The cyclic N-nitrosamine N-nitrosopyrrolidine (NPy) is a potent animal carcinogen that is found in mainstream and sidestream tobacco smoke. NPy is highly mutagenic in vitro in the Ames assay in the presence of an appropriate metabolizing enzyme system. Unlike acyclic N-nitrosamines, NPy does not yield a high level of DNA adducts' and to date no DNA adduct has been identified. Based on our observation that NPy induces single-strand breaks and/or alkali labile lesions, we have formulated the working hypothesis that NPy genotoxicity is derived from the formation of transient DNA adducts. We propose to test this hypothesis by: 1) determining if NPy is metabolized in vitro to N-nitroso-2-pyrrolidone (2-oxo- NPy) via an intermedite 2-hydroxy-NPy; 2) characterizing the in vivo genotoxicity of NPy with specific emphasis on the formation of DNA-protein and DNA-DNA crosslinks; 3) determining which metabolite of NPy is responsible for the crosslinking; 4) determining if NPy affords phosphotriesters and/or apurinic/apyrimidinic sites with any base or sequence specificity using DNA of known sequence; and 5) determining the chemical stability and reactivity of synthetic site specific phosphotriester-modified oligodeoxynucleotides that are suspected to be produced from NPy. The results of these studies will provide basic knowledge concerning the carcinogenicity of an N-nitroso compound that apparently does not yield stable DNA adducts.
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