A subcellular study on reactive oxygen species generation by PFAS in HepG2 cells.

Per- and polyfluoroalkyl substances (PFAS) hepatotoxicity is well documented, especially for legacy compounds such as PFHxS, PFOA, PFOS, and PFNA. However, the mechanism(s) involved are yet to be fully understood. The present study aims to investigate the origin of PFAS-induced formation of reactive oxygen species (ROS) and their relevance for the decrease of cell viability of HepG2 cells after exposure to PFASs. Moreover, a structure-activity relationship was assessed using PFASs with different headgroups (carboxylic, sulfonic, and alcoholic) and variable carbon-chain lengths (4-10 C). The link between ROS generation and cell viability was assessed using two antioxidants: quercetin, a generic antioxidant, and mito-tempo, a mitochondria-targeted antioxidant. Both antioxidants were demonstrated to be effective in reducing PFAS-induced ROS generation. The mechanism behind PFAS-induced ROS might be headgroup-dependent, as quercetin increased cell viability after both perfluoroalkyl carboxylic acids (PFCA) and perfluorosulfonic acids (PFSA) exposure, while mito-tempo only improved cell viability after PFCAs exposure. The two major sources of ROS generation in HepG2 cells are the peroxisomes and mitochondria. However, exposure to PFASs did not impact peroxisomal or mitochondrial activity after 24 h. Uncommon sources of ROS generation, such as lysosomal leakage or lipid peroxidation, have been demonstrated to result from previously generated ROS and not from PFASs exposure. Indeed, lysosomal leakage caused by PFASs exposure is negated by either quercetin or mito-tempo treatment, while lipid peroxidation only occurs after 24 h of exposure, long after the initial ROS generation by PFASs. This indicates that both events are a result of previously generated ROS. However, exposure to both PFOA and PFOS was demonstrated to reduce catalase activity in HepG2. In conclusion, the present study demonstrates that ROS generation after PFASs exposure might be due to inhibition of HepG2 endogenous antioxidants. Moreover, a headgroup-dependent mechanism of action has been observed, indicating that PFCAs and PFSAs exposure might lead to hepatotoxicity through different pathways.