NTE and non-NTE esterases in brain membrane: kinetic characterization with organophosphates

Some effects of organophosphorus compounds (OPs) esters cannot be explained by action on currently recognized targets. In this work, we evaluate and characterize the interaction (inhibition, reactivation and "ongoing inhibition") of two model compounds: paraoxon (non-neuropathy-inducer) and mipafox (neuropathy-inducer), with esterases of chicken brain membranes, an animal model, tissue and fractions, where neuropathy target esterase (NTE) was first described and isolated. Four enzymatic components were discriminated. The relative sensitivity of time-progressive inhibition differed for paraoxon and mipafox. The most sensitive component for paraoxon was also the most sensitive component for mipafox (EPα: 4.4-8.3% of activity), with I(50) (30 min) of 15-43 nM with paraoxon and 29 nM with mipafox, and it spontaneously reactivated after inhibition with paraoxon. The second most sensitive component to paraoxon (EPβ: 38.3% of activity) had I(50) (30 min) of 1540 nM, and was practically resistant to mipafox. The third component (EPγ: 38.6-47.6% of activity) was paraoxon-resistant and sensitive to micromolar concentrations of mipafox; this component meets the operational criteria of being NTE (target of organophosphorus-induced delayed neuropathy). It had I(50) (30 min) of 5.3-6.6 μM with mipafox. The fourth component (EPδ: 9.8-10.7% of activity) was practically resistant to both inhibitors. Two paraoxon-resistant and mipafox-sensitive esterases were found using the sequential assay removing paraoxon, but only one was paraoxon-resistant and mipafox-sensitive according to the assay without removing paraoxon. We demonstrate that this apparent discrepancy, interpreted as reversible NTE inhibition with paraoxon, is the result of spontaneous reactivation after paraoxon inhibition of a non-NTE component. Some of these esterases' sensitivity to OPs suggests that they may play a role in toxicity in low-level exposure to organophosphate compounds or have a protective effect related with spontaneous reactivation. The kinetic characterization of these components will facilitate further studies for isolation and molecular characterization.