Ultrashort-Chain Per- and Polyfluoroalkyl Substances and Cholestasis: In Vitro Evidence of Liver Dysfunction from Emerging Environmental Contaminants.

Over the past 75 years, fluorinated contaminants present in aquatic, dietary, and environmental matrices have increasingly raised scientific and public health concerns. Among these substances, ultrashort perfluoroalkyl substances (US-PFAS) demonstrate substantial environmental persistence and mobility, comparable to that of their long-chain counterparts. Reported human serum concentrations of individual US-PFAS reach up to 27.7 ng/mL, substantially exceeding established thresholds for total PFAS levels. Nevertheless, data on human toxicity of US-PFAS remain limited. The present study aimed to investigate the impact of US-PFAS on cholestatic features and to elucidate the underlying mechanisms using an adverse outcome pathway network as a mechanistic compass. Human HepaRG liver cell cultures were exposed to 4 US-PFAS at various concentrations for 1 to 72 h. Transcriptomic profiles were assessed by RNA sequencing analysis. Functional alterations in early and key events associated with cholestatic pathology were evaluated using fluorescently labeled probes, while bile canaliculi dynamics were examined through in situ immunostaining and phase-contrast imaging. This revealed significant perturbations of molecular pathways involved in both early and late events relevant for cholestatic liver injury. US-PFAS did not significantly affect hepatocellular transporter activity or bile canalicular integrity at early time points (≤6 h). In contrast, prolonged exposure (24-72 h) to several US-PFAS elicited hepatocellular stress responses, such as oxidative stress and endoplasmic reticulum stress, and promoted inflammatory cell adhesion. Collectively, these findings demonstrate that US-PFAS have the capacity to affect biliary homeostasis and trigger downstream cholestatic events, providing mechanistic insight relevant for hazard identification.