The effect of organophosphates on a chicken brain or sea urchin egg kinesin-driven microtubule motility assay

Abstract

The effect of neuropathic and non-neuropathic organophosphates (OPs) and acrylamide on an in vitro kinesin-driven microtubule (MT) motility assay was compared. The goal of the study was to determine whether this in vitro assay could confirm that a mechanism of action of neuropathic OPs was to impair kinesin activity and, therefore, possibly fast axonal anterograde transport (FAAT) in vivo. For our study, kinesin from chicken brain (CK) and sea urchin egg (SUK) was initially purified. Western immunoblotting confirmed the close antigenic homology between CK and SUK, using a mouse monoclonal sea urchin kinesin heavy chain-specific antibody (SUK 4). In the presence of microtubules (MTs) and MgATP, both CK- and SUK-driven MT movement was measured using a video-enhanced differential interference contrast microscope system with computer-assisted analysis. Using this assay system, we then tested separately the effect of two neuropathic OPs (diisopropylfluorophosphate (DFP) and phenyl saligenin phosphate (PSP)) and a non-neuropathic OP (paraoxon (PO)) each at a concentration of 10 −2 M at 27°C. Additionally, we tested acrylamide (10 −2 M), since it is one of the best-characterized neurotoxins impairing FAAT in vivo. Our results demonstrated that none of these compounds significantly affected kinesin-driven MT motility in vitro compared to the standard controls. Further, this assay system was thus not able to discriminate between the neuropathic and non-neuropathic effect of these OPs.