Chlorpyrifos developmental neurotoxicity: interaction with glucocorticoids in PC12 cells

Abstract

Prenatal coexposures to glucocorticoids and organophosphate pesticides are widespread. Glucocorticoids are elevated by maternal stress and are commonly given in preterm labor; organophosphate exposures are virtually ubiquitous. We used PC12 cells undergoing neurodifferentiation in order to assess whether dexamethasone enhances the developmental neurotoxicity of chlorpyrifos, focusing on models relevant to human exposures. By themselves, each agent reduced the number of cells and the combined exposure elicited a correspondingly greater effect than with either agent alone. There was no general cytotoxicity, as cell growth was actually enhanced, and again, the combined treatment evoked greater cellular hypertrophy than with the individual compounds. The effects on neurodifferentiation were more complex. Chlorpyrifos alone had a promotional effect on neuritogenesis whereas dexamethasone impaired it; combined treatment showed an overall impairment greater than that seen with dexamethasone alone. The effect of chlorpyrifos on differentiation into specific neurotransmitter phenotypes was shifted by dexamethasone. Either agent alone promoted differentiation into the dopaminergic phenotype at the expense of the cholinergic phenotype. However, in dexamethasone-primed cells, chlorpyrifos actually enhanced cholinergic neurodifferentiation instead of suppressing this phenotype. Our results indicate that developmental exposure to glucocorticoids, either in the context of stress or the therapy of preterm labor, could enhance the developmental neurotoxicity of organophosphates and potentially of other neurotoxicants, as well as producing neurobehavioral outcomes distinct from those seen with either individual agent.

Figure 1

Indices of ceil number, ceil growth and neurite formation in PC12 cells exposed simultaneously to 100 nM dexamethasone, with and without 10 or 50 μM chlorpyrifos during neurodifferentiation: (A) DNA, (B) total protein/DNA ratio, (C) membrane protein/DNA ratio, (D) membrane/total protein ratio. Bars represent mean ± SE obtained from 8–12 independent cultures for each condition. ANOVA for main treatment effects and interactions appears at the top of each panel and asterisks denote significant differences evoked by chlorpyrifos compared to the corresponding control condition. Abbreviation: CPF = chlorpyrifos.

Figure 2

Differentiation into dopamine and acetylcholine neuronal phenotypes in PC12 cells exposed simultaneously to 100 nM dexamethasone, with and without 10 or 50 μM chlorpyrifos during neurodifferentiation: (A) TH activity, (B), ChAT activity, (C) TH/ChAT ratio. Bars represent mean ± SE obtained from 8–12 independent cultures for each condition. ANOVA for main treatment effects and interactions appears at the top of each panel and asterisks denote significant differences evoked by chlorpyrifos compared to the corresponding control condition. Abbreviation: CPF = chlorpyrifos.

Figure 3

Indices of cell number, cell growth and neurite formation in PC12 cells exposed to 100 nM dexamethasone for the first 24 hr of neurodifferentiation, followed by treatment with and without 10 or 50 μM chlorpyrifos for an additional five days: (A) DNA, (B) total protein/DNA ratio, (C) membrane protein/DNA ratio, (D) membrane/total protein ratio. Bars represent mean ± SE obtained from 8–12 independent cultures for each condition. ANOVA for main treatment effects and interactions appears at the top of each panel and asterisks denote significant differences evoked by chlorpyrifos compared to the corresponding control condition. Abbreviation: CPF = chlorpyrifos.

Figure 4

Indices of cell number, cell growth and neurite formation in PC12 cells exposed to 100 nM dexamethasone for 24 hr in the undifferentiated state, followed by addition of NGF and treatment with and without 10 or 50 μM chlorpyrifos for an additional five days: (A) DNA, (B) total protein/DNA ratio, (C) membrane protein/DNA ratio, (D) membrane/total protein ratio. Bars represent mean ± SE obtained from 8–12 independent cultures for each condition. ANOVA for main treatment effects and interactions appears at the top of each panel and asterisks denote significant differences evoked by chlorpyrifos compared to the corresponding control condition. Abbreviation: CPF = chlorpyrifos.