Exposure Duration Shapes the Hepatic Response to GenX: Divergent Acute and Chronic Transcriptomic Profiles Reveal Non-Monotonic Dose Effects and Increased Sensitivity in Human Liver Spheroids

Hexafluoropropylene oxide dimer acid (GenX), a replacement for legacy per- and polyfluoroalkyl substances (PFAS), is increasingly detected in the environment, yet its chronic toxicity remains poorly characterized. Current safety assessments rely largely on short-term, high-dose studies that may not capture the biological consequences of long-term, low-dose exposure. To address this gap, we employed 3D human liver (HepG2/C3A) spheroids cultured in a continuously rotating bioreactor system (ClinoStar) to systematically evaluate dose- and time-dependent mRNA changes in response to GenX under environmentally relevant conditions. Spheroids were exposed to GenX (0.08-50 M, spanning environmentally relevant to mechanistically informative concentrations) for acute (4 days) and chronic (4 weeks) durations, followed by genome-wide TempO-Seq transcriptomic profiling and benchmark dose (BMD) modeling. GenX elicited pronounced non-monotonic mRNA changes in acute exposure conditions, with the greatest number of differentially expressed genes (DEGs) observed at an intermediate concentration (0.4 M). In contrast, chronic exposure exhibited a generally concentration-dependent increase in DEGs, except for the 10 M condition, indicating a more consistent dose-response relationship than acute exposure. Notably, acute and chronic exposures elicited qualitatively distinct mRNA changes with low concordance across matched concentrations, demonstrating that exposure duration was a major determinant of mRNA changes. Acute low-dose GenX exposure preferentially modulated mRNA encoding components of cell cycle-related pathways, whereas acute higher dose exposures suppress mRNA levels of the constituents of lipid metabolic pathways and increase expression of mRNA encoding proteins involved in stress- and toxicity-associated signaling. Chronic exposure revealed a different pattern of changes in mRNA expression not observed under acute exposure conditions, including suppression of cellular components involved in lipid-related pathways at the lowest concentration tested. At higher concentrations, mRNA levels of components of multiple metabolic pathways were altered. Benchmark dose modeling identified a significantly lower transcriptomic point of departure (tPOD) for chronic exposure as compared to acute exposure, suggesting increased cellular sensitivity to prolonged GenX exposure and supporting the relevance of chronic models for human exposure assessment. Collectively, these findings demonstrate that GenX elicits time-dependent and non-monotonic changes in mRNA levels of human liver (HepG2/C3A) spheroids, with distinct responses depending on the exposure duration and dose. This study, therefore, highlights the importance of incorporating chronic, human-relevant in vitro models and transcriptomic endpoints into PFAS risk assessment and suggests that conventional short-term assays may underestimate the biological impact of sustained low-dose exposure. Key message (Impact of the study)This study provides systematic comparisons of short term (4 day) versus longer term (4 weeks), environmentally relevant GenX exposure in human liver spheroids, revealing non-monotonic, time-dependent changes in mRNA levels encoding cellular components of lipid metabolism-related pathways with potential implications for appropriate dose and time exposure parameters for use in New Approach Methods to be applied in risk assessment.