Exposure duration shapes GenX-induced transcriptomics responses in 3D HepG2/C3A spheroids

Hexafluoropropylene oxide dimer acid (GenX), a replacement for legacy per- and polyfluoroalkyl substances (PFAS), is increasingly detected in the environment, yet its long-term toxicity remains poorly characterized. Current safety assessments rely largely on short-term, high-dose studies that may not capture the biological consequences of longer-term, low-dose exposure. To address this gap, we employed monocellular 3D HepG2/C3A hepatic 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 μM, spanning environmentally relevant to mechanistically informative concentrations) for acute (4 days) and long-term (4 weeks) durations, followed by genome-wide TempO-Seq transcriptomic profiling and benchmark dose (BMD) modeling. Following actue exposure, the greatest number of differentially expressed genes (DEGs) was observed at 0.4 μM, with fewer DEGs at higher concentrations. Long-term exposure produced a different concentration-dependent distribution of DEG with the greatest number observed at 50 μM. Notably, acute and long-term 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 transcripts encoding constituents of lipid metabolic pathways and increase expression of mRNA encoding proteins involved in stress- and toxicity-associated signaling. Long-term exposure revealed a different pattern of changes in mRNA expression not observed under acute exposure conditions, including suppression of mRNA expression levels of transcripts encoding cellular components involved in lipid-related pathways at the lowest concentration tested. At higher concentrations, mRNA levels encoding transcritps of components of multiple metabolic pathways were altered. Benchmark dose modeling identified a significantly lower transcriptomic point of departure (tPOD) for long-term exposure as compared to acute exposure, suggesting increased cellular response to prolonged GenX exposure and supporting the relevance of long-term models for human exposure assessment. Collectively, these findings demonstrate that GenX elicits distinct transcriptomic responses in HepG2/C3A spheroids depending on both concentration and exposure duration. This study, therefore, highlights the importance of incorporating long-term 3D hepatic 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 days) versus longer term (4 weeks), environmentally relevant GenX exposure in monocellular HepG2/C3A hepatic 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.