In vitro binding of human and rat PPAR alpha, beta/delta, and gamma receptors to PFAS, fatty acids, and clofibric acid

The molecular mechanism by which some per- and polyfluorinated alkyl substances (PFAS) exert toxicity has largely been attributed to activation of peroxisome proliferator-activated receptor alpha (PPARα). Less attention has been given to PPAR beta/delta (PPARβ/δ) or PPAR gamma (PPARγ). Further, it is unknown how, or if, PFAS activation of PPAR paralogs differs between rats and humans. We utilized in vitro luciferase reporter assays with either human or rat PPARα, β/δ, or γ ligand binding domains hybridized with a Gal4 DNA binding domain. We also tested for estrogen (ER), androgen (AR), and glucocorticoid receptor (GR) transcriptional activation using previously established stable transfection (ER) and viral transduction (AR, GR) luciferase reporter assays. Nine PFAS (hexafluoropropylene oxide-dimer acid ammonium salt (GenXAS) and free acid (GenXFA), nafion byproduct 2 (NBP2), perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), perfluorobutane sulfonate (PFBS), perfluorononanoic acid (PFNA), 6:2 fluorotelomer alcohol (6:2 FTOH), and perfluoromethoxyacetic acid (PFMOAA)), 3 endogenous fatty acids (oleic, linoleic, and octanoic), and the drug metabolite clofibric acid were assayed at 30 µM -1 mM. Thus far all compounds, except 6:2 FTOH, have activated both PPARα and PPARγ in both human and rat. Based on EC20 values, oleic and linoleic acid were the most potent human PPARα activators, while PFOS was the least potent. For rat PPARα, GenX was the most potent activator (no difference between GenXFA and GenXAS), while NBP2 and PFMOAA were the least with low fold induction (<10% of max). For PPARγ, all compounds had similar potency in the human and rat receptor assays (log EC20 ranges: -3.14 – -3.74 versus -3.05 – -3.53), except for PFMOAA which only reached 3% and 1% of max fold induction, respectively. In contrast, the only compounds that activated both human and rat PPARβ/δ were oleic and linoleic acid; however, NBP2 weakly activated (~10% of max) rat PPARβ/δ at 300 µM. No compounds displayed in vitro AR or GR transcriptional activation. Only 6:2 FTOH exhibited weak ER agonism. Overall, all PFAS studied, except 6:2 FTOH, displayed both human and rat PPARα and γ activity, with some having similar potency to endogenous free fatty acids. Thus, it is likely that both PPARα and γ activation contribute to the adverse in vivo effects observed for PFAS. Abstract does not necessarily reflect USEPA policy.

Impact/Purpose

Per- and poly-fluoroalkyl substance (PFAS) research is an area of critical need due to the extreme environmental persistence, widespread occurrence, long biological half-lives, and nearly ubiquitous human and environmental exposure to this chemical class. Perfluoroalkyl ether acids (PFEAs) are a sub-class of PFAS and include the compounds hexafluoropropylene oxide dimer acid (GenX) and Nafion byproduct 2 (NBP2). PFEAs are currently used in the production of fluoropolymers following the phase-out of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS), presumably due to more favorable fate and toxicity profiles. However, monitoring studies have detected GenX, NBP2, PFMOAA, and others in a wide range of matrices including surface water, drinking water, and/or human serum. Little to no published research is available regarding the potential toxicity of these compounds compared to the legacy PFAS. The data reported here indicate that all PFAS studied displayed both human and rat PPAR α and γ activity with some having similar potency to endogenous free fatty acids and clofibric acid. Thus, it is likely that both mechanisms contribute to the adverse effects observed for PFAS. Finally, this data will be highly informative for investigating and informing the development of AOPs that are relevant to the toxicity of PFAS. The data from this project will be useful to state, federal, and other regulatory agencies in the interpretation of toxicity data and the development of hazard/risk assessments for GenX and other PFAS.

Citation

Evans, N., M. Cardon, P. Hartig, E. Medlock Kakaley, V. Wilson, J. Conley, AND L. Gray. In vitro binding of human and rat PPAR alpha, beta/delta, and gamma receptors to PFAS, fatty acids, and clofibric acid. VIRTUAL-Society of Toxicology Annual Meeting, Anaheim, CA, April 30, 2020.