SOLID-PHASE ELECTROPHILE TRAPPING AGENTS

固相亲电捕获剂

• 批准号: 3195783
• 负责人: THOMAS J CURPHEY
• 金额: $ 9.49万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3195783
• 关键词: chemical carcinogen; chlorobenzenes; fluorescent dye /probe; gas chromatography; hamsters; high performance liquid chromatography; laboratory rat; nitrosamines; solid state; thiols; toxin metabolism

The goal of this proposal is to prepare a new tool for the study of metabolic activation of carcinogens. This tool will consist of a nucleophilic thiol supported on a solid phase, such a polystyrene, polyacrylamide, or controlled pore glass. The nucleophile will be anchored to the solid phase by means of a linkage which can be cleaved by a specific agent, such as hydrazine, to give products which can be analyzed by HPLC or GC. The supported nucleophile will be incubated with the carcinogen and cell or subcellular fraction to be studied. After the appropriate time period, the solid phase will be removed, washed free of all adhering material, the nucleophile cleaved from its support, and the products analyzed by the appropriate chromatographic technique. There are several advantages of this approach over the more usual one of adding a soluble nucleophile such as 3,4-dichlorobenzenethiol. First, the fraction containing the trapped electrophiles may be isolated from the complex incubation matrix by a simple filtration step, removing a variety of products which could potentially interfere with subsequent chromatographic analysis. Second, the structure of the nucleophile can be readily tailored to give optimal chromatographic properties to the trapped products. For example, halogen can be incorporated to facilitate GC analysis with a sensitive EC detector, or a fluorescent chromophore can be included to allow a fluorescence detector to be used with HPLC. Third, in incubations with whole cells, the supported nucleophiles would be unable to enter cells, and thus would detect only electrophiles stable enough to reach the extracellular medium. This would be especially useful in the study of carcinogens which are thought to require metabolic activation by one organ, usually liver, to produce an electrophile which targets some other organ. The new agents will be applied to study of metabolism of dimethylnitrosamine, since there are conflicting reports of the ability of the usual thiol nucleophile, 2,4-dichlorobenzenethiol, to trap a methylating species produced during metabolism of this nitrosamine. The new agents will also be used for study of species differences in metabolism of a series of beta-oxidized nitrosamines.

该提案的目标是为研究准备一个新工具 致癌物质的代谢激活。 该工具将包含一个 负载在固相例如聚苯乙烯上的亲核硫醇， 聚丙烯酰胺或可控孔玻璃。 亲核试剂将是 通过可通过以下方式裂解的连接锚定在固相上 特定的试剂，例如肼，可以产生可 通过 HPLC 或 GC 进行分析。 支持的亲核试剂将与 致癌物和待研究的细胞或亚细胞部分。 之后 适当的时间段，将固相除去，洗去 所有粘附材料，亲核试剂从其支持物上裂解，并且 通过适当的色谱技术分析产品。 与更常见的方法相比，这种方法有几个优点 添加可溶性亲核试剂，例如 3,4-二氯苯硫醇。 首先， 含有捕获的亲电子试剂的级分可以从 通过简单的过滤步骤即可去除复杂的孵化基质 可能会干扰后续的产品 色谱分析。 其次，亲核试剂的结构可以是 易于定制，为被捕获物提供最佳色谱特性 产品。 例如，可以加入卤素以促进GC 使用灵敏的 EC 检测器或荧光发色团进行分析 包括允许荧光检测器与 HPLC 一起使用。 第三，在 与整个细胞一起孵育时，支持的亲核试剂将无法 进入细胞，因此只能检测到足够稳定的亲电子试剂 到达细胞外介质。 这在以下情况下特别有用： 对被认为需要代谢激活的致癌物的研究 一个器官，通常是肝脏，产生一种针对某些 其他器官。 新试剂将应用于代谢研究 二甲基亚硝胺，因为关于其能力的报道相互矛盾 常见的硫醇亲核试剂 2,4-二氯苯硫醇，用于捕获 亚硝胺代谢过程中产生的甲基化物质。 这 新试剂还将用于研究物种差异 一系列β-氧化亚硝胺的代谢。