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

TL;DR

This study shows that combinations of common environmental pollutants can be more harmful to human cells than individual pollutants, emphasizing the need for better risk assessments.

Contribution

The study identifies synergistic effects of binary pollutant mixtures at environmentally relevant concentrations in human cells.

Findings

The BPA-DBP combination showed 31% cytotoxicity at 100 nM, indicating strong synergy.

BADGE-BPA also exhibited similar synergistic effects.

Reactive oxygen species were partially linked to the observed cytotoxicity in mixtures.

Abstract

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.

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