Synergistic neurotoxicity of mono-butyl phthalate and bisphenol A at environmentally relevant concentrations: Multi-Omics evidence for ferroptosis via glutathione metabolism disruption in neural stem cells and brain organoids.

Evaluating endocrine-disrupting chemicals (EDCs) risk such as mono-butyl phthalate (MBP) and bisphenol A (BPA) raises a pressing question: can doses deemed safe for each individual exposure become hazardous in combination? This study aimed to determine whether environmentally relevant concentrations of MBP and BPA exert synergistic neurotoxicity and to elucidate the underlying molecular mechanisms using an integrated multi-omics approach. Exposure concentrations (20 nM each) were derived from epidemiological biomonitoring data. Primary neural stem cells (NSCs) were used to detect CCK-8, reactive oxygen species (ROS), and apoptosis Synergy was evaluated using the Bliss independence model and two-way ANOVA. Transcriptomic (RNA-seq) and untargeted metabolomic (LC-MS) profiling were performed, followed by integrative pathway analysis. Three-dimensional (3D) brain organoids were used to validate the main findings. Co-exposure to MBP and BPA at equimolar concentrations synergistically reduced cell proliferation (BlissΔ >0, E_Observed = 0.371 vs. E_Bliss = 0.163) and synergistically elevated intracellular ROS (interaction index = 2.21, p = 0.0066; two-way ANOVA interaction p = 0.033). Transcriptomic and metabolomic analyses converged on glutathione metabolism as the most significantly dysregulated pathway in the co-exposure group. Integrated multi-omics analysis revealed ferroptosis may serve as the primary mode of synergistic cell death. Three-dimensional brain organoids confirmed the ferroptosis signature, with significantly decreased GPX4 and SLC7A11 protein expression and concomitantly increased ACSL4 in the co-exposure group. In summary, MBP and BPA co-exposure at human-relevant doses synergistically induces neurotoxicity in NSCs through glutathione metabolism disruption and subsequent ferroptosis. These findings underscore the importance of evaluating chemical mixtures rather than individual compounds in neurodevelopmental risk assessment.