Oxidative stress and inflammatory response of fibroblasts exposed to chlorpyrifos

Chlorpyrifos is a widely used insecticide that has been restricted by regulatory agencies, but still with significant environmental exposure. Nevertheless, few studies investigate the effect of this pesticide in fibroblasts. The aim of this work was to study the effect of chlorpyrifos in the viability of fibroblasts using concentrations detected for human skin exposure. Cellular mechanisms unleashed by chlorpyrifos were also explored.

The GM03349 fibroblast cell line derived from human skin was obtained from the Cell Bank (Coriell Institute) and exposed to different concentrations (0.36-250 µM) of chlorpyrifos and a commercial mixture (Lethal-20), for 6 days. For the viability assays, we used a resazurin assay. For oxidative stress evaluation, after 3h exposure, we measured ROS formation by DCF-DA microplate assay. The inflammatory cytokine IL-6 was measured in the supernatant by ELISA, after 6 days.

For the exposure to chlorpyrifos, only the highest concentration tested (250 µM) resulted in a decrease in cell viability to 18.98%. In the case of Lethal-20, at 125 µM, there was a reduction of cells’ viability to 15.82% and complete cell death at 250 µM. Moreover, ROS production was increased to 1.43 folds and 1.28 folds when the cells were exposed, respectively, to 250 µM chlorpyrifos pure or Lethal-20. To evaluate the inflammatory response, the secretion of IL-6 was assessed in culture supernatants. Exposure to 250 µM chlorpyrifos pure or Lethal-20 gave rise, respectively, to a 1.81 and a 2.43 fold increase of cytokine´s secretion.

In conclusion, prolonged exposure to concentrations of 125 µM in the case of Lethal-20 and to 250 µM in the case of chlorpyrifos caused a significant loss of fibroblasts' viability. Moreover, the toxicity of this pesticide in fibroblasts is evidenced by the induction of oxidative stress and the production of inflammatory cytokines.