Nitrosamines are a group of carcinogens widely occurring in the environment. It is known that metabolic activation is required for their carcinogenic actions. The enzymology of and factors affecting the metabolism of nitrosamines, however, are not clearly understood. Recent work in our laboratory has demonstrated the presence of a cytochrome P-450 (P-450)-mediated high affinity N-nitrosodimethylamine (NDMA) demethylase in rat liver microsomes inducible by fasting, diabetes, acetone, isopropanol, ethanol, and pyrazole. Our working hypothesis is that specific P-450 isozymes, inducible by the above factors, are responsible for the metabolism of NDMA and probably also other nitrosamines. In this proposal several lines of research will be pursued: 1. To purify the P-450 isozymes with high affinity and activity toward nitrosamines from rat liver microsomes. These P-450 isozymes will be characterized and compared with known P-450 isozymes in terms of their substrate specificities and other molecular properties. 2. To investigate the substrate specificity and mechanisms of nitrosamine metabolism (activation) by purified P-450 isozymes. The Km values of NDMA demethylase as well as the nature of the oxygenation, denitrosation and other reactions will be investigated. 3. To study the distribution and induction of the specific P-450 isozymes as well as their roles in nitrosamine metabolism in liver and nonhepatic tissues by immunochemical techniques and enzyme assays. 4. To illustrate the biological consequences of the induction of NDMA demethylase by measuring in vivo DNA alkylation in different organs. Nitrosamine metabolism in vivo and activation in model systems will be studied to provide additional mechanistic insights into the activation process. It is hoped that the proposed studies will yield definitive information on the enzymology and mechanisms of nitrosamine metabolism. Such information is crucial to our understanding of the carcinogenicity of nitrosamines and the modification of carcinogenesis by nutritional and environmental factors.
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