Disparate developmental neurotoxicants converge on the cyclic AMP signaling cascade, revealed by transcriptional profiles in vitro and in vivo

Abstract

Cell-signaling cascades are convergent targets for developmental neurotoxicity of otherwise unrelated agents. We compared organophosphates (chlorpyrifos, diazinon), an organochlorine (dieldrin) and a metal (Ni(2+)) for their effects on neuronotypic PC12 cells, assessing gene transcription involved in the cyclic AMP pathway. Each agent was introduced during neurodifferentiation at a concentration of 30 microM for 24 or 72 h and we assessed 69 genes encoding adenylyl cyclase isoforms and regulators, G-protein alpha-and beta,gamma-subunits, protein kinase A subtypes and the phosphodiesterase family. We found strong concordance among the four agents across all the gene families, with the strongest relationships for the G-proteins, followed by adenylyl cyclase, and lesser concordance for protein kinase A and phosphodiesterase. Superimposed on this pattern, chlorpyrifos and diazinon were surprisingly the least alike, whereas there was strong concordance of dieldrin and Ni(2+) with each other and with each individual organophosphate. Further, the effects of chlorpyrifos differed substantially depending on whether cells were undifferentiated or differentiating. To resolve the disparities between chlorpyrifos and diazinon, we performed analyses in rat brain regions after in vivo neonatal exposures; unlike the in vitro results, there was strong concordance. Our results show that unrelated developmental neurotoxicants can nevertheless produce similar outcomes by targeting cell signaling pathways involved in neurodifferentiation during a critical developmental period of vulnerability. Nevertheless, a full evaluation of concordance between different toxicants requires evaluations of in vitro systems that detect direct effects, as well as in vivo systems that allow for more complex interactions that converge on the same pathway.

Figure 1

Pairwise correlations of the effects of CPF, DZN, dieldrin and Ni²⁺ on expression of AC genes, calculated from Supplemental Tables 1 and 2 as the percent change from corresponding control values. Linear correlation coefficients are shown at the top of each panel and the line represents the least-squares fit of the data. NS, not significant.

Figure 2

Pairwise correlations of the effects of CPF, DZN, dieldrin and Ni²⁺ on expression of G-protein α-subunit genes, calculated from Supplemental Tables 1 and 2 as the percent change from corresponding control values. Linear correlation coefficients are shown at the top of each panel and the line represents the least-squares fit of the data. NS, not significant.

Figure 3

Pairwise correlations of the effects of CPF, DZN, dieldrin and Ni²⁺ on expression of G-protein β,γ-subunit genes, calculated from Supplemental Tables 1 and 2 as the percent change from corresponding control values. Linear correlation coefficients are shown at the top of each panel and the line represents the least-squares fit of the data. NS, not significant.

Figure 4

Pairwise correlations of the effects of CPF, DZN, dieldrin and Ni²⁺ on expression of protein kinase A genes, calculated from Supplemental Tables 1 and 2 as the percent change from corresponding control values. Linear correlation coefficients are shown at the top of each panel and the line represents the least-squares fit of the data. NS, not significant.

Figure 5

Pairwise correlations of the effects of CPF, DZN, dieldrin and Ni²⁺ on expression of phosphodiesterase genes, calculated from Supplemental Tables 1 and 2 as the percent change from corresponding control values. Linear correlation coefficients are shown at the top of each panel and the line represents the least-squares fit of the data. NS, not significant.

Figure 6

Correlations of the effects of CPF on undifferentiated versus differentiating cells for each the five classes of genes, calculated from Supplemental Tables 1 and 2 as the percent change from corresponding control values: AC (A), G-protein α-subunits (B), G-protein β,γsubunits (C), protein kinase A (D) and phosphodiesterase (E). Linear correlation coefficients are shown at the top of each panel and the line represents the least-squares fit of the data. NS, not significant.

Figure 7

Correlations of the effects of CPF (1 mg/kg) and DZN (1 or 2 mg/kg) on gene expression in vivo, evaluated on postnatal day 5 in forebrain and brainstem from neonatal rats given each agent daily from postnatal days 1–4: AC (A), G-protein α-subunits (B), G-protein β,γ-subunits (C), protein kinase A (D) and phosphodiesterase (E). Linear correlation coefficients are shown within each panel and the line represents the least-squares fit of the data.

Figure 8

Venn diagram illustrating the relationships among CPF, DZN, dieldrin and Ni²⁺ for their global effects on the five classes of genes as calculated in Table 1. (A) CPF and dieldrin have strong concordance, as do DZN and dieldrin, but the effects of CPF and DZN are only weakly related because the gene effects shared between CPF and dieldrin differ from those involved in the correlation of DZN and dieldrin. (B) CPF, dieldrin and Ni²⁺ all share common patterns of effects on gene expression.

Figure 9

Comparison of in vitro (A) and in vivo (B) correlations of gene expression values combined from all five classes. Linear correlation coefficients are shown at the top of each panel and the line represents the least-squares fit of the data. NS, not significant.