Evaluation of the Combined Toxicity of Pirimiphos-methyl and Piperonyl Butoxide: An In Vitro Study

Resumo

Introduction: Pirimiphos-methyl (PMM) and piperonyl butoxide (PBO) are widely used to control pests in various agricultural crops. While the toxic effects of PMM have been extensively documented, the safety of this pesticide when used in combination with PBO remains insufficiently explored. Objective: This study examined the cytotoxicity and genotoxicity of PMM and PBO combination in various cell lines (RAW 264-7, H9C2, HepG2, HEK 293) across different exposure times. Material and Methods: RAW 264-7, H9C2, HepG2, HEK 293 cells were seeded in 96-well plates and subjected to different PMM and PBO concentrations, at different concentrations (0.5; 1; 5; 10; 50 and 100 μg/mL) and at different exposure times (24, 48 and 72 h). Cytotoxicity was analyzed following the MTT assay protocol and genotoxicity using the Comet Assay. In addition, PMM and PBO effect were evaluated in LPS-stimulated RAW 264-7 cells. Results and Conclusion: RAW 264-7 cells showed sensitivity to high concentrations (50 and 100 μg/mL) after 72 hours. HepG2 cells were more affected by the combination at 100 μg/mL after 48 and 72 hours. HEK 293 cells showed cytotoxic effects at higher concentrations after 48 and 72 hours. No genotoxic effects were observed in HepG2 and mouse whole blood cells. The study also investigated the effects of PMF and PBO on RAW 264-7 cells treated with non-toxic concentrations and stimulated with LPS to evaluate the production of pro-inflammatory mediators. While individual concentrations of PMF or PBO showed no significant effects, the combination (MIX 5 μg/mL) inhibited 100% of NO production and significantly reduced TNF and IL-6 levels. Higher concentrations of PMF and PBO alone also reduced TNF and IL-6 production. The study suggests that low concentrations of PMF and PBO, individually and combined, have immunomodulatory effects without toxicity.