SOLID-PHASE ELECTROPHILE TRAPPING AGENTS

固相亲电捕获剂

• 批准号: 3195787
• 负责人: THOMAS J CURPHEY
• 金额: $ 10.32万
• 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 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3195787
• 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.

本提案的目的是为研究下列问题准备一个新的工具： 致癌物质的代谢活化。 该工具将包括一个 负载在固相上的亲核硫醇，例如聚苯乙烯， 聚丙烯酰胺或可控孔玻璃。 亲核试剂将是 通过连接键固定在固相上，该连接键可被 一种特定的试剂，如肼，以得到可以被 通过HPLC或GC分析。 支持的亲核试剂将与 致癌物质和细胞或亚细胞部分进行研究。 后 在适当的时间段内，将固相除去，洗涤除去， 所有粘附材料，亲核试剂从其载体上裂解， 通过适当的色谱技术分析产品。 这种方法比通常的方法有几个优点， 加入可溶性亲核试剂如3，4-二氯苯乙烷。 一是 可以将含有捕获的亲电体的级分从所述亲电体分离。 复杂的孵育基质通过简单的过滤步骤，去除各种 可能干扰后续生产的产品 色谱分析 第二，亲核试剂的结构可以是 易于定制，以使捕获的 产品. 例如，可以引入卤素以促进GC 可以使用灵敏的EC检测器或荧光发色团进行分析， 包括荧光检测器以允许荧光检测器与HPLC一起使用。 三是 与整个细胞孵育，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体， 到达细胞外介质。 这将是特别有用的， 研究被认为需要代谢活化的致癌物质， 一个器官，通常是肝脏，产生一种亲电体，针对一些 其他器官。 这些新的药物将被应用于药物代谢的研究。 二甲基亚硝胺，因为有相互矛盾的报告的能力， 通常的硫醇亲核试剂，2，4-二氯苯乙烷， 在亚硝胺代谢过程中产生的甲基化物质。 的 新的药剂也将用于研究 一系列β-氧化亚硝胺的代谢。