High-Content Genotoxicity Analysis reveals clastogenic effects of low-concentrations per- and polyfluoroalkyl substances mixtures in Human breast epithelial cells.

Although several chemicals are established carcinogens, the carcinogenic and genotoxic potential of many marketed compounds remains insufficiently characterized. We developed a high-content analysis protocol to assess genotoxicity in human breast epithelial MCF-10A cells by combining micronucleus detection with γH2AX foci quantification. Assay performance was first evaluated using established aneugenic and clastogenic positive controls and subsequently applied to the environmental contaminants perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), bisphenol A (BPA), and bisphenol F (BPF), individually and as binary mixtures, across concentrations from 1 pM to 100µM for 72h. These compounds have been detected in human breast milk and breast tissue, while epidemiological and experimental studies have raised concerns regarding their potential involvement in breast cancer, supporting the relevance of using a human mammary epithelial cell model. PFOS and PFOA individually increased cell proliferation at several concentrations without inducing significant genotoxic effects. In contrast, the PFOS/PFOA mixture increased micronucleus formation at 1nM and 100nM, as well as γH2AX foci at 100nM, demonstrating genotoxic effects at human-relevant concentrations. BPF showed a proliferative effect that was not observed with BPA or the BPA/BPF mixture, while neither compound alone induced significant genotoxic effects. The BPA/BPF mixture increased γH2AX foci at the highest concentration, which coincided with reduced cell number. Overall, these findings highlight the importance of considering mixture effects, as different chemical combinations induced distinct biological response patterns, and demonstrate the potential of high-content, human cell-based approaches for efficient and mechanistically informative genotoxicity assessment.