Integrated functional, metabolomic, and biotransformation profiling of mycotoxin hepatotoxicity in 2D and 3D human hepatic models.
Mycotoxins pose a major risk to food safety and human health, yet their hepatotoxic mechanisms remain incompletely characterized due to limitations in conventional in vitro models. In this study, we systematically compared mycotoxin-induced hepatotoxicity and metabolomic profiling across two human hepatic models cultured under 2D monolayer and 3D spheroid conditions. The various mycotoxins (Aflatoxin B1, Citrinin, Deoxynivalenol, Ochratoxin A, Patulin, and Zearalenone) exhibit distinct metabolic signatures, thereby serving as an appropriate panel for comprehensively evaluating diverse hepatotoxic mechanisms. Mycotoxin exposure induced concentration-dependent hepatotoxicity accompanied by functional impairment and structural disruption in hepatic models. Metabolomic profiling revealed distinctive regulatory patterns between 2D and 3D hepatic models, with 3D spheroids showing consistent down-regulation across multiple intracellular metabolic pathways and altered extracellular metabolite release, whereas 2D monolayers predominantly exhibited global metabolic activation. In silico-assisted MS/MS analysis further demonstrated that Phase I biotransformation was largely conserved across models, whereas Phase II conjugation reactions were more frequently detected and exhibited greater model specificity in 3D spheroids. Overall, these findings indicate that 3D hepatic spheroids capture more integrated and coordinated hepatotoxic and metabolic responses to mycotoxins compared with 2D monolayer systems. These distinctive regulatory dynamics support their value as a physiologically relevant platform for toxicity assessment and mechanistic investigation.