Cell type–specific toxicity of heptadecafluorooctanesulfonic acid (PFOS), pentadecafluorooctanoic acid (PFOA), and perfluorononanoic acid (PFNA): mechanistic insights into oral exposure pathways

Perfluorinated compounds (PFCs), including heptadecafluorooctanesulfonic acid (PFOS), pentadecafluorooctanoic acid (PFOA), and perfluorononanoic acid (PFNA), are persistent organic pollutants with human half-lives of up to four years, leading to progressive bioaccumulation. Exposure occurs predominantly through diet, with serum concentrations ranging from 1 to 256 µg/L in the general population and up to 5.2 mg/L in occupationally exposed workers. While PFCs have been linked to reproductive, metabolic, immunological, and developmental toxicity, their effects on intestinal epithelial cells and blood macrophages-two primary cellular targets of the oral exposure route- emain insufficiently characterised. Human intestinal epithelial (H4) and blood macrophage (TLT) cells were exposed to PFOS, PFOA, and PFNA for 24 h across a broad concentration range. Cytotoxicity was assessed using proliferation, Crystal Violet (CV), and MTT assays, and intracellular reactive oxygen species (ROS) production was measured using the DCFH-DA method. Mechanistic endpoints-including γ-H2AX activation, cell cycle distribution, apoptosis, and morphological alterations-were analyzed by imaging flow cytometry (ImageStream MK2) and the Muse Cell Analyzer. All three PFCs induced concentration-dependent and cell type-specific effects. PFNA showed the greatest cytotoxic potency, with ROS induction evident at 5 mg/L and significant loss of viability above 60 mg/L in both cell types. In H4 cells, PFNA strongly activated γ-H2AX, induced cell cycle arrest, morphological alterations, and apoptosis, consistent with genotoxic stress and downstream checkpoint engagement. PFOA in H4 cells similarly activated γ-H2AX, induced cell cycle arrest, and increased apoptotic frequency, while PFOS produced DNA damage and morphological changes without significant apoptosis. In TLT cells, PFNA induced cytotoxicity with limited apoptosis, PFOA caused cell cycle arrest in the absence of marked genotoxicity, and PFOS elicited minimal responses across all endpoints. These findings demonstrate that PFOS, PFOA, and PFNA elicit distinct, concentration-dependent toxic responses in a cell type-specific manner, with PFNA exhibiting the highest overall potency. The pronounced sensitivity of intestinal epithelial cells underscores the importance of oral exposure models and supports the need to evaluate both individual and mixture effects of PFCs to refine toxicological reference values.