This proof-of-concept project will conduct innovative research that leads to a comprehensive understanding of the bioaccumulation of per- and polyfluoroalkyl substances (PFAS) in lipid membrane and transporter proteins using mutually reinforcing in vitro and in silico techniques. A systematic investigation of factors that dictate the partitioning of PFAS (relevant to aqueous film-forming foam [AFFF]) in different biological environments will be determined. The overarching objective of this project is to develop new methodologies to assess the biological effects of PFAS (e.g., in silico and in vitro) at different levels of biological organization and under different exposure scenarios.

This project is aimed at addressing the following specific objectives:

1. Objective 1: Characterize the bioaccumulation properties of 11 different PFAS that are part of AFFF with lipid membranes and transporter proteins using in silico approaches. The following will be obtained: (i) molecular level insight into the PFAS uptake mechanism in both membrane and proteins and how these mechanisms vary with respect to the increasing chain length and functionalities of the PFAS; (ii) influence of anionic and zwitterionic nature of PFAS (single phase and mixtures) in controlling their partitioning at the lipid interface and binding affinity with proteins; (iii) effect of stereochemistry of PFAS in altering the partitioning characteristics, penetration pathways and binding energetics at both bio-interfaces. Here, classical and enhanced molecular dynamics (MD) simulations will be utilized.
2. Objective 2: Determine the lipid-water partition coefficient and the binding mode of 11 AFFF-specific PFAS to small unilamellar vesicles of controlled phospholipid content. ¹⁹F nuclear magnetic resonance (NMR) chemical shift perturbation and line broadening analysis will be utilized. These experiments will be correlated with MD simulations in Objective 1 to identify model shortcomings, inform experiments and iteratively generate data.
3. Objective 3: Conduct in vitro experiments using transporter proteins known to bind PFAS, to better understand the mechanisms of PFAS bioaccumulation in fish cell lines.

The bioaccumulation of PFAS molecules (found in AFFF) in lipid bilayer and transporter proteins will be examined using a combination of computational molecular modeling approaches and in vitro experiments. AlphaFold3 and homology modeling will be performed to build novel structures for the transporter proteins. Molecular docking studies will provide us with the best orientation of PFAS molecules within the binding site of the transporter proteins. Classical MD simulations will be performed to depict the interaction of PFAS within lipid bilayers and at different binding sites of three transporter proteins. Enhanced sampling methods will be utilized to determine the energetics of PFAS adsorption in the lipid bilayer and proteins. ¹⁹F NMR experiments will be conducted to determine lipid-water partition coefficients, elucidate interaction between PFAS and lipid vesicles, and reveal critical factors that govern the PFAS-liposome interactions. In vitro pharmacological and genetic perturbations experiments identify transporters involved in PFAS disposition in model zebrafish embryo fibroblast cell lines.

This research provides critical advancements in the understanding of the bioaccumulation behavior of PFAS, particularly those associated with AFFF, in aquatic organisms. By integrating complementary in vitro and in silico approaches, this work will deliver molecular-level insights into the mechanisms that govern PFAS interactions with lipid membranes and transporter proteins, key determinants of PFAS uptake, accumulation, and toxicity in aquatic systems. Both the passive partitioning and facilitated active transport mechanisms of PFAS will be addressed, since they are major determinants of bioaccumulation. The outcomes of this project will significantly improve risk assessments that consider chemical structure, stereochemistry, and mixture effects, factors often overlooked and contribute methodological innovation integrating molecular dynamics simulations with advanced NMR spectroscopy, to probe PFAS interactions at atomistic resolution. Successful completion of this proof-of-concept effort will ultimately lead to more cost effective PFAS management, directly benefiting the warfighter and installation communities. (Anticipated Project Completion - 2027)