Hydramethylnon induces synergistic toxicity of pyrethroid mixtures in human lung epithelial cells

Household insecticides are frequently used in combination indoors; however, the inhalation-relevant toxicological consequences of such co-exposure remain unclear. Therefore, in this study, we investigated whether co-exposure to hydramethylnon and representative pyrethroid insecticides (tetramethrin, prallethrin, and imiprothrin) induces synergistic toxicity in BEAS-2B human bronchial epithelial cells and explored a metabolic mechanism involving cytochrome P450 (CYP) inhibition. Cell viability was quantified via water-soluble tetrazolium salt-1 assay after 24 h exposure, and the half-maximal inhibitory concentration (IC₅₀) of individual chemicals were used to define toxic units for mixture design. Mixture interactions were evaluated by comparing experimentally observed mixture concentration-response relationships with predictions from concentration addition (CA) or independent action (IA) using the Open Mixture Risk Assessment (OpenMRA) platform, with interaction classification based on the model deviation ratio (MDR). Analyses of chemical category and mode of action (MoA) revealed that hydramethylnon mechanistically differed from pyrethroid insecticides, supporting the application of the IA model for mixture toxicity evaluation. Although mixtures containing only pyrethroids showed additive effects consistent with the CA model (MDR, 0.95), every mixture containing hydramethylnon exhibited significant synergism, with MDRs of 2.62-5.80 under the IA model. Mechanistically, hydramethylnon significantly reduced CYP3A4 and CYP3A5 mRNA expression levels in BEAS-2B cells and inhibited recombinant CYP3A4 activity in a dose-dependent manner (IC₅₀, 3.30 µg/mL). Molecular docking further confirmed stable binding of hydramethylnon within CYP3A4/3A5 active sites. Collectively, these findings suggest that hydramethylnon acts as a synergy-inducing co-exposure factor for pyrethroid mixtures in the bronchial epithelium, possibly via suppression of CYP-mediated detoxification, underscoring the need to incorporate metabolic interactions into inhalation-relevant mixture risk assessment.

The online version contains supplementary material available at https://doi.org/10.1007/s43188-026-00354-y.