Evaluation of the individual and combined toxicity of perfluoroalkyl substances to human liver cells using biomarkers of oxidative stress.

Although human exposure is to mixtures of per- and polyfluoroalkyl substances (PFAS), their combined effects and underlying mechanisms remain largely unknown. In this study, the combined effects of PFAS was investigated by treating human liver cells (HepG2) with various concentrations of perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), perfluorodecanoic acid (PFDA), perfluorononanoic acid (PFNA), and perfluorohexanoic acid (PFHxS) individually or in binary combinations (PFOS + PFOA, PFOS + PFDA, PFOS + PFNA, PFOS + PFHxS, PFOA + PFDA, PFOA + PFNA, and PFOA + PFHxS) for 24 h using an orthogonal design. The individual and binary combination effects of PFAS on the cytotoxicity, intracellular reactive oxygen species (ROS) production, and glutathione (GSH) levels were determined by MTS assay, dichlorofluorescein diacetate assay, and GSH-Glo™ Glutathione assay, respectively. The results showed that exposure to PFOA, PFOS, PFDA, PFNA, and PFHxS individually and in binary combinations caused concentration-dependent cytotoxicity to HepG2 cells. Also, intracellular ROS production was not significantly induced in both the individual and co-treatment groups, indicating that ROS production may not be likely influencing the combined cytotoxicity of PFAS to HepG2 cells. However, the depletion of the intracellular glutathione levels was correlated with cytotoxicity. Moreover, the factorial analysis results showed no significant interactive effects between PFOS + PFOA, PFOS + PFDA, PFOS + PFNA, PFOS + PFHxS, PFOA + PFDA, PFOA + PFNA, and PFOA + PFHxS. Taken together, the results showed that both individual and combined PFAS could induce concentration-dependent cytotoxicity and depletion of GSH levels, but could not induce significant increases in ROS production at the concentration range tested. Overall, these results provided valuable toxicological data on the combined effects of mixed PFAS that may help to better assess their human health risk.