ID AND DEV OF BIOLOGICAL MARKERS OF HUMAN EXPOSURE TO THE INSECTICIDE PERMETHRI

人类接触杀虫剂氯菊酯的生物标志物的识别和开发

• 批准号: 8362754
• 负责人: BRUCE D HAMMOCK
• 金额: $ 16.38万
• 依托单位: UNIVERSITY OF CALIF-LAWRNC LVRMR NAT LAB
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8362754
• 关键词: Acids; Agricultural Workers; Alcohols; Antibodies; Biological Assay; Biological Markers; Biological Markers; Blood; C14 isotope; Chemicals; Clinical; Collection; Data; Dermal; Detection; Development; Dose; Exposure to; Funding; Glucuronides; Glycine; Grant; Half-Life; Haptens; Health; High Pressure Liquid Chromatography; Hour; Human; Immunoassay; Insect Repellents; Insecticides; Knowledge; Lice; Liquid Chromatography; Liquid substance; Manuscripts; Mass Spectrum Analysis; Measures; Methods; Military Personnel; Monitor; National Center for Research Resources; Pattern; Permethrin; Pesticides; Preparation; Principal Investigator; Publications; Radiolabeled; Research; Research Infrastructure; Resource Development; Resources; Review Literature; Route; Saliva; Sampling; Scintillation Counting; Soaps; Source; System; United States National Institutes of Health; Urine; Validation; accelerator mass spectrometry; analytical method; base; cost; exposed human population; liquid chromatography mass spectrometry; metabolic abnormality assessment; prevent; pyrethroid; radiotracer; tool; ultraviolet; urinary

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Agricultural workers, gardeners and homeowners are routinely exposed to the insecticide permethrin. Also, military personnel are exposed to the permethrin when using the DOD Insect Repellent System and over-the-counter lice soaps use permethrin as the active ingredient. A urinary metabolite of permethrin, that is in high abundance and is relatively stable, may be an ideal biomarker of exposure to this pesticide. In addition, the ratio of one metabolite to another may vary, according to the route of administration. The results of this study would be used to identify candidates for the development of a rapid, sensitive immunochemical based analytical method that can be used to routinely monitor human exposure to permethrin. Objectives: The purpose of this study is to determine the human metabolite(s) of permethrin in urine following dermal exposure that are in greatest abundance and are the most stable. Accelerator mass spectrometry is a method for measuring levels of 14C several orders of magnitude more sensitive than liquid scintillation counting. With this high sensitivity we will conduct human metabolism studies at biologically relevant doses. SPECIFIC AIMS: I. Develop an LC/MS method for separation of permethrin and its putative human metabolites. II. Determine the human metabolite profile of permethrin using accelerator mass spectrometry (AMS). III. Develop an immunoassay to the key metabolite identified in Objective II as a biomarker of human exposure to permethrin. METHODOLOGY: For specific aim I, synthesize metabolite standards; develop an HPLC method to separate the putative pyrethroid metabolites using ultraviolet detection; determine the feasibility of an HPLC/mass spectrometry method for analysis of pyrethroid metabolites. For specific aim II, clinical exposure of humans to radiolabeled permethrin dermally and collection of urine, blood and saliva; separation of samples by methods developed in specific aim I and analysis of separated samples by accelerator mass spectrometry; identification of most prevalent metabolite from resultant data. For specific aim III, synthesis of haptens; development of antibodies; use of the haptens and antibodies in the development of an immunoassay for the most prevalent metabolite; validation of immunoassay. EXPECTED PRODUCTS (MILESTONES): Literature review of putative human metabolites of permethrin; small quantities of synthesized metabolites of permethrin; an HPLC method for separating permethrin metabolites in human urine or saliva; identification of the most abundant human metabolite(s) by accelerator mass spectrometry; an immunoassay to detect the targeted human metabolite of permethrin STATUS/RESULTS TO DATE: Literature review has been completed and putative major metabolites identified. All of the major metabolites have been synthesized or acquired. Using the metabolite standards a high performance liquid chromatography method for their analysis has been developed. This method will later be used to identify metabolites found in human urine samples and the liquid chromatography-mass spectrophotometric method used for validation. Using the chemical knowledge from the preparation of metabolites, synthesis of haptens for immunoassay detection of these molecules is complete. Immunoassays for 3-phenoxybenzoic acid, the glycine conjugate of 3-phenoxybenzoic acid, the glycine conjugate of dichlorovinylchrysanthemic acid (DCCA) and the glucuronide conjugate of 3-phenoxybenzyl alcohol (see publication below) have been developed. An assay for free DCCA is in progress (manuscript in preparation). The assay for 3-phenoxybenzoic acid has been adapted to a sensitive, high throughput method (see publication below). Clinical exposures have been completed. All samples have been measured by AMS for total carbon-14. An estimate of the total dose absorbed (for 4 subjects) ranged from 0.06 to 0.27%. The permethrin is eliminated from the blood with a half life of about 12-24 hours hours. The urinary half life averaged 24 hours. Saliva was sampled, but permethrin does not appear to be excreted by that route. Liquid chromatography analysis of the metabolite pattern in urine is underway. Conclusion: The results of this study will be used to identify candidates for the development of a rapid, sensitive immunochemical based analytical method that can be used to routinely monitor human exposure to permethrin. The ability to carefully monitor the presence of absorbed doses of permethrin will be a useful tool to prevent the possibility of human health effects due to permethrin exposure.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 农业工人、园丁和房主经常接触杀虫剂氯菊酯。 此外，军事人员在使用国防部驱虫系统时会接触氯菊酯，而非处方除虱皂则使用氯菊酯作为活性成分。氯菊酯的尿液代谢物丰度高且相对稳定，可能是接触该农药的理想生物标志物。 此外，一种代谢物与另一种代谢物的比例可以根据给药途径而变化。这项研究的结果将用于确定开发快速、灵敏的基于免疫化学的分析方法的候选者，该方法可用于常规监测人体接触氯菊酯的情况。 目的：本研究的目的是确定皮肤接触后尿液中氯菊酯的人体代谢物，其含量最多且最稳定。加速器质谱法是一种测量 14C 水平的方法，比液体闪烁计数灵敏几个数量级。凭借这种高灵敏度，我们将以生物学相关剂量进行人体代谢研究。 具体目标： I. 开发一种 LC/MS 方法来分离氯菊酯及其假定的人体代谢物。 二. 使用加速器质谱 (AMS) 确定氯菊酯的人体代谢谱。 三． 对目标 II 中确定的关键代谢物进行免疫测定，作为人类接触氯菊酯的生物标志物。 方法论：针对特定目标I，合成代谢物标准品；开发一种 HPLC 方法，利用紫外线检测来分离假定的拟除虫菊酯代谢物；确定 HPLC/质谱法分析拟除虫菊酯代谢物的可行性。 对于具体目标 II，人类临床暴露于放射性标记的氯菊酯皮肤并收集尿液、血液和唾液；通过特定目标 I 中开发的方法分离样品，并通过加速器质谱分析分离的样品；从结果数据中识别最常见的代谢物。对于具体目标III，半抗原的合成；抗体的开发；使用半抗原和抗体开发最常见代谢物的免疫测定；免疫测定的验证。 预期产品（里程碑）：氯菊酯假定人体代谢物的文献综述；少量的氯菊酯合成代谢物；一种分离人尿液或唾液中氯菊酯代谢物的HPLC方法；通过加速器质谱法鉴定最丰富的人类代谢物；检测氯菊酯的目标人体代谢物的免疫测定法 迄今为止的状态/结果：文献综述已经完成，并确定了假定的主要代谢物。 所有主要代谢物均已合成或获得。 使用代谢物标准品，开发了一种用于分析的高效液相色谱方法。 该方法随后将用于鉴定人类尿液样本中发现的代谢物，并用于验证液相色谱-质谱法。利用代谢物制备中的化学知识，完成了用于免疫分析检测这些分子的半抗原的合成。 已经开发出 3-苯氧基苯甲酸、3-苯氧基苯甲酸的甘氨酸缀合物、二氯乙烯基菊酸 (DCCA) 的甘氨酸缀合物和 3-苯氧基苯甲醇的葡萄糖醛酸苷缀合物（参见下面的出版物）的免疫测定法。 游离 DCCA 的测定正在进行中（手稿正在准备中）。 3-苯氧基苯甲酸的测定已采用灵敏、高通量的方法（参见下面的出版物）。临床暴露已完成。所有样品均已通过 AMS 测量了总碳 14。吸收的总剂量（4 名受试者）的估计范围为 0.06% 至 0.27%。氯菊酯从血液中消除，半衰期约为 12-24 小时。尿半衰期平均为24小时。 采集了唾液样本，但氯菊酯似乎并未通过该途径排出。尿液中代谢物模式的液相色谱分析正在进行中。 结论：本研究的结果将用于确定开发快速、灵敏的免疫化学分析方法的候选者，该方法可用于常规监测人体接触氯菊酯的情况。仔细监测吸收剂量的氯菊酯的存在的能力将是防止由于氯菊酯暴露而对人类健康造成影响的有用工具。