Sub-lethal concentrations of the
organophosphate phenyl saligenin phosphate (PSP) inhibited the outgrowth of axon-like processes in differentiating mouse N2a
neuroblastoma cells (IC(50) 2.5 microM). A transient rise in the phosphorylation state of neurofilament heavy chain (NFH) was detected on Western blots of
cell extracts treated with 2.5 microM 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-
alpha-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).