FB) and zearalenone (ZEN) are regulated in food and feed. Fusarium fungi produce other toxins such as beauvericin, enniatins, apicidin and aurofusarin, less described and called “emerging” mycotoxins (EM). They are neither routinely determined, nor legislatively regulated. Little is known on their occurrence even if preliminary results suggest a high prevalence.
In vitro, beauvericin and enniatins induce cytotoxicity, oxidative stress and apoptosis. In vivo, no conclusion can be drawn on chronic exposure due to the lack of relevant data. Aurofusarin and apicidin have been poorly investigated with less than 30 publications on their toxic effects. On the intestine, the first barrier against food contaminants, DON, FB and ZEN alter cell proliferation and differentiation, nutrient absorption, immunity and barrier function while the effects of EM are unknown. Mycotoxins, as well as antibiotics, are secondary metabolites of fungi. While the effects of the latter are well described on the gut microbiome, the impact of Fusarium toxin on intestinal microbiota are limited and the impact of EM totally unknown.
This project aims to fill gaps in the knowledge of EM. It will covers both occurrence and toxicity of EM and more precisely proposes (i) to perform a large-scale survey on the occurrence of EM in French cereals and to determine the relation between the concentration of EM and agricultural practices and (ii) to assess the impact of EM alone or combined with regulated mycotoxins in human and pigs. We will focus on intestinal functions (barrier, nutrients absorption, immunity) and use in parallel in vitro, ex vivo and in vivo models. Omics approaches (transcriptomics, proteomics, and metabolomics) will allow obtaining a global view of the toxicological effects of pigs orally exposed to EM. Intestinal and fecal samples will be used to assess by 16S gene sequencing and metatranscriptomic the effects of EM on the gut microbiota. The modified forms of EM produced by metabolization will be identified by mass spectrometry. These animal studies also aim to establish, a tolerable daily intake (TDI)
To the best of our knowledge, this project which compares in a one health approach human and pig, is unique in its strategy. The complementarity of the approaches (from field survey and to omics methods) will allow performing a global study on occurrence and toxicity of four-selected EM. This innovative project relies on the involvement of 6 partners with complementary expertise, from agronomy to toxicology. It will benefit from the well-established relations between them.