Effects of chlorpyrifos and chlorpyrifos-methyl on the outgrowth of axon-like processes, tubulin, and GAP-43 in N2a cells

The aim of this work was to study the neurodegenerative effects of the organophosphate (OP) pesticides chlorpyrifos (CPF) and chlorpyrifos-methyl (CHM) on cultured mouse N2a neuroblastoma cells. CPF or CHM, at a subcytotoxic concentration of 3 μM, were added to the cells either at the time of the induction of cell differentiation (codifferentiation) or 16 h after the induction of differentiation (postdifferentiation). CPF and CHM were similar in inhibiting significantly the outgrowth of axon-like processes from N2a cells after only 4 h exposure under both co- and postdifferentiation exposure conditions. Densitometric scanning of Western blots of extracts of cells treated with CPF or CHM for 4 h revealed significantly decreased cross-reactivity with a monoclonal antibody recognizing the protein GAP-43 under post- but not under codifferentiation exposure conditions. Exposure to CPF or CHM for 4 h under postdifferentiation conditions also resulted in reduced fluorescence of N2a cell body staining with anti-GAP-43. Cross-reactivity of Western blots with a monoclonal antibody recognizing -tubulin was not significantly affected by OP treatment. These data indicate that a disturbance in GAP-43 may be important in the retraction of axons in predifferentiated N2a cells and support the notion that the mechanisms involved in CPF-and CHM-induced inhibition of axonal outgrowth may be different under co- and postdifferentiation exposure conditions

The toxicity of chlorpyrifos towards differentiating mouse N2a neuroblastoma cells

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 (post-differentiation) the induction of cell differentiation. The outgrowth of axon-like processes was impaired following 4 or 8 h exposure to CPF in both co- and post-differentiation experiments. Western blotting analysis revealed reduced levels of neurofilament heavy chain (NF-H) following 8 h of exposure but no significant effect at 4 h under both co- and post-differentiation 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 h of 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

Inhibition of neurite outgrowth in differentiating mouse N2a neuroblastoma cells by phenyl saligenin phosphate: Effects on MAP kinase (ERK 1/2) activation, neurofilament heavy chain phosphorylation and neuropathy target esterase activity

Sub-lethal concentrations of the organophosphate phenyl saligenin phosphate (PSP) inhibited the outgrowth of axon-like processes in differentiating mouse N2a neuroblastoma cells (IC50 2.5 μM). A transient rise in the phosphorylation state of neurofilament heavy chain (NFH) was detected on Western blots of cell extracts treated with 2.5 μM PSP for 4 h compared to untreated controls, as determined by a relative increase in reactivity with monoclonal antibody Ta51 (anti-phosphorylated NFH) compared to N52 (anti-total NFH). However, cross-reactivity of PSP-treated cell extracts was lower than that of untreated controls after 24 h exposure, as indicated by decreased reactivity with both antibodies. Indirect immunofluorescence analysis with these antibodies revealed the appearance of neurofilament aggregates in the cell bodies of treated cells and reduced axonal staining compared to controls. By contrast, there was no significant change in reactivity with anti-a tubulin antibody B512 at either time point. The activation state of the MAP kinase ERK 1/2 increased significantly after PSP treatment compared to controls, particularly at 4 h, as indicated by increased reactivity with monoclonal antibody E-4 (anti-phosphorylated MAP kinase) but not with polyclonal antibody K-23 (anti-total MAP kinase). The observed early changes were concomitant with almost complete inhibition of the activity of neuropathy target esterase (NTE), one of the proposed early molecular targets in organophosphate-induced delayed neuropathy (OPIDN)

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 (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

Diazoxon disrupts the expression and distribution of βIII-tubulin and MAP 1B in differentiating N2a cells

This study aimed at assessing the effects of diazoxon (DZO), a major metabolite of the insecticide diazinon (DZ), on key cytoskeletal proteins in differentiating N2a neuroblastoma cells. Initial experiments established that sub-lethal concentrations of 1, 5 and 10 μM DZO produced profound inhibition of neurite outgrowth. Densitometric scanning of probed immunoblots of N2a cell lysates demonstrated that DZO had no effect on total β-tubulin levels. However, probing with a monoclonal antibody that recognised specifically the βIII-tubulin isotype revealed that 10 μM DZO induced a significant reduction in the levels of this particular form. Levels of polyglutamylated tubulin were not altered. Exposure to 10 μM DZO also decreased the expression of microtubule associated protein 1B (MAP 1B). However, DZO had no effect on the expression of MAP tau. DZO also failed to affect the levels neurofilament light (NFL) and neurofilament medium (NFM) chain levels. Indirect immunofluorescence demonstrated that the staining of neurites in treated cells was weaker than in the controls for βIII-tubulin. In conclusion, DZO disrupts the microtubule (MT) network affecting the expression and distribution of two specific MT proteins known to be important in neuritogenesis. DZO may contribute to the developmental neurotoxicity seen following exposure to DZ