Toxicity assessment of three emerging bisphenols (BPA, BPAP and BPC) and their binary mixtures in an advanced in...

Bisphenols (BPs) are industrial chemicals extensively used in polycarbonate plastics and epoxy resins for everyday products, including food and beverage containers, toys, and thermal paper, representing major sources of human exposure. Bisphenol A (BPA) is the most prevalent; however, due to its endocrine-disrupting, reproductive, and genotoxic effects, it is classified as a substance of high concern by the European Chemicals Agency. Regulatory restrictions have prompted the use of structural analogues, including bisphenol AP (BPAP) and bisphenol C (BPC); however, emerging evidence suggests they may pose similar or greater health risks. Comprehensive data on their toxicity and human exposure, especially in mixtures, remain limited. This study assessed the cytotoxic and genotoxic effects of BPA, and less studied analogues BPAP, BPC, and their binary mixtures using a human-relevant 3D HepG2 spheroid model, representing an advanced in vitro system. Spheroids were exposed for 24 and 96 h, and effects were evaluated by ATP-based viability assays, comet assay, and targeted transcriptomics. None of the bisphenols induced significant cytotoxicity, although slight reductions in viability were observed for BPAP, BPC, and mixtures. DNA strand breaks were detected after exposure to BPA, BPC, and mixtures. Transcriptomic analysis revealed modest stress responses and strong upregulation of xenobiotic metabolism genes (CYP1A1, CYP1A2, CYP3A4, UGT1A1, NAT2), indicating cellular recognition of the tested bisphenols as bioactive xenobiotics. Antioxidant and DNA repair genes were largely unchanged, except for upregulation of oxidative stress markers HMOX1 and SRXN1 and a slight increase in OGG1, indicating oxidative DNA damage. Importantly, mixture effects were predominantly additive, with no evidence of synergistic interactions under the tested conditions. Overall, bisphenol exposure primarily triggered oxidative stress and metabolic responses rather than robust DNA damage signalling, suggesting that genotoxicity is largely mediated by reactive oxygen species. By integrating multiple endpoints in a 3D liver model, this study provides a more comprehensive assessment of bisphenol toxicity and highlights potential risks associated with BPA analogues and their mixtures, supporting the need for a comprehensive safety assessment to evaluate their suitability as replacements in consumer products.