Restoring Mitochondrial Function After Pesticide Exposure

The effects of the compounds 1–5 and resveratrol used as the reference on mitochondria were evaluated as for their capacity to restore mitochondrial function after organophosphate pesticide (glyphosate and chlorpyrifos) treatment. Briefly, BEAS-2B cells (3 × 10⁴ cells/well in a 96-well) were treated with either glyphosate or chlorpyrifos and at two concentration levels (10 and 100 µM) in the presence or absence of the test compounds (50 µM). After 24 h of incubation, the changes in mitochondrial potential and mitochondrial reducing activity (MRA) were evaluated. The changes in the mitochondrial potential were detected by 5,5′,6,6′-tetrachloro-1,1′,3,3′tetraethylbenzimidazolylcarbocyanine iodide/chloride (JC-1), a cationic dye that exhibits potential-dependent accumulation in mitochondria, indicated by fluorescence emission shift from red (∼590 nm) to green (∼525 nm). MRA was assessed by the resazurin assay. To this aim, cells were incubated with resazurin (6 µM) in the presence and absence of the compounds (50 µM) and the fluorescence intensity evaluated over time (0–240 min), in a plate reader (Infinite F200 PRO, Sunrise, Tecan, Männedorf, Swiss), and the results were normalized to the total protein using the Bradford assay (Sigma) [39 (link)]. Each experimental step was carried out in six replicates.