Per and poly-fluoroalkyl substances (PFAS) are a group of synthetic chemical compounds created for their ability to repel water. Despite their usefulness in commercial and industrial products, PFAS exposure has been implicated in a number of human health conditions, including numerous cancers, birth defects, and endocrine disruptions. Creating sound, empirically based regulations for PFAS contamination is therefore of the utmost importance to public health. Since various PFAS are almost always detected in water as a mixture, understanding the implications of PFAS mixture toxicity is integral in establishing reasonable regulations. To accomplish this, we used an MTT cell viability assay to establish the toxicity of eight PFAS commonly detectable in contaminated groundwater (PFBA, PFBS, PFHxA, PFHxS, PFOA, PFNA, PFOS, and GenX) on HepG2 cells, with the liver being a common target for PFAS induced cellular injury. Using the combination index equation method, we then quantitatively determined the antagonistic, additive, or synergistic effects of PFAS combinations at varying concentrations. Additionally, we considered the tendency of PFAS to adsorb onto plastic materials, and performed control experiments to determine the adsorption of PFAS onto the 96 well plates during toxicity treatment. This was done in order to ensure that toxicity was not being underestimated in our MTT results. To further understand the role of isomers in induced toxicity, the concentration of both linear and branched isomers of PFOS was analyzed over the course of treatment. Finally, we performed genotoxicity analysis of the PFAS of greatest demonstrated cytotoxicity (as per the MTT assay) PFOA, PFOS, and PFNA alone, and PFOA and PFOS in combination using the single cell electrophoresis assay (comet assay). Ultimately, individual PFAS HepG2 cytotoxicity had a significant range which was highly dependent on perfluorinated chain length and hydrophobicity of the molecule, with IC50 values ranging from 5700 µM in the case of PFBA, to as low as 26 µM for PFOS. Combination effects were less clear, with most combinations skewing toward synergism at lower concentrations and antagonism at higher concentrations. PFAS induced HepG2 genotoxicity was not directly correlated with cytotoxicity, demonstrating the need for several measures of toxicity in conjunction in order to best inform PFAS regulations.

Cytotoxicity and genotoxicity of mixtures of per- and polyfluoroalkyl substances (PFAS) to HepG2 cells

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ACS Fall 2022

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