Investigations of Oxidative Stress, Antioxidant Response, and Protein Binding in Chlorpyrifos Exposed Rat Neuronal PC12 Cells

ABSTRACT Chlorpyrifos (CPF) is a widely used organophosphate insecticide. In addition to its known properties of cholinesterase inhibition, the production of reactive oxygen species (ROS) has been suggested as a possible toxic mechanism. To investigate CPF-generated ROS, rat neuronal PC12 cells were exposed to CPF concentrations of 0 to 5000 μg/mL in Krebs buffered media (KRH), KRH + 4% bovine serum albumin (BSA), and KRH + 25 μM of the antioxidant Trolox for 0 to 5 h. Paraquat served as a positive control for ROS. The fluorescent probe 2,7-dichlorodihydro-fluorescein and the MTS assay were used to measure ROS and cytotoxicity, respectively. Examinations into CPF-albumin binding were also conducted. CPF was not strongly cytotoxic to PC12 cells, causing only mild cytotoxicity at 5000 μg/ml. In KRH media, CPF-generated ROS was observed at 4 and 5 h at 500 and 1000 μg/mL, and at 1 to 5 h at 5000 μg/mL CPF. In KRH + 4% BSA, ROS was seen only at 5 h in 5000 μg/mL CPF. Trolox significantly reduced CPF- and paraquat-induced ROS. Calculated CPF-albumin binding at 1, 10, and 100 μg/mL CPF in 4% BSA was 96%, 75%, and 15%. These data show CPF at ≥500 μg/mL induced ROS in PC12 cells, but the addition of the antioxidant Trolox and 4% BSA dramatically reduced ROS levels.