Does chlorpyrifos neurotoxicity involve developing glial cells? An in vitro and in vivo analysis

The widespread use of the organophosphate pesticide chlorpyrifos (CPF) has raised major biomedical and societal concerns regarding its potential to cause fetal or neonatal neurobehavioral damage, even at doses that do not elicit acute toxicity. Chlorpyrifos disrupts the developing brain both before and after the close of neurogenesis, corresponding to glial proliferation and differentiation. Given the critical roles glial cells play in the proper development and function of the brain, we hypothesized that the neurotoxicity of chlorpyrifos involves alteration of glial cell development. C6 glioma cells were used to determine whether chlorpyrifos effects in an isolated, homogenous population of glial-type cells mirrored effects in the intact brain. Indeed, as in neonatal rats, CPF inhibited DNA synthesis in replicating C6 cells independently of cholinergic stimulation since cholinergic antagonists did not block the effects of CPF. Further, CPF interfered with adenylyl cyclase cell signaling at the level of G-proteins in replicating cells as well as in differentiating cells, albeit to a lesser extent. On the other hand, CPF enhanced the formation of reactive oxygen species and obstructed DNA binding to Sp-1, a transcription factor essential to differentiation, only in differentiating C6 cells. To determine whether CPF effects on glial-type cells in vitro translate to effects in vivo, CPF was administered subcutaneously to late gestational (GD17--20, 1,2,5,10,20,40 mg/kg/d) and neonatal rats (PN1--4, 1 mg/kg/d; PN11--14, 5 mg/kg/d) and immunoblotted for markers of astrocytes (glial fibrillary acidic protein, GFAP), oligodendrocytes (myelin basic protein, MBP) and neurons (neurofilament subunits 68kD and 200kD, NF68 and NF200). Chlorpyrifos-induced alterations of all four neuroproteins depended upon gender and age, indicating a shift in both gender and the neural populations targeted. The most widespread effects occurred during peak gliogenesis, myelination, and axon formation (PN11--14). In general, males were preferentially targeted during postnatal exposures while females experienced delayed effects following prenatal exposure, findings that are in agreement with behavioral consequences. In conclusion, alterations in glial cell development are at least partially responsible for the developmental neurotoxicity of CPF, extending the window of vulnerability to late events in development, including myelination, synaptic plasticity, and architectural modeling of the brain.