Mycotoxins, produced by fungi, frequently occur at different stages in the food supply chain between pre- and postharvest. Globally produced cereal crops are known to be highly susceptible to contamination, thus constituting a major public health concern. Among the encountered mycotoxigenic fungi in cereals, Fusarium spp. are the most frequent and produce both regulated (i.e., T-2 toxin, deoxynivalenol -DON-, zearalenone -ZEA-) and emerging (i.e., enniatins -ENNs-, beauvericin -BEA-) mycotoxins. In this study, we investigated the in vitro cytotoxic effects of regulated and emerging fusariotoxins on HepaRG cells in 2D and 3D models using undifferentiated and differentiated cells. We also studied the impact of ENN B1 and ENN B exposure on gene expression of HepaRG spheroids. Gene expression profiling pinpointed the differentially expressed genes (DEGs) and overall similar pathways were involved in responses to mycotoxin exposure. Complement cascades, metabolism, steroid hormones, bile secretion, and cholesterol pathways were all negatively impacted by both ENNs. For cholesterol biosynthesis, 23/27 genes were significantly down-regulated and could be correlated to a 30% reduction in cholesterol levels. Our results show the impact of ENNs on the cholesterol biosynthesis pathway for the first time. This finding suggests a potential negative effect on human health due to the essential role this pathway plays.

Enniatins (ENNs) are emerging mycotoxins mainly produced by Fusarium species. They frequently contaminate cereals and cereal‐based products. Despite their widespread occurrence, these mycotoxins are not yet regulated, but concerns about their potential health effects due to dietary exposure exist. The most prevalent and well‐characterized are ENN A, A1, B, and B1. Their ionophoric nature enables them to disrupt cellular ion homeostasis, leading to potent cytotoxic effects. This review is structured into three major sections. First, we provide a comprehensive meta‐analysis based on ENNs occurrence in 6916 food samples collected worldwide, between 2004 and 2024, including cereals, products derived from cereals, other products, and baby food and discuss mycotoxin mitigation. Second, we indicate known ENN‐producing Fusarium species and performed a gene sequence analysis suggesting that a dedicated biosynthetic gene cluster exists. Finally, we provide an in‐depth overview of ENNs toxicity, ranging from cytotoxicity studies to mechanistic insights into their cellular effects. By consolidating current knowledge, this review highlights critical gaps in ENN research and emphasizes the need for further investigations to better understand their health impacts in a regulatory framework.

Enniatins (ENNs) are emerging Fusarium mycotoxins detected in food and feed. Despite their widespread occurrence, their toxicity remains poorly understood; thus, advanced in vitro systems that can mimic human physiology are of interest. We evaluated the cytotoxic and genotoxic effects of ENN A1 and ENN B1 exposure on differentiated (DIFF) and undifferentiated (UND) HepaRG liver cells cultured as 2D monolayers and 3D spheroids. Cytotoxicity, assessed by ATP-based luminescence, revealed a time-dependent decrease in inhibitory concentration 50 (IC₅₀) values between 24 h and 48 h across all models. In DIFF HepaRG cells, ENN A1 IC₅₀ values in 3D spheroids decreased from 14.4–18.2 µM at 24 h to 2.2–3.0 µM at 48 h, reaching values comparable to those measured in 2D DIFF cells at 48 h (2.2–2.6 µM), while no IC₅₀ could be determined in 2D at 24 h. For ENN B1, a pronounced time-dependent toxicity was observed, with IC₅₀ values in 3D DIFF spheroids decreasing from 4.1–6.6 µM at 24 h to 1.3–1.6 µM at 48 h, remaining lower than those measured in 2D DIFF cells at 48 h (2.4–3.0 µM). ENN A1 primarily induced apoptotic responses, whereas both ENN A1 and B1 were associated with necrotic responses, and ENN B1 induced a transient and limited autophagic signal, suggesting a minor role for autophagy. To further characterize cellular responses to ENN exposure, spheroids cultured in microfluidic chips were sectioned, and proliferation (Ki67), DNA damage (γH2AX), and apoptosis (cleaved caspase-3) was assessed. Immunostaining revealed no proliferative response, whereas significant DNA damage was detected, particularly in DIFF spheroids. At low, sub-cytotoxic concentrations (~5 µM, 24 h), ENN A1 induced significant DNA damage, as shown by increased γH2AX levels, while cytotoxic effects were only observed at higher concentrations (IC₅₀ ~ 18 µM, 24 h), supporting a potential genotoxic effect independent of cytotoxicity. Despite the structural similarities between ENN A1 and ENN B1, our results highlighted distinct cell death pathways between the two analogues. Both ENNs were detected throughout spheroids without evidence of peripheral restriction, although a homogeneous functional test could not be conclusively demonstrated. Overall, the 3D HepaRG spheroid model proved to be a more physiologically relevant system, offering differential sensitivity, as well as enhanced mechanistic insight, compared to 2D cultures.

Les mycotoxines, métabolites secondaires produits par des champignons filamenteux, représentent une problématique majeure de sécurité des aliments du fait de leurs potentielles toxicités. Elles contaminent les denrées destinées à la consommation humaine mais aussi animale et sont présentes tout au long de la chaîne agroalimentaire, « du champ à la fourchette ». Parmi elles, les mycotoxines émergentes, telles que les enniatines (ENNs) et la beauvéricine (BEA), figurent parmi les plus prévalentes. Cependant, malgré certaines avancées récentes sur leurs effets toxiques, le manque de données toxicologiques in vitro et in vivo empêche encore toute réglementation définissant des seuils maximaux d'exposition. Dans ce contexte, et en accord avec le principe des 3R (Remplacer, Réduire, Raffiner), cette thèse vise à évaluer la toxicité des ENNs et BEA seules ou en mélanges avec des modèles in vitro innovants. Des lignées intestinales (Caco-2 et HT29-MTX-E12) et hépatiques (HepaRG) ont été utilisées et une cytotoxicité de l'ordre du micromolaire a été observée selon la mycotoxine et/ou le mélange testé ainsi que des voies de mort cellulaire variables. Une génotoxicité de l'ENN A 1 et une modulation transcriptionnelle de voies clés liées au métabolisme lipidique, au cycle cellulaire et à la réponse immunitaire ont également été mises en évidence avec l'ENN B et B1. Enfin, le développement d'un modèle complexe de triculture intestin-foie a permis d'étudier la co-exposition aux ENNs et à la BEA ainsi que leur métabolisation alors que l'utilisation d'un modèle de sphéroïdes a permis d'observer et déterminer l'impact de la pénétration des mycotoxines au sein de ce structure 3D.