Insecticide Interactions With Acetylcholinesterase

说明（由申请人提供）：本项目的长期目标是提供有助于评估神经毒性有机磷杀虫剂对人类健康构成的风险的信息。它试图通过调查初步观察来实现这一目标，这些观察表明，目前关于这些有毒杀虫剂如何抑制乙酰胆碱酯酶的观点不足以描述杀虫剂-酶在广泛杀虫剂浓度范围内的相互作用。初步数据表明，某些有机磷抑制乙酰胆碱酯酶的能力随抑制剂浓度的变化而变化。此外，证据表明，有机磷对氧磷引起乙酰胆碱酯酶在抑制之前的短暂激活。总的来说，这些数据表明某些有机磷酸盐在人重组乙酰胆碱酯酶上存在二级结合位点。因此，本应用的总体假设是，有机磷酸盐及其亲本杀虫剂与人重组乙酰胆碱酯酶上不同于催化三联体的位点结合，并且占领该位点改变了活性位点的事件。由于这种结合，少量接触某些有机磷杀虫剂可能造成比目前根据使用较高接触水平的剂量-反应关系所估计的更大的健康风险。本申请的具体目的如下：1)确定在广泛的抑制剂浓度范围内，测试有机磷对人重组乙酰胆碱酯酶的双分子抑制率常数&；2)确定所测试的有机磷酸盐及其母体化合物是否能在通过磷酸化抑制酶之前短暂地增强重组乙酰胆碱酯酶对底物乙酰硫胆碱的水解；3)确定被试有机磷及其对应的亲本杀虫剂是否与人重组乙酰胆碱酯酶外周阴离子位点结合；4)确定被有机磷酸盐抑制的人重组乙酰胆碱酯酶的去磷酸化（再激活）速率是否会因外周阴离子位点或其他有机磷酸盐的二级位点被占领而改变。

DESCRIPTION (provided by applicant): The long-term objective of this project is to provide information that will be useful in assessing the risks posed to human health by the neurotoxic organophosphorus insecticides. It seeks to accomplish this objective by investigating preliminary observations that suggest the current view of how these toxic insecticides inhibit acetylcholinesterase is inadequate to describe the insecticide-enzyme interactions over a wide range of insecticide concentrations. Preliminary data indicates that the capacity of certain organophosphates to inhibit acetylcholinesterase changes as a function of inhibitor concentration. Moreover, evidence presented indicates that the organophosphate paraoxon causes a transient activation of acetylcholinesterase prior to inhibition. Collectively, these data suggest the presence of a secondary binding site on human recombinant acetylcholinesterase for certain organophosphates. Therefore, the overall hypothesis of this application is that organophosphates and their parent insecticides bind to a site on human recombinant acetylcholinesterase distinct from the catalytic triad, and that occupation of this site alters events at the active site. As a consequence of such binding, low exposures to certain organophosphorus insecticides could pose a greater health risk than is currently estimated based on dose-response relationships using higher exposure levels. The specific aims of this application are as follows: 1) To determine the bimolecular inhibition rate constant &,, for the inhibition of human recombinant acetylcholinesterase by the test organophosphates over a wide range of inhibitor concentrations; 2) To determine if the test organophosphates and their parent compounds can transiently enhance the hydrolysis of the substrate acetylthiocholine by human recombinant acetylcholinesterase prior to inhibition of the enzyme through phosphorylation; 3) To determine if the test organophosphates and their corresponding parent insecticides bind to the peripheral anionic site on human recombinant acetylcholinesterase; and 4) To determine if the rate of dephosphorylation of organophosphate- inhibited human recombinant acetylcholinesterase (reactivation) is altered by the occupation of the peripheral anionic site or some other secondary site for organophosphates.