Nonenzymatic functions of acetylcholinesterase splice variants in the developmental neurotoxicity of organophosphates: chlorpyrifos, chlorpyrifos oxon, and diazinon

Abstract

Background: 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.

Objectives: We exposed differentiating PC12 cells, a model for developing neurons, to 30 microM 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.

Results: 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 isoform. 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.

Conclusions: 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.

Figure 1

AChE isoforms resulting from alternative splicing of the AChE pre-mRNA. Abbreviations: E, exon; For, forward; I, intron; Rev, reverse. The AChE-R variant mRNA is the product of splicing and includes intron (I) 4 and omits exon (E) 6. AChE-S omits intron 4 and extends through exon 6. Arrows represent primers used in PCR detection of each splice variant.

Figure 2

Sample gel lanes from a representative PC12 cell determination of AChE-R, AChE-S, and a DNA standard ladder. The upper band in the sample lanes is the 18S ribosomal control; the standard DNA ladder consists of increments of 100 base pairs.

Figure 3

Effects of organophosphate treatment (CPF, DZN, CPO) on AChE mRNA splice variants in differentiating PC12 cells. Cells were treated for 48 hr in the presence of NGF. Data represent means and SEs obtained from 6–12 cultures for each condition, presented as the percent change from control values. Across both variants, ANOVA indicates a significant main treatment effect (p < 0.0001) and a difference in treatment effects between the two variants (treatment × subtype interaction, p < 0.0005). For each variant, there was a main treatment effect (p < 0.002 for AChE-R and p < 0.0001 for AChE-S). *Differs significantly from the corresponding control.

Figure 4

Effects of in vivo organophosphate treatment on AChE mRNA splice variants. (A) AChE-R. (B) AChE-S. Animals were treated with CPF or DZN at the indicated doses on PND1–4, and samples were obtained on PND5. Data represent mean and SE obtained from six animals of each sex in each treatment group, presented as the percent change from the corresponding control values. Across both variants, ANOVA indicates a significant main treatment effect (p < 0.005), which was also significant for each variant separately (p < 0.03 for AChE-R; p < 0.02 for AChE-S). Differences in males were statistically significant (p < 0.006), whereas those in females were not. Statistics for individual treatments whose main effects differ from the corresponding control values are shown at the bottom of each panel; tests for individual regions were not conducted because of the absence of treatment × region interactions. Note the difference in scales in (A) and (B). NS, not significant.