NITROSAMINE ACTIVATION AND EXTRAHEPATIC P450S

DESCRIPTION: Nitrosamines are strongly carcinogenic in animals and are believed to be carcinogenic in humans as well. In rats, the esophagus is the most susceptible organ to tumor induction by nitrosamines. The specificity of tumor induction of nitrosamines is believed to be due to tissue-specific activation (a-hydroxylation) by P-450 enzymes localized in the esophagus, as shown by the efficient activation of both NNN and NBzMA (N'-Nitrosonornicotine and N-Nitrosobenzylmethylamine, respectively). The goal of this project is to isolate and characterize the P450 enzyme(s) in rat esophagus that are specific for nitrosamine activation. Preliminary studies with human P450s, rat nasal mucosa and rat esophageal microsomes, indicate that P450 2A enzymes play an important role in the activation of nitrosamines. The working hypothesis in this project is that nitrosamine activation in rat esophagus and nasal tissue is due to P450s of the 2A family. A cDNA library from rat esophagus and nasal mucosa tissue will be generated, and the library will be screened with one or more P450 2A gene fragments. The resulting cDNAs will be sequenced and will be expressed in insect cells using the baculovirus expression system. The expressed P450s will be tested for NNN and NBzMA hydroxylation activity. Using these P450s and various related nitrosamines, the hypothesis will be tested that nitrosamines which are strong esophageal carcinogens are good substrates for the tissue specific P450s that have been isolated. According to this hypothesis, a change in the structure which decreases the carcinogenicity, will also result in a decreased P450-mediated activation. Finally, human esophagus will be analyzed to determine if a similar P450 enzyme specific for nitrosamine activation is present.