The effects of diazinon and cypermethrin on the differentiation of neuronal and glial cell lines

Flaskos, John; Harris, Wayne C.; Sachana, Magdalini; Muñoz, David; Tack, J.-C.; Hargreaves, Alan J.

doi:10.1016/j.taap.2006.10.033

Cited by 46 publications

(47 citation statements)

References 39 publications

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“…This finding is in agreement with previous studies which employed similar concentrations (1-10 µM) of CPF and other OPs such as diazinon and diazinon oxon that were also found to be non-cytotoxic towards differentiating N2a cells for exposure periods of up to 24 h (Flaskos et al 2007;Sachana et al 2001Sachana et al , 2003Sachana et al , 2005Sidiropoulou et al 2009). However, in those studies exposure was initiated at the point of induction of cell differentiation (i.e.…”

Section: Discussionsupporting

confidence: 93%

“…2006; Harris et al 2009b;Flaskos et al 2007;2011). The trend of greater reduction in the number of long neurites and the maximum neurite length observed in Ta51-stained CPO-treated cells is consistent with the possibility that the oxon metabolite may have a greater impact or a more Although inhibition of neurite outgrowth from N2a cells has been previously investigated after CPF exposure (Sachana et al 2001), and other OPs such as leptophos, phenyl saligenin phosphate (PSP), diazinon and CPO using Coomassie blue staining (Sachana et al 2003;Hargreaves et al 2006;Harris et al 2009b;Flaskos et al 2007;2011), this is the first time that CFSE has been used to measure the growth or retraction of neurites in cultured N2a cells. Since it provides similar results to our previous work with CPF, morphometric analysis of CFSE-labeled N2a cells can be considered as a novel and reliable approach to assess cell differentiation and neurite outgrowth.…”

Section: Discussionmentioning

confidence: 99%

“…Cell differentiation was induced by serum withdrawal and the addition of 0.3 mM dibutyryl cyclic AMP (Flaskos et al 2007). Prior to induction of differentiation, a viable cell count was determined using a haemocytometer chamber and Trypan blue exclusion assay (Bhuyan et al, 1976).…”

Section: Cell Maintenance and The Induction Of Cell Differentiationmentioning

confidence: 99%

“…Cell pellets were then resuspended in ice cold 200 mM sodium phosphate buffer (pH 7.4) containing Triton X100 (0.1% v/v) at 4°C. AChE was then determined according to the assay of Ellman et al (1961) modified for a microtitre plate format, as described by Flaskos et al (2007).…”

Section: Acetylcholinesterase Activity Assaymentioning

confidence: 99%

“…N2a cells were fixed for 10 min with 3.7% w/v formaldehyde in PBS, extracted in 0.5% w/v Tween-20 in Tris buffered saline (TBS: 10 mM Tris, 140 mM NaCl; pH 7.4) for 15 min and blocked with 3 % w/v bovine serum albumin (BSA) in TBS (BSA/TBS) for 45 min at room temperature. They were then incubated with primary antibodies against NFH, p-NFH and β-tubulin followed by Alexa Fluor 488 rabbit anti-mouse IgG labeled secondary antibody, as described previously (Hargreaves et al 2006;Harris et al 2009b;Flaskos et al 2007Flaskos et al , 2011. After washing, to remove unbound secondary antibodies, the chamber slide was carefully dismantled and the cell monolayer mounted under a glass coverslip using VectaShield mounting medium for fluorescence (Vector Laboratories Ltd., Peterborough, UK) containing DAPI counterstain for nuclei visualization.…”

Section: Indirect Immunofluorescencementioning

confidence: 99%

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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

Sindi

Harris

Arnott

et al. 2016

Toxicology and Applied Pharmacology

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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 20 h before exposure to CPF or CPO for 2-8 h. 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 2 h 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 2 h exposure and reduced levels of reactivity of the same antibody following 8 h treatment with both compounds, whereas reactivity with antibodies to anti-total NFH or anti-tubulin was not affected. The alteration in NFH phosphorylation at 2 h exposure was associated with increased activation of extracellular signal-regulated protein kinase ERK 1/2. However, increased levels of phosphatase activity were observed following 8 h 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.

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Section: Discussionsupporting

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Section: Cell Maintenance and The Induction Of Cell Differentiationmentioning

confidence: 99%

Section: Acetylcholinesterase Activity Assaymentioning

confidence: 99%

Section: Indirect Immunofluorescencementioning

confidence: 99%

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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

Sindi

Harris

Arnott

et al. 2016

Toxicology and Applied Pharmacology

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Developmental Neurotoxicity of Anticholinesterase Pesticides

Flaskos

Sachana

2011

Anticholinesterase Pesticides

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Evaluation of developmental toxicity induced by anticholinesterase insecticide, diazinon in female rats

ElMazoudy

Attia

Abdel‐Gawad

2011

Birth Defects Research Pt B

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Developmental toxicities, including birth defects, are significant public health problems. This study was planned to assess the cholinergic and developmental potentials of diazinon that is widely used as an organophosphate insecticide. Pregnant female Sprague-Dawley rats were given diazinon orally at doses of 0, 1.9, 3.8, and 7.6 mg/kg body weight (b.w.)/day on gestation days 6 to 15. Maternal brain acetylcholinesterase activities, measured on gestation day20, were significantly decreased at 3.8 and 7.6 mg/kg b.w./day, but fetal acetylcholinesterase activity was not altered. Maternal toxicities, as evidenced by cholinergic symptoms including diarrhea, tremors, weakness, salivation, and decreased activities, were observed at the 3.8 and 7.6 mg/kg b.w./day dose groups. Net gravid uterine weight was decreased at a dose of 7.6 mg/kg b.w./day. No maternal effects were apparent in the 1.9 mg/kg b.w./day dose group. Maternal toxicity at a dose of 3.8 mg/kg b.w./day did not induce fetotoxicity or teratogeneicity. However, 7.6 mg/kg b.w./day doses significantly resulted in fetal toxicity and malformations in addition to maternal toxicity in animals. In conclusion, teratogenic disorders only outlined by doses that produced marked maternal toxicity. Since the malformations were not morphologically related, they were considered to be secondary to maternal toxicity; hence, the malformations were not related to cholinesterase inhibition.

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The effects of diazinon and cypermethrin on the differentiation of neuronal and glial cell lines

Cited by 46 publications

References 39 publications

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

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

Developmental Neurotoxicity of Anticholinesterase Pesticides

Evaluation of developmental toxicity induced by anticholinesterase insecticide, diazinon in female rats

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