Transcriptional profiles for glutamate transporters reveal differences between organophosphates but similarities with unrelated neurotoxicants

Abstract

The developmental neurotoxicity of organophosphates involves mechanisms other than their shared property as cholinesterase inhibitors, among which are excitotoxicity and oxidative stress. We used PC12 cells as a neurodevelopmental model to compare the effects of chlorpyrifos and diazinon on the expression of genes encoding glutamate transporters. Chlorpyrifos had a greater effect in cells undergoing nerve growth factor-induced neurodifferentiation as compared to undifferentiated PC12 cells, with peak sensitivity at the initiation of differentiation, reflecting a global upregulation of all the glutamate transporter genes expressed in this cell line. In differentiating cells, chlorpyrifos had a significantly greater effect than did diazinon and concordance analysis indicated no resemblance in their expression patterns. At the same time, the smaller effects of diazinon were highly concordant with those of an organochlorine pesticide (dieldrin) and a metal (divalent nickel). We also performed similar evaluations for the cystine/glutamate exchanger, which provides protection against oxidative stress by moving cystine into the cell; again, chlorpyrifos had the greatest effect, in this case reducing expression in undifferentiated and differentiating cells. Our results point to excitotoxicity and oxidative stress as major contributors to the noncholinesterase mechanisms that distinguish the neurodevelopmental outcomes between different organophosphates while providing a means whereby apparently unrelated neurotoxicants may produce similar outcomes.

Figure 1

Effects of chlorpyrifos exposure on expression of genes for glutamate transporters in undifferentiated PC12 cells. Multivariate ANOVA appears at the top of the 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. Expression ratios in the control group were: slc1a1, 1.12 ± 0.05 at 24h, 1.25 ± 0.06 at 72h; slc1a2, 1.04 ± 0.08 at 24h, 0.99 ± 0.12 at 72h; slc1a3, 0.72 ± 0.06 at 24h, 1.28 ± 0.09 at 72h; slc1a4, 0.80 ± 0.07 at 24h, 0.96 ± 0.08 at 72h; slc1a5, 0.84 ± 0.01 at 24h, 0.90 ± 0.01 at 72h; slc1a6, 0.86 ± 0.07 at 24h, 1.03 ± 0.06 at 72h; slc1a7, 0.99 ± 0.06 at 24h, 1.04 ± 0.06 at 72h; vglut3, 1.18 ± 0.09 at 24h, 1.05 ± 0.10 at 72h.

Figure 2

Effects of different neurotoxicants on genes for glutamate transporters in differentiating PC12 cells: (A) chlorpyrifos, (B) diazinon, (C) dieldrin, (D) Ni²⁺. Multivariate ANOVA appears at the top of each panel. Where there was a significant difference in the treatment effects on the various genes (B,C,D), 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. Where there was only a treatment effect and interaction of treatment × time (A), the treatment effects for each time are shown within the legend box. Expression ratios in the control group were: slc1a1, 0.79 ± 0.08 at 24h, 0.94 ± 0.10 at 72h; slc1a2, 1.16 ± 0.09 at 24h, 1.36 ± 0.10 at 72h; slc1a3, 0.57 ± 0.06 at 24h, 0.95 ± 0.08 at 72h; slc1a4, 0.83 ± 0.09 at 24h, 1.09 ± 0.08 at 72h; slc1a5, 1.13 ± 0.04 at 24h, 0.99 ± 0.03 at 72h; slc1a6, 0.87 ± 0.09 at 24h, 0.84 ± 0.02 at 72h; slc1a7, 0.98 ± 0.03 at 24h, 0.96 ± 0.04 at 72h; vglut3, 1.12 ± 0.06 at 24h, 0.93 ± 0.08 at 72h.

Figure 3

Pairwise correlations of the effects of chlorpyrifos in undifferentiated vs. differentiating cells (A), and in differentiating cells for chlorpyrifos vs. diazinon (B), dieldrin (C) or Ni²⁺ (D). In (A), a single point (slc1a3 at 1h, arrow) is responsible for the significant correlation; without that point, R = 0.20, not significant (NS).

Figure 4

Pairwise correlations of the effects of diazinon vs. dieldrin (A), diazinon vs. Ni²⁺ (B) and dieldrin vs. Ni²⁺ (C) in differentiating cells. Linear correlation coefficients are shown at the top of each panel along with the least-squares fit.

Figure 5

Effects of neurotoxicants on expression of the cystine/glutamate exchanger, slc7a11. Multivariate ANOVA indicates a significant main treatment effect (p < 0.0007) and a treatment × time interaction (p < 0.02). Asterisks denote treatments showing a main effect compared to control; the dagger denotes a significant treatment × time interaction and shows the individual time for which the treatment effect was present. Expression ratios in the control group were: undifferentiated cells, 2.09 ± 0.10 at 24h, 1.43 ± 0.15 at 72h; differentiating cells, 1.47 ± 0.12 at 24h, 0.92 ± 0.09 at 72h.