Co-exposure to polystyrene nanoparticles and tetrabromobisphenol A induces genotoxicity and interferes with intestinal barrier differentiation

Tetrabromobisphenol A (TBBPA) and polystyrene nanoparticles (PSNPs) are contaminants of emerging concern frequently detected in environmental matrices, with dietary exposure representing a major route. Despite their co-occurrence, data on combined toxicity remain limited. This study evaluated the effects of TBBPA and PSNPs individual and combined exposure on human intestinal barrier integrity, differentiation and function using differentiated Caco-2 cells and Caco-2/HT29-MTX co-cultures as validated in vitro models of increasing complexity. Cells were exposed to TBBPA (1–150 µM) and PSNPs (0.1–50 µg/mL) individually or in combination, either for 24 h in fully differentiated monolayers or continuously during the 21-day differentiation period. In differentiated Caco-2 cells, combined exposure altered cytoskeletal organization, with PSNPs being internalized and accumulating as large intracellular aggregates that persisted for at least 144 h post-exposure. Ultrastructural analysis revealed microvilli alterations and multilamellar bodies formation, although tight junction architecture remained intact and cellular adhesion structures recovered progressively following contaminant removal, suggesting retained barrier repair capacity. During the differentiation period, TBBPA was the primary cytotoxic agent and PSNPs alone showed no cytotoxicity at the concentrations tested, as confirmed by benchmark dose analysis, which yielded low relative potency factor values for PSNPs across all tested combinations. Genotoxicity assessment revealed oxidative DNA damage in selected TBBPA and PSNPs combinations using the FPG-modified comet assay, with gene expression analysis indicating activation of DNA damage response and oxidative stress pathways. Co-exposure modulated the expression of intestinal differentiation markers, including ALPI, suggesting interference with epithelial maturation processes. The complexity of the dose–response relationships observed underscores the importance of comprehensive dose-range testing and the use of physiologically relevant differentiated models in the risk assessment of contaminant mixtures. Graphical Abstract Graphical headlights • TBBPA is the main contributor to cytotoxicity during co-exposure with PSNPs. • PSNPs persist within intestinal epithelial cells up to 144 h after exposure. • Co-exposure induces oxidative stress and DNA damage in intestinal epithelial cells. • Combined exposure alters intestinal epithelial differentiation and barrier maturation.