The mechanism of tumor induction by unsymmetric nitrosamines is poorly understood. Alpha-hydroxylation activates these carcinogens to DNA alkylating agents by generating a metabolite that decomposes to an aldehyde and a diazohydroxide. The diazohydroxide, presumably via a diazonium ion, alkylates DNA. Unsymmetric nitrosamines have two activation pathways that lead to two different types of DNA adducts. Interactions between these two pathways likely contribute to the carcinogenic activity of the nitrosamine. In addition, the aldehyde metabolites are themselves chemically reactive and may contribute to the toxicological properties of nitrosamines. Interactions between the various reactive nitrosamine metabolites will be explored with 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK). This tobacco-specific nitrosamine selectively induces lung tumors in laboratory animals and is a possible human carcinogen. NNK is activated to either a methylating agent or a pyridyloxobutylating agent. Experimental data support a carcinogenic mechanism for this compound in which the formation and persistence of O6-methylguanine (O6-mG) is important for the initiation of tumorigenesis by NNK. Pyridyloxobutylating agents and NNK-derived aldehydes, formaldehyde and 4-oxo-1-(3-pyridyl)-1-butanone, are capable of interfering the repair of O6-mG by O6-alkylguanine-DNA alkyltransferase (AGT). In vivo levels of the AGT substrate pyridyloxobutyl DNA adduct, O6-[4-oxo-4-(3-pyridyl)butyl] - guanine, are insufficient to explain the extent of O6-mG persistence in NNK-treated mice. Therefore, there appears to be other mechanisms by which nitrosamine metabolites interfere with O6-mG repair. The objective of this application is to explore the mechanisms by which nitrosamine metabolites can reduce the repair of O6-mG by AGT. The central hypothesis is that nitrosamine metabolites can inactivate DNA repair pathways, leading to increased persistence of promutagenic adducts. These mechanisms differ from a mechanism involving competition between O6-alkylguanine adducts for repair by AGT. We plan to test our central hypothesis by pursuing the following specific aims: 1) Determine the importance of direct alkylation of AGT by diazohydroxides on AGT depletion; 2) Determine the contribution of aldehydes to the depletion of AGT following nitrosamine exposure; 3) Determine the effect of nitrosamine metabolites on the degradation rate of inactivated AGT; 4) Determine the ability of nitrosamine metabolites to influence the rate of AGT protein synthesis. The results of our studies will lead to a better understanding of mechanisms of lung cancer induction. In addition, this mechanism is likely applicable to other unsymmetric nitrosamines as well as carcinogenic mixtures such as tobacco smoke, a known human carcinogen.
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