title: Chlorpyrifos- and chlorpyrifos oxon-induced neurite retraction in pre-differentiated N2a cells is associated with transient hyperphosphorylation of neurofilament heavy chain and ERK 1/2.
abstract: Chlorpyrifos (CPF) and CPF-oxon (CPO) are known to inhibit neurite outgrowth but little is known about their ability to induce neurite retraction in differentiating neuronal cells. The aims of this study were to determine the ability of these compounds to destabilize neurites and to identify the key molecular events involved. N2a cells were induced to differentiate for 20h before exposure to CPF or CPO for 2-8h. Fixed cell monolayers labeled with carboxyfluorescein succinimidyl ester or immunofluorescently stained with antibodies to tubulin (B512) or phosphorylated neurofilament heavy chain (Ta51) showed time- and concentration-dependent reductions in numbers and length of axon-like processes compared to the control, respectively, retraction of neurites being observed within 2h of exposure by live cell imaging. Neurofilament disruption was also observed in treated cells stained by indirect immunofluorescence with anti-phosphorylated neurofilament heavy chain (NFH) monoclonal antibody SMI34, while the microtubule network was unaffected. Western blotting analysis revealed transiently increased levels of reactivity of Ta51 after 2h exposure and reduced levels of reactivity of the same antibody following 8h treatment with both compounds, whereas reactivity with antibodies to anti-total NFH or anti-tubulin was not affected. The alteration in NFH phosphorylation at 2h exposure was associated with increased activation of extracellular signal-regulated protein kinase ERK 1/2. However, increased levels of phosphatase activity were observed following 8h exposure. These findings suggest for the first time that organophosphorothionate pesticide-induced neurite retraction in N2a cells is associated with transient increases in NFH phosphorylation and ERK1/2 activation.
authors: Interdisciplinary Biomedical Research Centre, School of Science and Technology, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK; School of Applied Medical Sciences, Umm Al-Qura University, Makkah, Saudi Arabia. Electronic address: ramya.sindi2010@my.ntu.ac.uk.
authors: Ramya A
authors: RA
authors: Sindi
authors: Interdisciplinary Biomedical Research Centre, School of Science and Technology, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK.
authors: Wayne
authors: W
authors: Harris
authors: School of Science and Technology, Nottingham Trent University, Nottingham NG11 8NS, UK.
authors: Gordon
authors: G
authors: Arnott
authors: Laboratory of Biochemistry and Toxicology, School of Veterinary Medicine, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece.
authors: John
authors: J
authors: Flaskos
authors: School of Science and Technology, Nottingham Trent University, Nottingham NG11 8NS, UK.
authors: Chris
authors: C
authors: Lloyd Mills
authors: Interdisciplinary Biomedical Research Centre, School of Science and Technology, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK. Electronic address: alan.hargreaves@ntu.ac.uk.
authors: Alan J
authors: AJ
authors: Hargreaves
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D004390:Chlorpyrifos / Q000633:toxicity*; D007306:Insecticides / Q000633:toxicity*; D020935:MAP Kinase Signaling System; D051379:Mice; D016501:Neurites / Q000187:drug effects*; D016900:Neurofilament Proteins / Q000378:metabolism*; D010766:Phosphorylation
chemical_list: D007306:Insecticides; D016900:Neurofilament Proteins; D004390:Chlorpyrifos
keywords: Chlorpyrifos; Microtubules; Mouse N2a neuroblastoma cells; Neurite outgrowth; Neurofilaments; Neurotoxicity; Organophosphate toxicity