Cereal and crop production throughout the world is facing contamination by natural substances such as mycotoxins. Consequently to climate change and its impact on the development of toxin-producing fungus, mycotoxin contamination of food and feed is increasing in the last decades, raising consumer health concern worldwide. The genus Fusarium can produce a wide range of mycotoxins. Among the main Fusarium mycotoxins, deoxynivalenol (DON), has been found in a broad panel of foodstuffs while the enniatins, with enniatin B (ENNB) as the major produced variant, are considered as emerging mycotoxins. Enniatins can be found in mixture with DON in food. However, if DON is regulated in Europe, no regulation for enniatins is currently available due to a lack of information on toxicity. Nevertheless, the mechanisms of action involved in DON toxicity and its combined effect with enniatins are not well characterized. Therefore, the aim of this study was to characterize the toxic effects of these two mycotoxins, alone and in combination, on the HepaRG liver cell model using a multiomic approach.

First, the toxicity of the 2 mycotoxins (ENNB and DON) alone and in mixture at equivalent concentration was assessed on HepaRG cells by High Content Analysis. Several toxicological endpoints were investigated such as viability, DNA damage, apoptosis, cell cycle analysis, and pro-inflammatory response. Proteomic and metabolomic analyses were performed, in order to decipher the mechanisms involved in their toxicity.

DON induced a dose-dependent decrease of cell number, and increased caspase 3 and γH2AX as well as IL-8 release. In contrast to DON, ENNB decreased the viability only of 20 % and did not affect caspase 3 and γH2AX staining. However, similarly to DON, ENNB increased IL-8 release. The combination of these two mycotoxins rescues HepaRG from DON-induced cell death and inhibited the DON-increase of γH2AX and caspase 3 induced. The mixture slightly decreased the IL-8 release provoked by each toxin. The molecular mechanisms involved in the single toxins and the mixture responses were compared using proteomics (on cell content) and metabolomics (on cell culture medium). We showed that the protective effect of ENNB observed on DON mortality involved lipids and mitochondria. Indeed, DON treatment increased the abundance of long chain saturated dicarboxylic acids, signs of a defective fatty acid oxidation (FAO). DON also increased the abundances of histones and proteins associated to mitochondrial respiration and decreased specifically abundances of proteins implicated in FAO. These effects were alleviated by ENNB in the cells treated with the mixture, suggesting opposite effects of the two toxins on HepaRG cells.

This study provided new findings on the molecular effects of DON and ENNB on liver cells and highlighted that their combined effects should not be ruled out in risk assessment.