Dose- and duration-dependent hepatotoxicity of PFO4DA: Divergent PPARα responses and conserved glucocorticoid receptor suppression

Per- and polyfluoroalkyl substances (PFAS) alternatives, such as per- and polyfluoroalkyl ether carboxylic acids (PFECA), have been widely detected in environmental and biological samples; however, their hepatic toxicity mechanisms remain poorly understood. This study focused on a representative PFECA compound, perfluoro-3,5,7,9-tetraoxadecanoic acid (PFO4DA), and investigated its hepatotoxic effects and underlying molecular mechanisms by integrating multi-omics analyses with in vitro assays. The results demonstrated that PFO4DA is a weak peroxisome proliferator-activated receptor α (PPARα) agonist and exhibits dose-dependent regulation of PPARα signaling in vivo. Specifically, subacute high-dose exposure (0.4, 2, and 10 mg/kg/day for 28 days) activated PPARα signaling, whereas chronic low-dose environmentally relevant exposure (2 and 10 μg/kg/day for 140 days) indirectly suppressed PPARα activity, likely due to reduced availability of endogenous ligands. Metabolomic and lipidomic analyses revealed that chronic low-dose PFO4DA exposure induced broad depletion of multiple lipid species, particularly free fatty acids (FFAs) and their derivatives, thereby substantially reshaping hepatic metabolic homeostasis. Regardless of exposure scenario, glucocorticoid receptor (GR) signaling was consistently suppressed, suggesting that GR inhibition is a conserved toxicological feature of PFECA exposure. Moreover, amino acid profiles were broadly disturbed, accompanied by dysregulation of nutrient-sensing regulators, including ATF4 and FGF21. These findings extend the current understanding of PFECA-induced hepatotoxicity beyond classical nuclear receptor pathways and highlight the critical involvement of nutrient-sensing and metabolic regulatory networks. Collectively, this work identifies exposure-dependent divergence in PPARα signaling but conserved GR suppression as central features of PFO4DA hepatotoxicity, providing mechanistic insight essential for environmental health risk assessment of emerging PFAS alternatives.