Extracellular Vesicle (<scp>EV</scp>) Mechanisms of Toxicity for Per and Polyfluoroalkyl Substances: Comparing Transcriptomic Points of Departure Across Global Versus <scp>EV</scp> Regulatory Gene Sets

Extracellular vesicles (EVs) are emitted from cells throughout the body and serve as signaling molecules that mediate disease development. Emerging evidence suggests that per- and polyfluoroalkyl substances (PFAS) impact EV release and content, influencing liver toxicity. Still, the upstream regulators of EV changes affected by PFAS exposure remain unclear. This study evaluated the hypothesis that PFAS exposures, individually and in a mixture, alter the expression of genes involved in EV regulation at concentrations comparable to genes involved in global biological response mechanisms. HepG2 liver cells were treated at multiple concentrations with individual PFOS, PFOA, or PFHxA, in addition to an equimolar PFAS mixture. Gene expression data were analyzed using three pipelines for concentration-response modeling, with results compared against empirically derived datasets. Final benchmark concentration (BMC) modeling was conducted via Laplace model averaging in BMDExpress (v3). BMCs were derived at an individual gene level and across different gene sets, including Gene Ontology (GO) annotations as well as a custom EV regulation gene set. To determine relative PFAS contributions to the evaluated mixture, relative potency factors were calculated across resulting BMCs using PFOS as a standard reference chemical. Results demonstrated that PFAS exposures altered the expression of genes involved in EV regulation, particularly for genes overlapping with endoplasmic reticulum stress. EV regulatory gene changes occurred at similar BMCs as global gene set alterations, supporting concurrent regulation and the role of EVs in PFAS toxicology. This application of transcriptomics-based BMC modeling further validates its utility in capturing both established and novel pathways of toxicity.