| Chlorpyrifos Affects Phenotypic Outcomes in a Model of Mammalian Neurodevelopment: Critical Stages Targeting Differentiation in PC12 Cells Ruth R. Jameson, Frederic J. Seidler, Dan Qiao, and Theodore A. Slotkin Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA Abstract
The organophosphate insecticide chlorpyrifos (CPF) adversely affects mammalian brain development through multiple mechanisms. To determine if CPF directly affects neuronal cell replication and phenotypic fate, and to identify the vulnerable stages of differentiation, we exposed PC12 cells, a model for mammalian neurodevelopment, to CPF concentrations spanning the threshold for cholinesterase inhibition (5-50 µM) and conducted evaluations during mitosis and in early and mid-differentiation. In undifferentiated cells, exposure to 5 µM CPF for 1-3 days reduced DNA synthesis significantly without eliciting cytotoxicity. At the same time, CPF increased the expression of tyrosine hydroxylase (TH) , the enzymatic marker for the catecholamine phenotype, without affecting choline acetyltransferase (ChAT) , the corresponding marker for the cholinergic phenotype. Upon exposure to nerve growth factor (NGF) , PC12 cells developed neuritic projections in association with vastly increased TH and ChAT expression accompanying differentiation into the two phenotypes. CPF exposure begun at the start of differentiation significantly reduced ChAT but not TH activity. In contrast, when CPF was added in mid-differentiation (4 days of NGF pretreatment) , ChAT was unaffected and TH was increased slightly. Thus, CPF exerts stage-specific effects, reducing DNA synthesis in the undifferentiated state, impairing development of the cholinergic phenotype at the start of differentiation, and promoting expression of the catecholaminergic phenotype both in undifferentiated and differentiated cells. CPF administration in vivo produces deficits in the number of neurons and cholinergic function, and because we were able to reproduce these effects in vitro, our results suggest that CPF directly influences the phenotypic fate of neuronal precursors. Key words: acetylcholine, brain development, catecholamines, chlorpyrifos, DNA synthesis, organophosphate insecticides, PC12 cells. Environ Health Perspect 114:667-672 (2006) . doi:10.1289/ehp.8750 available via http://dx.doi.org/ [Online 29 December 2005]
Address correspondence to T.A. Slotkin, Box 3813 DUMC, Duke University Medical Center, Durham, NC 27710 USA. Telephone: (919) 681-8015. Fax: (919) 684-8197. E-mail: t.slotkin@duke.edu Research was supported by grants from the National Institutes of Health (ES10356 and ES07031) . The authors declare they have no competing financial interests. Received 17 October 2005 ; accepted 29 December 2005.
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