PFAS exposure modulates mitochondrial susceptibility in steatotic hepatocytes

Per- and polyfluoroalkyl substances (PFAS) are highly persistent environmental pollutants that bioaccumulate in living organisms and are increasingly implicated in metabolic dysfunction-associated steatotic liver disease (MASLD). Mitochondria, as central regulators of energy and lipid metabolism, represent key subcellular targets of PFAS toxicity. While emerging studies have examined PFAS-induced mitochondrial dysfunction using whole-cell analyses, studies focusing on mitochondria-enriched cellular fractions and their functional alterations under steatotic conditions, despite their clinical significance, remain limited. Here, we analyzed mitochondria-enriched cellular fractions and the extracellular secretome from human HepaRG hepatocytes exposed to a PFAS mixture under both steatotic and non-steatotic conditions by using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS). The PFAS mixture composition and concentrations reflected those commonly reported in human epidemiological data. We observed PFAS- and steatosis-driven effects on metabolism, including lipid buildup in mitochondria-rich fractions due to mitochondrial-lipid droplet contact, disturbances in cardiolipin levels, and increased extracellular abundance of acylcarnitines. Notably, PFAS-induced impact on mitochondrial metabolism was exacerbated in steatotic hepatocytes. Pathway analysis identified disturbances in lipid metabolism and inflammation-related pathways, including leukotriene metabolism, squalene and cholesterol biosynthesis, highlighting a shared metabolic signature across the PFAS-steatosis axis. Functional mitochondrial assays further showed that PFAS exposure suppressed respiratory capacity under both conditions, whereas steatosis alone increased basal mitochondrial activity but nearly abolished spare respiratory capacity. Together, these findings indicate that steatosis is associated with greater PFAS-related alterations in mitochondrial respiration and metabolic composition within the mitochondria-enriched fraction, supporting heightened susceptibility of steatotic hepatocytes to acute PFAS exposure.