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 bioaccumulation. Exposure occurs mainly through diet, with serum concentrations ranging from 1–256 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 key targets of oral exposure—remain insufficiently characterized. Human intestinal epithelial (H4) and blood macrophage (TLT) cells were exposed to PFOS, PFOA, and PFNA (5–500 mg/L) for 24 h. Cytotoxicity was assessed using Crystal Violet (CV), MTT, and ROS assays. Mechanistic endpoints included γ-H2AX activation, cell cycle progression, apoptosis, and morphological alterations, analysed by imaging flow cytometry (ImageStream MK2). All three PFCs induced concentration-dependent and cell-type-specific effects. PFOS and PFOA triggered ROS generation and transient increases in MTT activity, reflecting mitochondrial compensation, followed by sharp declines in both MTT and CV values at higher concentrations. PFNA showed the greatest potency, with ROS induction at 5 mg/L and significant loss of viability above 60 mg/L. In H4 cells, PFNA strongly activated γ-H2AX, induced cell cycle arrest, morphological changes, and apoptosis, consistent with genotoxic and epigenetic disruption. PFOA in H4 cells activated γ-H2AX and apoptosis, while PFOS promoted DNA damage and morphological alterations but favoured non-apoptotic pathways. In TLT cells, PFNA induced cytotoxicity with limited apoptosis, PFOA caused cell cycle arrest without strong genotoxicity, and PFOS elicited minimal responses. PFCs exert distinct, concentration-dependent toxic effects, with PFNA being most potent. Intestinal epithelial cells were particularly sensitive, underscoring the importance of exploring oral exposure pathways of PFCs and the need to evaluate their toxicity, mixture effects, which may refine toxicological reference values.