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.

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 农业工人、园丁和房主经常接触杀虫剂氯氰菊酯。此外，军事人员在使用国防部驱蚊系统和非处方药虱子肥皂时会接触到氯菊酯，而非处方药中使用的是氯菊酯作为活性成分。氯菊酯的尿代谢物丰度高且相对稳定，可能是接触这种杀虫剂的理想生物标志物。此外，根据给药途径的不同，一种代谢物与另一种代谢物的比例可能会有所不同。这项研究的结果将被用来确定开发一种快速、灵敏的基于免疫化学的分析方法的候选对象，该方法可用于常规监测人类接触二氯氰菊酯。 目的：确定人体皮肤暴露后尿中含量最高、最稳定的二氯氰菊酯代谢物S。加速器质谱仪是一种测量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，我们将在生物相关剂量下进行人体新陈代谢研究。 具体目的：1.建立氯氰菊酯及其可能的人体代谢物的LC/MS分离方法。使用加速器质谱仪(AMS)测定二氯氰菊酯的人体代谢物分布。建立目标II中确定的关键代谢物的免疫分析方法，作为人类接触氯氰菊酯的生物标记物。 方法：针对特定目标I，合成拟除虫菊酯类代谢物标准品；建立拟拟除虫菊酯类代谢物的紫外检测高效液相色谱分离方法；确定拟除虫菊酯类代谢物的高效液相色谱/质谱联用分析方法的可行性。针对特定目标二，人体经皮下暴露于放射性标记的氯菊酯，并采集尿液、血液和唾液；采用针对特定目标I开发的方法分离样品，并用加速器质谱仪分析分离样品；从所产生的数据中确定最普遍的代谢物。针对特定目的III，半抗原的合成；抗体的开发；半抗原和抗体在开发最普遍代谢物的免疫分析中的使用；免疫分析的验证。 预期产品(里程碑)：氯菊酯可能的人体代谢物的文献综述；少量合成的氯菊酯代谢物；高效液相色谱法分离人体尿液或唾液中的氯菊酯代谢物；加速器质谱仪鉴定最丰富的人体代谢物(S)；检测目标人体代谢物的免疫测定法 到目前为止的状况/结果：已经完成了文献综述，并确定了可能的主要代谢物。所有的主要代谢物都已合成或获得。利用这些代谢物标准品，建立了其分析的高效液相色谱方法。这种方法稍后将用于鉴定人类尿样中发现的代谢物，并将用于验证的液-质联用方法。利用制备代谢物的化学知识，合成用于免疫分析检测这些分子的半抗原是完全的。3-苯氧基苯甲酸、3-苯氧基苯甲酸的甘氨酸结合物、二氯乙烯菊酸(DCCA)的甘氨酸结合物和3-苯氧基苯甲醇的葡萄糖醛酸化物结合物(见下文)已经建立了免疫分析方法。对游离DCCA的检测正在进行中(手稿正在准备中)。3-苯氧基苯甲酸的测定已经适应了一种灵敏的、高通量的方法(见下文)。临床暴露已经完成。所有样品都已用AMS测量了总碳14。对总吸收剂量的估计(4名受试者)从0.06%到0.27%不等。氯氰菊酯从血液中清除，半衰期约为12-24小时。排尿半衰期平均为24小时。对唾液进行了采样，但二氯苯醚菊酯似乎不是通过这条路线排出的。目前正在对尿液中的代谢物模式进行液相色谱分析。 结论：这项研究的结果将被用来确定开发一种快速、灵敏的基于免疫化学的分析方法的候选对象，该方法可用于常规监测人类接触氯氰菊酯。能够仔细监测二氯氰菊酯吸收剂量的存在，将是防止因接触二氯氰菊酯而可能对人类健康造成影响的有用工具。