The toxicity of chlorpyrifos towards differentiating mouse N2a neuroblastoma cells

Abstract

The aim of this work was to study the effects of chlorpyrifos (CPF) on the outgrowth of axons by differentiating mouse N2a neuroblastoma cells. This was achieved by morphological, western blotting and enzymatic analyses of cells induced to differentiate in the presence and absence of CPF added either at the same time (co-differentiation) or 16 h after (postdifferentiation) the induction of cell differentiation. The outgrowth of axon-like processes was impaired following 4 or 8 hours exposure to CPF in both co-and post-differentiation experiments. Western blotting analysis revealed reduced levels of neurofilament heavy chain (NF-H) following 8h exposure but no significant effect at 4h under both co-and postdifferentiation conditions. By contrast, levels of the heat shock protein HSP-70 were raised at both time points but only in co-differentiation experiments. Neuropathy target esterase (NTE) activity was lower than controls following 4 or 8 hours exposure under co-differentiation conditions but not under any post-differentiation conditions. The results suggest that the inhibition of axon production and maintenance by CPF in differentiating N2a cells may involve multiple targets, which are different under co-and post-differentiation conditions.

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

“…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.…”

“…under postdifferentiation exposure conditions) is in good agreement with that noted in the same culture system using a similar concentration of CPF under both post-and co-differentiation conditions (Sachana et al 2001). In addition, the current study has revealed for the first time the potential neurotoxic ability of CPO to induce the retraction of axon-like processes from pre-differentiated N2a cells, and that this effect appears to be stronger than that of CPF with respect to longer (axon-like) neurites.…”

“…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.…”

“…In a preliminary study we found that CPF at a non-cytotoxic concentration of 3μM had the ability to induce retraction of approximately 50 % of axon-like processes formed by differentiating N2a cells, as determined by Coomassie Brilliant Blue staining (Sachana et al 2001). However, the effects of CPO on pre-formed neurites had not been previously tested.…”

“…It has been shown that CPF and CPO can inhibit the outgrowth of neurites and disrupt the levels of cytoskeletal proteins in differentiating neuronal cell lines (Sachana et al 2001(Sachana et al , 2005Flaskos et al 2011). It is also known that the induction of developmental neurotoxicity may depend on the developmental stage at which exposure occurs.…”