Nonenzymatic Functions of Acetylcholinesterase Splice Variants in the Developmental Neurotoxicity of Organophosphates: Chlorpyrifos, Chlorpyrifos Oxon, and Diazinon

Abstract

Organophosphate pesticides affect mammalian brain development through mechanisms separable from the inhibition of acetylcholinesterase (AChE) enzymatic activity and resultant cholinergic hyperstimulation. In the brain, AChE has two catalytically similar splice variants with distinct functions in development and repair. The rare, read-through isoform, AChE-R, is preferentially induced by injury and appears to promote repair and protect against neurodegeneration. Overexpression of the more abundant, synaptic isoform, AChE-S, enhances neurotoxicity. We exposed differentiating PC12 cells, a model for developing neurons, to 30 μM chlorpyrifos (CPF) or diazinon (DZN), or CPF oxon, the active metabolite that irreversibly inhibits AChE enzymatic activity, in order to determine whether they differentially induce the formation of AChE-S as a mechanistic predictor of developmental neurotoxicity. We then administered CPF or DZN to neonatal rats on postnatal days 1–4 using daily doses spanning the threshold for AChE inhibition (0–20%); we then evaluated AChE gene expression in forebrain and brainstem on post-natal day 5. In PC12 cells, after 48 hr of exposure, CPF, CPF oxon, and DZN enhanced gene expression for AChE-R by about 20%, whereas CPF and DZN, but not CPF oxon, increased AChE-S expression by 20–40%. Thus, despite the fact that CPF oxon is a much more potent AChE inhibitor, it is the native compound (CPF) that induces expression of the neurotoxic AChE-S iso-form. For in vivo exposures, 1 mg/kg CPF had little or no effect, but 0.5 or 2 mg/kg DZN induced both AChE-R and AChE-S, with a greater effect in males. Our results indicate that nonenzymatic functions of AChE variants may participate in and be predictive of the relative developmental neurotoxicity of organophosphates, and that the various organophosphates differ in the degree to which they activate this mechanism.