Weakened serum albumin binding-to-activated EGFR internalization in cellular accumulation for the synergistic toxicity of tetrabromobisphenol A and perfluorooctanoic acid

Brominated flame retardants (BFRs) and per-fluoroalkyl substances (PFAS), are globally concerned persistent toxic substances (PTS) with high co-exposure risks due to their coexistence in the environment matrices and even in humans. However, their combined toxicity hasn't been explored yet. The respective representatives tetrabromobisphenol A (TBBPA) and perfluorooctanoic acid (PFOA) share similar exposure routes and properties of substantial bioaccumulation and environmental persistence. To investigate the combined toxicity of BFRs and PFAS with TBBPA and PFOA as the research subjects, and elucidate the mechanisms responsible for the combined toxicity. Combined toxicity were evaluated after co-exposure with TBBPA and PFOA at non-toxic and even environmentally relevant low doses in vitro. Cellular accumulation of TBBPA/PFOA was detected by UPLC-MS/MS. Serum albumin binding constant of TBBPA or PFOA under single and co-existence was detected by fluorescence spectrometric titration, and the effects of varied albumin binding affinity on cellular accumulation were evaluated. Then, combined methods of transcriptomics, Q-PCR, Western blot, immunofluorescence imaging, gene knockdown and molecular docking were employed to detect epidermal growth factor receptor (EGFR) expression, internalization and its role in contributing to the cellular uptake of the pollutants. Cell viability under co-exposure with 100 μM TBBPA/250 μM PFOA at non-toxic concentration alone reduced to 65.55 %, while the ratio of apoptotic and necrotic cells enhanced to 63.93 %. The combined toxicity was demonstrated to be synergism. Increased cellular accumulation of each pollutant was responsible for the synergistic toxicity. We next found TBBPA/PFOA co-existence can weaken each other's serum albumin binding ability, leading to higher free TBBPA and PFOA levels in medium that were easier for cellular uptake than albumin-bounded ones, thereby resulting in the enhanced cellular levels of both compounds. Endocytosis was then demonstrated to contribute to the cellular uptake of free TBBPA/PFOA molecules. Thereafter, a non-canonical EGFR internalization was activated and upregulated due to the cellular oxidative stress, which further mediated more cellular uptake of TBBPA/PFOA. Taken together, the effects of weakened serum albumin binding-to-activated EGFR internalization contributed to the enhanced cellular accumulation of each pollutant in a cascade mode, thereby resulting in the combined cytotoxicity. Our findings represent the first experimental study on BFRs and PFAS co-exposure risks with TBBPA/PFOA, and propose a new mechanism for the combined toxicity, which may have broad environmental, chemical, and biomedical significance for future research on the other environmental pollutants and their analogs. • Synergistic cytotoxicity was found between TBBPA and PFOA. • TBBPA/PFOA co-existence weakened each other's BSA binding ability. • EGFR internalization was activated to mediate more cellular uptake of TBBPA/PFOA.