ENN A1 and B1 In Vitro Toxicological Effects on 2D and 3D Organ-on-Chip HepaRG Liver Cells

Enniatins (ENNs) are emerging <i>Fusarium</i> 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.