Repeated in vitro exposure to PFOA impairs intestinal barrier integrity and leads to cytosolic accumulation as detected by subcellular chemical imaging

Perfluorooctanoic acid (PFOA) is a persistent organic pollutant recognised for its environmental presence and bioaccumulation, despite regulatory restrictions under the Stockholm Convention. While systemic effects of PFOA have been studied extensively, its interaction with the first biological barrier encountered after oral exposure, the gastrointestinal barrier, remains poorly characterised. In this study, we evaluated the fate, uptake and effects of PFOA in human intestinal cell culture models after acute (24 h) and repeated (11-day) exposures. Using radiolabelled PFOA profiling, we found that PFOA was not metabolised by Caco-2 cells nor by HT29-MTX mucus-producing cells, in monocultures or in co-culture. Mass balance studies revealed higher basolateral passage of PFOA in Caco-2 monocultures compared to Caco-2/HT29-MTX co-cultures, suggesting that the mucus in the co-culture may trap PFOA and limit its passage. These observations led us to conduct further toxicological evaluations in the Caco-2 monoculture model. Acute exposure, while having no effect on cell viability, increased cellular ATP at highest exposure concentration. Repeated exposure led to a progressive concentration-dependent decrease in trans-epithelial electrical resistance, indicating a compromised barrier function. This effect was not linked to changes in tight junction gene expression, but rather attributed to cell death. High-resolution chemical imaging revealed intracellular accumulation of PFOA in Caco-2 cell cytosol after repeated exposure. These findings highlight the potential of repeated exposure to PFOA, even at low concentrations, to impair intestinal barrier integrity, which may have implications for systemic absorption and toxicity. • Intestinal co-culture with mucus cells reduces basolateral [ 14 C]-PFOA passage. • Acute PFOA exposure increases ATP production by intestinal cells at high concentrations. • Repeated PFOA exposure progressively impairs intestinal barrier integrity even at low concentrations. • Subcellular chemical imaging enables PFOA localisation in cytosol of cells.