Bisphenol A and Its Substitutes Bisphenol S/Bisphenol F in Combined Exposure Collectively Exacerbate Hepatotoxicity by Triggering Oxidative Stress and Inflammatory Responses via the PI3K-Akt/NF-κB Axis

Abstract Bisphenol A (BPA) and its substitutes bisphenol S (BPS) and bisphenol F (BPF) are environmental endocrine-disrupting chemicals that frequently co-occur. However, the hepatotoxic effects and underlying mechanisms of their combined exposure remain unclear. This study integrated network toxicology, bioinformatics, molecular modeling, and in vivo/in vitro experiments to investigate how combined exposure to BPA, BPS, and BPF collectively exacerbates hepatotoxicity in association with the PI3K-Akt/NF-κB signaling pathway. Network toxicology identified 127 common targets of BPA, BPS, and BPF, of which 19 were closely associated with liver injury. KEGG enrichment analysis significantly pointed to the PI3K-Akt and NF-κB pathways. Molecular docking and dynamics simulations revealed that each individual compound exhibited favorable binding energy to core targets, forming stable complex conformations. In vivo experiments demonstrated that continuous gavage administration of BPA, BPS, BPF, or their mixture induced liver tissue damage, oxidative stress, and inflammatory responses in mice, with the mixture group showing the most pronounced effects. In vitro experiments using primary hepatocytes and HepG2 cells further confirmed that combined exposure additively inhibited cell viability, increased reactive oxygen species (ROS) levels, activated the PI3K-Akt/NF-κB pathway, and upregulated the expression of pro-inflammatory factors. Although formal synergy assessment is lacking, this study reveals that combined exposure to BPA, BPS, and BPF produces greater-than-single-compound toxicity, which is associated with oxidative stress and inflammatory responses alongside the PI3K-Akt/NF-κB signaling pathway. These findings provide a new theoretical basis for the risk assessment of combined exposure to bisphenol analogues.