Is oxidative stress involved in the developmental neurotoxicity of chlorpyrifos?

The increasing use of chlorpyrifos (CPF) has elicited concern about neurotoxic effects on the fetus and neonate. CPF targets a number of events specific to brain development, over and above the ability of its active metabolite, CPF oxon, to inhibit cholinesterase. We used PC12 cells, a model system which displays many of the neurodevelopmental effects of CPF, in order to examine whether oxidative stress underlies the direct effects of CPF on development. Production of reactive oxygen species (ROS) was measured with a fluorescent intracellular dye. When PC12 cell suspensions were treated acutely with CPF for 10 min, ROS generation was increased in a concentration-dependent manner; CPF oxon was much less effective than the native compound. CPF also increased the ROS production in response to an acute sodium nitroprusside challenge, indicating sensitization of the cells to other oxidant stressors. Next, PC12 cells were grown in an undifferentiated state in the presence of CPF or CPF oxon for extended time periods, under conditions in which CPF inhibits mitosis, and the cells were then washed and ROS production measured. Neither compound elicited a significant change in ROS production. Finally, differentiation was initiated with nerve growth factor and the cells were exposed continuously to CPF or CPF oxon over a 72 h period; under these conditions, CPF inhibits neurite outgrowth. When the cells were washed and evaluated for ROS production, no significant differences were seen. These results indicate that CPF, but not CPF oxon, has the ability to elicit acute increases in ROS production. However, the effect disappears immediately once CPF exposure is terminated, possibly reflecting cellular defense mechanisms that lessen the impact of oxidant injury.