In-Vitro and In-Silico Assessment of Per- and Polyfluoroalkyl Substances (PFAS) in Aqueous Film-Forming Foam (AFFF) Binding to Human Serum Albumin

Abstract

Drinking water contaminated by fluorosurfactant-based aqueous film-forming foams (AFFF) is a source of human exposure to poly- and perfluoroalkyl substances (PFAS). However, assessment of bioaccumulation potentials of diverse PFAS in commercial products such as AFFF have been insufficient and challenging, especially due to a lack of analytical standards. Here we explore the value of suspect screening, equilibrium dialysis, and molecular-docking simulations to identify potentially bioaccumulative PFAS. We exposed human serum albumin (HSA) protein to dilutions of a legacy AFFF produced by 3M in 1999 using equilibrium dialysis and screened in-vitro protein-binding affinities using high-resolution mass spectrometry (HRMS). Through suspect screening, we identified 32 PFAS and 18 hydrocarbon surfactants in the AFFF that bound to HSA. Quantification of noncovalent association constants for 26 PFAS standards confirmed that many PFAS, including the short-chain perfluoropropane sulfonic acid (log Ka= 4.1 ± 0.2 M−1), exhibit strong binding affinities with HSA. At least five PFAS in AFFF (including three PFAS with less than five perfluorocarbons) remained bound to the precipitated HSA pellet after extensive solvent washing—an indication of high PFAS binding potential. Three PFAS (PFBS, PFOS, and PFOA) were confirmed in the protein pellet with analytical standards and quantified after acid digestion—this sample fraction accounted for 5 to 20% of each compound mass in the sample. We calculated pseudo-bioconcentration factors (BCFpseudo) for PFAS that suspect screening flagged as noncovalently bound or potentially covalently bound. Most PFAS exhibiting high BCFpseudo, especially those with seven perfluorocarbons, contained a carboxylic acid or a sulfonic acid. Finally, we used molecular docking to simulate HSA binding affinities for 62 ligands (26 PFAS targets, 18 PFAS qualified in AFFF, and 18 hydrocarbon surfactants qualified in AFFF). We found that molecular docking can effectively separate HSA-binding and -nonbinding compounds in AFFF. In-vitro and in-silico approaches described in this study provide replicable, high-throughput workflows for assessing bioaccumulation potentials of diverse PFAS in commercial products.

Widening the Lens on PFASs: Direct Human Exposure to Perfluoroalkyl Acid Precursors (pre-PFAAs)

“…This has been a serious challenge for pre-PFAA research because humans are exposed to a wide range of pre-PFAAs, some with unknown structures or no available neat standards. To circumvent this issue, a handful of studies have used commercial products, such as AFFF formulations, to dose in vivo − and in vitro , models and measure known and novel transformation products. Others have used environmental samples containing complex PFAS mixtures .…”

Proteins as adsorbents for PFAS removal from water

“…Indeed, there are reports of binding interactions between C8 PFAS and proteins, such as human serum albumin (HSA) and bovine serum albumin (BSA). [29][30][31][32][33] However, very little is known about the interactions of other proteins with these contaminants. Moreover, even serum albumins have not been evaluated for their ability to remove PFOA and PFOS from water samples, and their ability to bind to shorter chain PFAS (<C8) has not been explored.…”

“…Serum albumins are known to bind to both PFOA and perfluorooctane sulfonic acid PFOS, enabling their prolonged circulation in humans and animals. 26,[29][30][31][32][33][34][35][36][37][38][39] Chen et al observed that PFOA binds preferably to sub-domain IIA in BSA, while PFOS binds preferably to sub-domain IIIA. 38 Also, PFAS accumulates and distributes differently across several species, 2,[40][41][42][43] while ionic strength and pH can modulate PFAS binding to these proteins, too.…”

Correction: Li et al. In-Vitro and In-Silico Assessment of Per- and Polyfluoroalkyl Substances (PFAS) in Aqueous Film-Forming Foam (AFFF) Binding to Human Serum Albumin. Toxics 2021, 9, 63

“…The authors wish to make the following corrections to this paper [1]: There was an error in the original publication. In Section 4.2, the article stated, "HSA binding affinities of perfluorohexanesulfonic acid (PFHxS) and perfluoroheptanoic acid (PFHpA) were exceptionally high (Log K A : 5.89 ± 0.55 and 5.74 ± 0.38, respectively)".…”