Receptor-Bound Perfluoroalkyl Carboxylic Acids Dictate Their Activity on Human and Mouse Peroxisome Proliferator-Activated Receptor γ

In in vitro cell assays, nominal concentrations of a test chemical are most frequently used in the description of its dose-response curves. Although the biologically effective concentration (BEC) is considered as the most relevant dose metric, in practice, it is very difficult to measure. In this work, we attempted to determine the BEC of long-chain perfluoroalkyl carboxylic acids (PFCAs) in peroxisome proliferator-activated receptor gamma (PPAR gamma) activity assays. In both adipogenesis and transcriptional activity assays with human and mouse cells, PPAR gamma activity of 7 PFCAs first increased and then decreased with their carbon chain length. The binding affinity of these PFCAs with the ligand-binding domain of PPAR gamma was measured by fluorescence competitive binding assay and showed very poor correlation with their receptor activity (r(2) = 0.002-0.047). Internal concentrations of the PFCAs in the cells were measured by LC-MS/MS. Although their correlation with the receptor activity increased significantly, it is still low (r(2) = 0.41-0.82). Using the binding affinity constant, internal concentration, and PPAR gamma concentration measured by immunoassays, concentrations of receptor-bound PFCAs in cells were calculated, which exhibited excellent correlation with PPARy activity in both adipogenesis and transcriptional activity assays (r(2) = 0.91-0.93). These results demonstrate that the concentration of receptor-bound PFCA is the BEC that dictates its activity on human and mouse PPAR gamma in cell assays. In the absence of any direct detection method, our approach can be used to calculate the target-site concentration of other ligands.