Biomonitoring-informed neonicotinoid mixture exposure reveals modest cytotoxicity and cell-type-specific molecular responses in human cell lines

BACKGROUND: Neonicotinoids (NEOs) are widely detected in environmental matrices and in humans, raising concerns about potential health risks. However, most toxicological studies have focused on single compounds and/or concentrations far exceeding biomonitoring-relevant exposure ranges. METHODS: We developed a UPLC-MS/MS method to quantify eight NEOs and five metabolites in human urine. Using environmental monitoring and urinary biomonitoring data from Hainan Island, China, we constructed exposure-informed NEO mixture profiles based on surface water, tap water, and urinary concentrations, and evaluated their cytotoxicity in four human cell lines (A2780, HEK293T, HeLa, and HepG2). Toxicity was assessed via cell viability, oxidative stress, DNA fragmentation, and integrated multi-omics analyses, with the urinary-biomonitoring-informed mixture evaluated across four cell lines and the surface/tap-water mixtures assessed as an initial transcriptomic screen in HEK293T cells. RESULTS: Environmentally relevant mixtures (surface/tap water) elicited only limited transcriptomic changes (≤ 0.34% of genes). In contrast, biomonitoring-informed mixtures (≈ 0.1 µM ΣNEOs) induced modest but detectable dose-dependent cytotoxicity, with viability reductions of 3-8%, modest oxidative stress, and limited increases in DNA fragmentation, with effects distinct from individual compounds at matched concentrations. Transcriptomic analysis revealed cell-type-specific responses converging on three core modules: innate immune-like inflammatory signaling, ER stress/unfolded protein response, and metabolic reprogramming. Metabolomic profiling identified two dominant perturbation patterns-arginine/nitrogen-centered rewiring (A2780 and HEK293T) versus membrane lipid remodeling (HeLa and HepG2). CONCLUSIONS: Biomonitoring-informed NEO/metabolite mixtures elicited measurable, cell-specific molecular perturbations within biomonitoring-relevant concentration ranges, engaging coordinated stress and adaptation programs. These findings support the incorporation of mixture composition and biomonitoring-relevant concentration ranges into future toxicological evaluation.