Diisopropylfluorophosphate Impairs the Transport of Membrane-Bound Organelles in Rat Cortical Axons

Abstract

The extensive use of organophosphates (OPs) is an ongoing environmental health concern due to multiple reports of OPrelated neurologic abnormalities. The mechanism of the acute toxicity of OPs has been attributed to inhibition of acetylcholinesterase (AChE), but there is growing evidence that this may not account for all the long-term neurotoxic effects of OPs. In previous experiments (using ex vivo and in vitro model systems) we observed that the insecticide OP chlorpyrifos impaired the movements of vesicles and mitochondria in axons. Here, using a time-lapse imaging technique, we evaluated the OP-nerve agent diisopropylfluorophosphate (DFP) across a wide range of concentrations (subnanomolar to micromolar) for effects on fast axonal transport of membrane-bound organelles (MBOs) that contain the amyloid precursor protein (APP) tagged with the fluorescent marker Dendra2 (APPDendra2). Both 1 and 24 hours of exposure to DFP and a positive control compound, colchicine, resulted in a decrease in the velocity of anterograde and retrograde movements of MBOs and an increase in the number of stationary MBOs. These effects occurred at picomolar (100 pM) to low nanomolar (0.1 nM) concentrations that were not associated with compromised cell viability or cytoskeletal damage. Moreover, the effects of DFP on axonal transport occurred at concentrations that did not inhibit AChE activity, and they were not blocked by cholinergic receptor antagonists. Given the fundamental importance of axonal transport to neuronal function, these observations may explain some of the long-term neurologic deficits that have been observed in humans who have been exposed to OPs.

Pharmacologically increasing microtubule acetylation corrects stress‐exacerbated effects of organophosphates on neurons

“…Mitochondria are the primary site of energy production and have been suggested to be the target of noncholinergic toxicity of OPs . Past studies have indicated a link between exposure to higher concentrations of OP and oxidative stress and transport deficits after OP exposure . Consistent with previous work, when neurons were treated with the DFP, transport of mitochondria was impaired and mitochondrial length increased, suggesting an increase in mitochondrial fusion.…”

Models to identify treatments for the acute and persistent effects of seizure‐inducing chemical threat agents

“…Since there was no reliable literature data on the stability of DFP in DMSO or in aqueous buffers used for tissue culture or imaging experiments, we also investigated the stability of DFP in solution and the rate at which it hydrolyses into diisopropyl phosphoric acid through nucleophilic substitution of the F. This hydrolysis can be easily monitored by the diminishing octet at 4.77 ppm and the appearance of a shifted octet at 4.51 ppm in the 800‐Hz 1 H‐NMR spectrum or the change of the 31 P signal from a doublet induced by the coupling to the F in DFP to a singlet in the hydrolysis product that no longer contains F. While DFP was highly stable in DMSO and showed no signs of hydrolysis even after 19 days, it hydrolyzed quickly in unbuffered deionized water, which is typically acidic. DFP was reasonably stable in phosphate‐buffered solutions, where it showed no signs of hydrolysis over 20 h, suggesting that it would be sufficiently stable in tissue culture experiments or imaging experiments that last less than 24 hours . Therefore, we believe that DFP is stable in the Ca 2+ ‐imaging experiments described above.…”

“…DFP was reasonably stable in phosphate-buffered solutions, where it showed no signs of hydrolysis over 20 h, suggesting that it would be sufficiently stable in tissue culture experiments or imaging experiments that last less than 24 hours. 54 Therefore, we believe that DFP is stable in the Ca 2+ -imaging experiments described above.…”

Neurotoxicity in Preclinical Models of Occupational Exposure to Organophosphorus Compounds

“…CPF-induced impairments in axonal transport can partially be explained by altered motility of mitochondria and changes in motor proteins like kinesin and microtubules, as well as motor-associated proteins like tubulin (Gearhart et al, 2007; Prendergast et al, 2007; Grigoryan and Lockridge, 2009; Middlemore-Risher et al, 2011). Similar impairments in motor proteins are induced by DFP exposure, which may also impair axonal transport (Gearhart et al, 2007; Gao et al, 2016). …”