SOLID-PHASE ELECTROPHILE TRAPPING AGENTS

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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.