Insecticide Interactions With Acetylcholinesterase

说明(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，这些初步观察表明，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在广泛的杀虫剂浓度范围内杀虫剂与酶的相互作用。初步数据表明，某些有机磷酸盐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，现有证据表明，有机磷对氧磷会在抑制之前引起乙酰胆碱酯酶的短暂激活。总而言之，这些数据表明，某些有机磷化合物在人重组乙酰胆碱酯酶上存在一个二级结合位点。因此，这一应用的总体假设是，有机磷及其母体杀虫剂与人重组乙酰胆碱酯酶上的一个不同于催化三联体的位点结合，该位点的占据改变了活性位点上的事件。由于这种结合，对某些有机磷杀虫剂的低暴露可能会造成比目前根据较高暴露水平的剂量-反应关系估计的更大的健康风险。本申请的具体目的如下：1)测定受试有机磷对人重组乙酰胆碱酯酶抑制的双分子抑制速率常数，在较宽的抑制浓度范围内；2)测定受试有机磷及其母体化合物在通过磷酸化抑制底物乙酰胆碱酯酶之前是否能瞬时增强底物乙酰胆碱酯酶的水解度；3)测定受试有机磷及其相应的母体杀虫剂是否结合到重组人重组乙酰胆碱酯酶的外周阴离子部位；以及4)确定有机磷抑制的重组人乙酰胆碱酯酶的去磷酸化速率(重新激活)是否因占据外周阴离子部位或有机磷的其他次级部位而改变。

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.