Broad Profiling of Hepatic and Immunotoxic Effects of Per- and Polyfluoroalkyl Substances (PFASs) Across Sexes and Exposure Durations in C57/BL6 Mice

Per and Poly-fluorinated alkyl substances (PFAS) are a class of chemicals commonly referred to as “forever chemicals”. Of this class, perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) are frequently found in the environment and blood of Canadians. Here, we sought to broadly characterize the effect of PFOS and PFOA on liver toxicity and immunotoxicity in both male and female mice. Experimental mice were exposed to PFOA or PFOS for 28 or 56 days, followed by a comprehensive evaluation that included 6 organ weights, 8 serum biomarkers of liver toxicity, and transcriptomic analysis across 15 treatment conditions. This revealed that both compounds caused hepatomegaly, increased serum liver enzymes, pancreatic atrophy, and reduced serum triglycerides (TGL) regardless of sex or duration of treatment. Transcriptomics identified enrichment of PPAR signaling, lipid metabolism dysfunction, and AGE-RAGE pathways across groups which supports a role for metabolic and oxidative stress in PFAS-induced hepatotoxicity. To characterize immunotoxicity 19 cytokine profiles, and 3 tissue types including the thymus, spleen, and blood were assessed for changes in population dynamics. Immune effects were distinctly sex specific. In males, PFOS led to broad cytokine suppression (IL-4, IL-17a, TNF-a, MCP-1) and a non-significant increase in peripheral T cells following PFOA exposure. In contrast, females exhibited minimal cytokine changes but showed significant alterations in thymocyte development. Together, these findings reveal three key outcomes: (1) both PFASs cause liver and pancreatic toxicity, (2) hepatic effects are largely sex-independent, and (3) immune perturbations are highly sex-dependent. These results underscore the importance of evaluating sex-specific effects in immunotoxicology studies of persistent environmental contaminants. Overall design: Male and female C57BL/6-Elite mice (6–9 weeks old) were acclimated for two weeks, individually housed (males) or paired (females), with controlled diet and water. Starting at 8–11 weeks old, mice were orally dosed daily via gavage with either PFOA or PFOS at varying doses (0.166 to 1.5 mg/kg/day) for 28 or 56 days. Control groups included vehicle (0.5% Tween 20) and naïve (no gavage) mice. Body weight was recorded daily, and water consumption was measured weekly. After the dosing period, mice were euthanized 24 hours post-final dose for blood and organ collection (liver, pancreas, kidneys, GI tract), which were weighed for analysis.