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. 2007 Sep;115(9):1306-13.
doi: 10.1289/ehp.10194.

Ameliorating the developmental neurotoxicity of chlorpyrifos: a mechanisms-based approach in PC12 cells

Theodore A Slotkin  1 , Emiko A MacKillop, Ian T Ryde, Frederic J Seidler
Affiliations

Ameliorating the developmental neurotoxicity of chlorpyrifos: a mechanisms-based approach in PC12 cells

Theodore A Slotkin et al. Environ Health Perspect. 2007 Sep.

Abstract

Background: Organophosphate developmental neurotoxicity involves multiple mechanisms converging on neural cell replication and differentiation.

Objectives: We evaluated mechanisms contributing to the adverse effects of chlorpyrifos (CPF) on DNA synthesis, cell number and size, and cell signaling mediated by adenylyl cyclase (AC) in PC12 cells, a neuronotypic cell line that recapitulates the essential features of developing mammalian neurons.

Results: In undifferentiated cells, cholinergic receptor antagonists had little or no protective effect against the antimitotic actions of CPF; however, when nerve growth factor was used to evoke differentiation, the antagonists showed partial protection against deficits in cell loss and alteration in cell size elicited by CPF, but were ineffective in preventing the deterioration of AC signaling. Nicotine, which stimulates nicotinic acetylcholine receptors but also possesses a mixture of prooxidant/antioxidant activity, had adverse effects by itself but also protected undifferentiated cells from the actions of CPF and had mixed additive/protective effects on cell number in differentiating cells. The antioxidant vitamin E also protected both undifferentiated and differentiating cells from many of the adverse effects of CPF but worsened the impact on AC signaling. Theophylline, which prevents the breakdown of cyclic AMP, was the only agent that restored AC signaling to normal or supranormal levels but did so at further cost to cell replication.

Conclusions: Our results show definitive contributions of cholinergic hyperstimulation, oxidative stress, and interference with AC signaling in the developmental neurotoxicity of CPF and point to the potential use of this information to design treatments to ameliorate these adverse effects.

Keywords: adenylyl cyclase; brain development; chlorpyrifos; cholinergic antagonists; neurotoxicity; nicotine; organophosphate insecticides; oxidative stress; theophylline; vitamin E.

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Figures

Figure 1
Figure 1
Effects of CPF (30 or 50 μM) with and without cotreatment with 30 μM atropine (ATR) plus 30 μM mecamylamine (MEC) on DNA content [A; ANOVA: treatment, p < 0.0001 (n = 12–24)] and total protein/DNA ratio [B; ANOVA: treatment, p < 0.0001 (n = 11–12)] in differentiating PC12 cells. Cells were treated with the indicated agents along with NGF for a total of 6 days. *Significantly different from controls. †Cotreated values significantly different from CPF alone. #Cotreated values significantly different from ATR plus MEC.
Figure 2
Figure 2
Effects of 30 μM CPF with and without cotreatment with 30 μM atropine (ATR) plus 30 μM mecamylamine (MEC) on basal AC activity [A; ANOVA: treatment, p < 0.0001 (n = 9–11)], fluoride-stimulated AC activity [B; ANOVA: treatment, p < 0.0003 (n = 9–11)], and forskolin-stimulated AC activity [C; ANOVA: treatment, p < 0.004 (n = 10–11)] in differentiating PC12 cells. Cells were treated with the indicated agents along with NGF for a total of 6 days. *Significantly different from controls. #Cotreated values significantly different from ATR plus MEC .
Figure 3
Figure 3
Effects of 30 μM or 50 μM CPF with and without cotreatment with 10 μM nicotine (NIC) on DNA content [A; ANOVA: treatment, p < 0.0001 (n = 12–30)] and total protein/DNA ratio [B; ANOVA: treatment, p < 0.0002 (n = 16–18)] in differentiating PC12 cells. Cells were treated with the indicated agents along with NGF for a total of 6 days. *Significantly different from controls. #Cotreated values significantly different from NIC alone.
Figure 4
Figure 4
Effects of 30 μM CPF with and without cotreatment with 10 μM nicotine (NIC) on basal AC activity [A; ANOVA: treatment, p < 0.0001 (n = 10–12)] , fluoride-stimulated AC activity [B; ANOVA: treatment, not significant (n = 11–12)], and forskolin-stimulated AC activity [C; ANOVA: treatment, p < 0.004 (n = 10–12)] in differentiating PC12 cells. Cells were treated with the indicated agents along with NGF for a total of 6 days. *Significantly different from controls. #Cotreated values significantly different from NIC alone.
Figure 5
Figure 5
Effects of 30 μM CPF with and without cotreatment with 10 or 30 μM vitamin E (VitE) on DNA synthesis in undifferentiated PC12 cells. ANOVA: treatment, p < 0.0001 (n = 5–16). Cells were treated with the indicated agents for 24 hr, and then [3H]thymidine incorporation into DNA was measured for 1 hr. *Significantly different from controls. †Cotreated values significantly different from CPF alone.
Figure 6
Figure 6
Effects of 30 or 50 μM CPF with and without cotreatment with 10 or 30 μM vitamin E (VitE) on DNA content [A; ANOVA: treatment, p < 0.0001 (n = 8–16)] and total protein/DNA ratio [B; ANOVA: treatment, p < 0.0004 (n = 8)] in differentiating PC12 cells. Cells were treated with the indicated agents along with NGF for a total of 6 days. *Significantly different from controls. †Cotreated values significantly different from CPF alone. #Cotreated values significantly different from VitE alone.
Figure 7
Figure 7
Effects of 30 μM CPF with and without cotreatment with 10 or 30 μM vitamin E (VitE) on basal AC activity [A; ANOVA: treatment, p < 0.0001 (n = 7–8)], fluoride-stimulated AC activity [B; ANOVA: treatment, p < 0.01 (n = 6–8)], and forskolin-stimulated AC activity [C; ANOVA: treatment, p < 0.0001 (n = 7–9)] in differentiating PC12 cells. Cells were treated with the indicated agents along with NGF for a total of 6 days. *Significantly different from controls. †Cotreated values significantly different from CPF alone. #Cotreated values significantly different from VitE alone.
Figure 8
Figure 8
Effects of 30 or 50 μM CPF with and without cotreatment with 1 or 10 mM theophylline (Theo) on DNA content [A; ANOVA: treatment, p < 0.0001 (n = 11–22)] and total protein/DNA ratio [B; ANOVA: treatment, p < 0.0001 (n = 11–12)] in differentiating PC12 cells. Cells were treated with the indicated agents along with NGF for a total of 6 days. Note the difference in scale from the other figures. *Significantly different from controls. †Cotreated values significantly different from CPF alone. #Cotreated values significantly different from Theo alone.
Figure 9
Figure 9
Effects of 30 μM CPF with and without cotreatment with 1 mM theophylline (Theo) on basal AC activity [A; ANOVA: treatment, p < 0.0001 (n = 8–10)], fluoride-stimulated AC activity [B; ANOVA: treatment, p < 0.0001 (n = 8–10)], and forskolin-stimulated AC activity [C; ANOVA: treatment, p < 0.0001 (n = 9–10)] in differentiating PC12 cells. Cells were treated with the indicated agents along with NGF for a total of 6 days. *Significantly different from controls. †Cotreated values significantly different from CPF alone. #Cotreated values significantly different from Theo alone.
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