Project 4: Genetic Susceptibility

建议的研究旨在开发高通量的协议，通过表征 血液中的特定生物标记蛋白，并利用这些更准确的暴露于 研究与对氧磷酶(PONI)基因遗传变异相关的基因/环境相互作用， 尤其是发生在发育早期的OP暴露。具体目标1是验证High 基于免疫磁珠(1MB)的快速纯化和分析方法 红细胞酰基多肽水解酶和血浆丁酰胆碱酯酶两种蛋白质的活性部位肽 (BChE)，作为OP化合物暴露的生物标志物。一种基于微毛细管的靶向蛋白质组学策略 高效液相色谱-选择性反应监测-质谱法(LC-SRM-MS)将得到验证和 用于量化社区存档样本中BCHE和APH的修改百分比 参与式研究项目。来自生物标志物实验的数据将与其他数据相关联 可用于尿代谢物水平、APH和/或BChE的抑制以及PONI状态。具体目标2和3 使用基因敲除和人性化的小鼠模型来确定PONI状态对OP暴露的重要性 发生在早期发育的关键时期。具体目标2是确定APH和APH的程度 在怀孕小鼠暴露于毒死蜱(CP)或CPO的试验中，BChE被共价修饰。特定目标 3是确定新生小鼠暴露后BChE和APH共价修饰的程度 致CP或CPO。这些目标将评估敏感性、暴露和反应的生物标记物之间的相互作用 体内系统，使我们能够确定OP化合物的全剂量响应并生成定量 生物标志物数据。

The proposed studies aim to develop high throughput protocols for assessing OP exposures by characterizing specific biomarker proteins in blood, and to make use of these more accurate measures of exposure to investigate gene/environment interactions related to genetic variability in the paraoxonase (PONI) gene, particularly with respect to OP exposures that occur during early development. Specific Aim 1 is to validate high throughput immunomagnetic bead (1MB) based isolation protocols for rapid purification and analysis of the active site peptides of two proteins, red cell acyl peptide hydrolase (APH) and plasma butyrylcholinesterase (BChE), as biomarkers of exposure to OP compounds. A targeted proteomics strategy based on microcapillary liquid chromatography-selected reaction monitoring-mass spectrometry (¿mu¿LC-SRM-MS) will be validated and used to quantify the percentage modification of BChE and APH in archived samples from the Community Based Participatory Research Project. Data from the biomarker experiments will be correlated with other data available on urinary metabolite levels, inhibition of APH and/or BChE, and PONI status. Specific Aims 2 and 3 use knockout and humanized mouse models to determine the importance of PONI status for OP exposures that occur during critical periods of early development. Specific Aim 2 is to determine the extent to which APH and BChE are covalently modified following test exposure of pregnant mice to chlorpyrifos (CP) or CPO. Specific Aim 3 is to determine the extent to which BChE and APH are covalently modified following exposure of neonatal mice to CP or CPO. These aims will evaluate interactions among biomarkers of sensitivity, exposure and response in an in vivo system, allowing us to determine full dose responses of the OP compounds and generate quantitative biomarker data.