Investigating the cytotoxicity of per- and polyfluoroalkyl substances in HepG2 cells: A structure-activity relationship approach.

Per- and polyfluoroalkyl substances (PFAS) are a family of man-made chemicals with currently over 4'700 compounds identified. While toxicological data are available for some of the legacy PFAS, such as PFOA and PFOS, a knowledge gap remains concerning both emerging and legacy PFAS' toxicity due to the diversity of the PFAS. Therefore, a better understanding of the PFAS structure-activity relationship may prove helpful. The present study investigated a potential structure-activity relationship between PFAS and hepatotoxicity. As such, the effects of thirteen PFAS with varying carbon chain-length and functional head-groups (in a concentration range of 0-800 µM) on the cell viability of HepG2 cells and intracellular reactive oxygen species formation have been tested using the MTT and DCFH assay, respectively. The exposure times were either 3 or 24 h. In addition, intracellular PFAS levels were determined in HepG2 after 24 h exposure. The present study demonstrated that the cytotoxicity of PFAS is dependent on their chain-length as cell viability decreased with increasing chain-length at both exposure times. Calculated Relative Potency Factors (RPF), based on the TC50 values, were used for a tentative ranking of PFAS regarding their hepatotoxicity: PFNA ˃ PFDA ˃ PFOS ≥ PFOA ˃ PFHxS ˃ PFBS ˃˃ PFHpA = PFHxA = PFBA = PFPrA = 6:2 FTOH = 4:2 = FTOH = 3:1 FTOH. Similar results were observed regarding intracellular reactive oxygen species generation at both exposure times, with a tentative ranking of: PFNA ˃ PFOS ˃ PFOA ≥ PFDA ˃ PFHxS ˃ PFBS ˃ PFBA ˃ PFHpA ≥ PFHxA ˃ PFPrA ˃ 6:2 FTOH = 4:2 FTOH = 3:1 FTOH. Moreover, a concentration-dependent reactive oxygen species generation has been observed for all PFSAs and PFCAs, but not for the FTOHs. In conclusion, the carbon chain-length and functional head-group of a PFAS determine their in vitro toxicity for the two toxicological endpoints assessed in the present study. Moreover, no effects were observed for the tested FTOHs. As such, the present study established a potential structure-activity relationship that opens the possibility of developing a predictive model to help with the risk assessment of PFAS in the future.

The impact of legacy and novel perfluoroalkyl substances on human cytochrome P450: An in vitro study on the inhibitory potential and underlying mechanisms.

Per- and polyfluoroalkyl substances (PFASs) are a group of synthetic compounds with a wide range of industrial applications. PFOA and PFOS have been the most extensively studied and have been associated with hepatotoxicity. Recently, the interaction with cytochrome P450 (CYP) has been proposed as a potential key molecular event leading to PFAS-induced hepatotoxicity. In the present study, we aimed to determine a structure-activity relationship between thirteen PFASs and their inhibitory potential on the activities of four CYPs (CYP2E1, CYP2D6, CYP3A4 and CYP2C19). The influence of PFASs (5-3200 µM) on CYP enzyme activities was measured using the Vivid® P450 metabolism assays. Using the same assays, Michaelis-Menten saturation curves were determined to explore the type of PFAS-induced CYP inhibition. Most PFASs were capable of inhibiting activity of the tested CYPs, as shown by their IC50 values. CYP2E1 is particularly inhibited by 3:1 FTOH, PFOA, and PFOS, whereas CYP2D6 is inhibited by PFHxS, PFHpA, PFOA, PFOS, PFNA, and PFDA. Additionally, CYP3A4 is most strongly inhibited by PFHxS, PFOA, PFOS, PFNA, and PFDA. Finally, CYP2C19 is inhibited by PFBS, PFHxS, PFHpA, PFOA, PFOS, PFNA, and PFDA. Interestingly, PFHxA and PFHxS induced an increase in CYP2E1 activity, whereas 4:2 FTOH strongly induced CYP2D6 activity. The mechanism of inhibition of CYPs by PFASs differed per CYP isoenzyme. CYP3A4 was competitively inhibited by PFBS, PFHxS, PFOS, PFNA and PFDA and non-competitively by PFOA. Additionally, CYP2C19 was competitively inhibited by PFHxA, PFOS and PFNA, whereas PFBS and PFHxS induced a mixed inhibition. Inhibition of CYP2C19 by PFHpA was atypical with an increased Vmax and a decreased Km. Finally, PFHxS competitively inhibited CYP2D6, whereas PFBS, PFOA, PFOS, PFDA and PFNA induced an atypical inhibition. Our results show that CYP inhibition by PFASs appears to be structure-dependent as well as CYP dependent. Inhibition of CYP2D6, CYP2C19 and CYP3A4 increased with increasing chain-lengths between six and nine carbons. The PFTOHs were only able to inhibit CYP2E1 and did not affect any of the other CYPS. Some PFASs remarkably induced the enzyme activity of CYPs. These results indicate that in addition to PFOA and PFOS, multiple novel PFASs may alter drug metabolism by the interference with CYPs.