Cytotoxic and Synergistic Effects of Environmentally Relevant Binary Pollutant Mixtures in a Human Lymphoblast Cell Line

Environmental pollutants are persistent chemicals that pose substantial risks to human health, contributing to global mortality and economic burden. In real-world situations, exposure rarely occurs to single compounds; instead, people are chronically exposed to complex mixtures at low concentrations. However, most regulatory frameworks still rely on single-substance risk assessments, potentially underestimating the hazards associated with combined exposures. This study investigated the cytotoxic interactions of binary mixtures of five environmentally relevant pollutants: bisphenol A (BPA), bisphenol A diglycidyl ether (BADGE), dibutyl phthalate (DBP), di(2-ethylhexyl) phthalate (DEHP), and perfluorooctanoic acid (PFOA), using the human lymphoblast cell line NALM-6. Cells were exposed for 72 h to each compound individually and to all possible binary combinations, reflecting concentrations reported in human plasma or serum. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and interactions were analyzed using the Bliss model of independence and two-way analysis of variance (ANOVA). Intracellular reactive oxygen species were measured using the 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) probe to explore the involvement of oxidative stress. Synergistic interactions were observed under specific conditions, although not all statistically identified interactions corresponded to biologically significant effects. The BPA-DBP combination produced the highest cytotoxicity when both pollutants were present at 100 nM (31%), consistent with a strong synergistic effect. A similar pattern was observed for BADGE-BPA. ROS production was partially associated with cytotoxicity in these selected mixtures. Overall, these findings highlight the importance of distinguishing statistical synergy from toxicological relevance.