Diverse neurotoxicants converge on gene expression for neuropeptides and their receptors in an in vitro model of neurodifferentiation: effects of chlorpyrifos, diazinon, dieldrin and divalent nickel in PC12 cells

Abstract

Unrelated developmental neurotoxicants can produce similar neurobehavioral outcomes. We examined whether disparate agents affect neuromodulators that control numerous neurotransmitters and circuits, employing PC12 cells to explore the targeting of neuroactive peptides by organophosphates (chlorpyrifos, diazinon), an organochlorine (dieldrin) and a metal (Ni(2+)); we utilized microarrays to profile gene expression for the peptides and their receptors. Chlorpyrifos evoked robust upregulation of cholecystokinin, corticotropin releasing hormone, galanin, neuropeptide Y, neurotensin, preproenkephalin and tachykinin 1; this involved a critical period at the commencement of neurodifferentiation, since the effects were much less notable in undifferentiated PC12 cells. Diazinon targeted a similar but smaller repertoire of neuropeptide genes and the magnitude of the effects was also generally less. Surprisingly, dieldrin shared many of the same neuropeptide targets as the organophosphates and concordance analysis showed significant overlap among all three pesticides. However, dieldrin had more notable effects on neuropeptide receptors, and overlap between diazinon and dieldrin for the receptors led to a stronger resemblance of these two agents than of chlorpyrifos and dieldrin. Ni(2+) was unique, evoking upregulation of only one of the peptides affected by the other agents, while causing downregulation of several others. Nevertheless, there was still significant concordance between Ni(2+) and either diazinon or dieldrin, reflecting similarities toward the receptors. Our results show that neuropeptides are likely to be a prominent target for the developmental neurotoxicity of organophosphates and other neurotoxicants, and further, that the convergence of disparate agents on the same genes and pathways may contribute to similar neurobehavioral outcomes.

Figure 1

Effects of neurodifferentiation on neuropeptide gene expression. Each panel shows the expression ratio at 24h and 72h in undifferentiated cells differentiating cells. Values represent means and standard errors, normalized by setting the value for all genes on the array to 1.0. Asterisks below each panel denote genes showing a significant difference between undifferentiated and differentiating cells. Note the different scale for crh and npy.

Figure 2

Effects of neurodifferentiation on neuropeptide receptor gene expression. Each panel shows the expression ratio at 24h and 72h in undifferentiated cells and differentiating cells. Values represent means and standard errors, normalized by setting the value for all genes on the array to 1.0. Asterisks below each panel denote genes showing a significant difference between undifferentiated and differentiating cells.

Figure 3

Effects of chlorpyrifos exposure on expression of genes for neuropeptides (A) and neuropeptide receptors (B) in undifferentiated PC12 cells. Multivariate ANOVA appears at the top of each panel and asterisks shown below each gene denote a significant main treatment effect; daggers denote genes for which a treatment × time interaction was detected and show the individual times for which treatment effects were present.

Figure 4

Effects of chlorpyrifos exposure on expression of genes for neuropeptides (A) and neuropeptide receptors (B) in differentiating PC12 cells. Multivariate ANOVA appears at the top of each panel and asterisks shown below each gene denote a significant main treatment effect; daggers denote genes for which a treatment × time interaction was detected and show the individual times for which treatment effects were present. Testing for individual gene effects were not carried out for neuropeptide receptors because the ANOVA failed to show significant treatment effects or interactions of treatment with other variables.

Figure 5

Effects of diazinon exposure on expression of genes for neuropeptides (A) and neuropeptide receptors (B) in differentiating PC12 cells. Multivariate ANOVA appears at the top of each panel and asterisks shown below each gene denote a significant main treatment effect; daggers denote genes for which a treatment × time interaction was detected and show the individual times for which treatment effects were present. Testing for individual gene effects were not carried out for neuropeptide receptors because the ANOVA failed to show significant treatment effects or interactions of treatment with other variables.

Figure 6

Effects of dieldrin exposure on expression of genes for neuropeptides (A) and neuropeptide receptors (B) in differentiating PC12 cells. Multivariate ANOVA appears at the top of each panel and asterisks shown below each gene denote a significant main treatment effect; daggers denote genes for which a treatment × time interaction was detected and show the individual times for which treatment effects were present.

Figure 7

Effects of Ni²⁺ exposure on expression of genes for neuropeptides (A) and neuropeptide receptors (B) in differentiating PC12 cells. Multivariate ANOVA appears at the top of each panel and asterisks shown below each gene denote a significant main treatment effect; daggers denote genes for which a treatment × time interaction was detected and show the individual times for which treatment effects were present. Testing for individual gene effects were not carried out for neuropeptide receptors because the ANOVA failed to show significant treatment effects or interactions of treatment with other variables.

Figure 8

Pairwise correlations of the effects of chlorpyrifos in undifferentiated vs. differentiating cells (A,B,C), and in differentiating cells for chlorpyrifos vs. diazinon (D,E,F), including all the genes in the study (A,D), just the neuropeptide genes (B,E) or just the neuropeptide receptor genes (C,F). Values were taken from Figures 3, 4 and 5. The line in each panel shows the least-squares fit of the data. The corresponding equation and correlation coefficient are shown at the top of each panel, along with the significance of the correlation.

Figure 9

Pairwise correlations in differentiating cells of the effects of chlorpyrifos vs. dieldrin (A,B,C), and chlorpyrifos vs. Ni²⁺ (D,E,F), including all the genes in the study (A,D), just the neuropeptide genes (B,E) or just the neuropeptide receptor genes (C,F). Values were taken from Figures 4, 6 and 7. The line in each panel shows the least-squares fit of the data. The corresponding equation and correlation coefficient are shown at the top of each panel, along with the significance of the correlation.

Figure 10

Pairwise correlations in differentiating cells of the effects of diazinon vs. dieldrin (A,B,C), diazinon vs. Ni²⁺ (D,E,F) and dieldrin vs. Ni²⁺ (G,H,I), including all genes in the study (A,D,G), just the neuropeptide genes (B,E,H) or just the neuropeptide receptor genes (C,F,I). Values were taken from Figures 5, 6 and 7. The line in each panel shows the least-squares fit of the data. The corresponding equation and correlation coefficient are shown at the top of each panel, along with the significance of the correlation.

Figure 11

Venn diagram showing overall concordance among neurotoxicants. (A) Chlorpyrifos, diazinon and dieldrin are concordant with each other but the relationship between dieldrin and diazinon is stronger than that between chlorpyrifos and diazinon or between chlorpyrifos and dieldrin; this reflects the greater correlation of neuropeptide receptor genes for diazinon and dieldrin. (B) Chlorpyrifos is concordant with diazinon and diazinon with Ni²⁺; nevertheless, chlorpyrifos and Ni²⁺ are dissimilar because the genes accounting for the relationship between chlorpyrifos and diazinon differ from those accounting for the similarities of diazinon to Ni²⁺.