The object of this proposal is to elucidate the mechanisms by which carcinogenic tobacco alkaloid derived nitrosamines are metabolized to intermediates which damage DNA and initiate the carcinogenic process. These studies are focused on N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAl), and N'-nitrosoanatabine (NAT). Human exposure to these carcinogens exceeds that of any other nitrosamine in nonoccupational environments. In studies carried out to date, metabolic Alpha-hydroxylation has been shown to be an important activation process for NNN and NNK. The proposed experiments are designed to conform the role of Alpha-hydroxylation in the activation of NNN and NNK and to elucidate the metabolic activation and detoxification pathways of NNAl and NAT. The target tissue DNA adducts formed from NNN and NNK will be identified. To accomplish this, the products obtained upon reaction of deoxyribonucleosides with diazohydroxides formed in the metabolism of NNN and NNK will be structurally characterized and used as markers for the identification of DNA adducts formed in vivo upon treatment of rats with high specific activity 3H or 14C-labelled NNN or NNK. The persistence of adducts in target and non-target tissues will be compared. The role of NNAl in carcinogenesis by NNK will be assessed through bioassays in rats and mice, and by identification of metabolites and DNA adducts of NNAl in vivo and in target tissues. The DNA adducts from NNAl and NNK in target tissues will be compared. To establish the metabolic activation and detoxification pathways for NAT, [6'-(14)C]NAT will be synthesized and its metabolism studied in vivo in the rat. Bioassays of NAT metabolites retaining the N-nitroso group will be carried out and the metabolic pathways of [6'-(14)C]NAT in target tissues determined. These studies are focused on a significant group of carcinogens which are likely to play a major role in tobacco-related cancers. By understanding their mechanisms of action, a scientific basis for assessing their role in human cancer will be established.
