The aim of the research is to understand the underlying reactions responsible for the carcinogenicity of N-nitroso-compounds and to determine whether DNA repair enzymes play a role in counteracting the initiation of neoplastic growth by these agents. The studies will focus on dimethylnitrosamine and its methylation of DNA. The methylated bases will be quantitated after separation by HPLC using radioimmunoassay techniques with specific antibodies, by fluorescence detection, or by incorporation of radioactivity. The formation and persistence in DNA of methylated bases will be studied in various tissues of rats and hamsters over a range of doses of dimethylnitrosamine. The effects of ethanol, low protein diets, and other physiological changes which alter the ability to metabolize dimethylnitrosamine on the distribution of methylation will be examined. Particular attention will be paid to the role of the liver in metabolizing orally administered dimethylnitrosamine virtually completely in a """"""""first pass effect"""""""" and thus protecting other organs from interaction with the carcinogen. The antibodies specific for methylated bases will be used to examine the distribution of methylation within the liver. The extent to which methylation of DNA can occur by chemical reaction with S-adenosylmethionine, its decarboxylated derivative, and other potential methylating agents will be examined. In parallel to the studies on the persistence of methylated bases in vivo, investigations will be carried out on enzymes removing methylated bases from DNA. The O(6)-methylguanine-DNA transmethylase from rat liver which has been purified 10000-fold will be purified to homogeneity and monoclonal antibodies produced to it and used to set up a radioimmunoassay for the protein. This will be used to investigate the regulation of this activity, the degree to which the methyl accepting protein is in the fully methylated form and the induction of activity in response to methylating agents. An ultrasensitive assay will be developed using a tetranucleotide as substrate. The activities of glycosylases removing ring N-methyl purines and derivatives from DNA will be measured and possible changes in these activities under conditions known to alter the transmethylase will be investigated. Finally, assays will be devised to look for repair activity directed towards alkylated pyrimidines and used to characterize these enzymes.
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