Cyclic nitrosamines are an important class of potent carcinogens which occur in the human environment and can form endogenously in humans. These compounds demonstrate a high degree of organospecificity for tumor formation in laboratory animals. For example, N-nitrosopyrrolidine (NPYR) is an effective liver carcinogen in the rat, but never induces tumors of the esophagus whereas the next higher homologue, N-nitrosopiperidine (NPIP), is a potent esophageal carcinogen. We hypothesize that the formation and persistence of specific DNA adducts of NPYR and NPIP are responsible for their organospecific carcinogenic activity in rats. Substantial evidence indicates that both NPYR and NPIP form endogenously in humans, but rigorous demonstration of this concept has been elusive because there is no appropriate biomarker. We propose to use DNA adducts of cyclic nitrosamines as biomarkers of their formation and metabolic activation in humans. Research on cyclic nitrosamine metabolic activation has resulted in the identification of exocyclic DNA adducts of derived electrophiles such as the carcinogen 2-butenal (crotonaldehyde). Crotonaldehyde DNA adducts are present as endogenous adducts in human tissues but our ongoing studies suggest that these adducts are only a minor proportion of the total. Thus, we hypothesize that substantial amounts of crotonaldehyde-derived paraldol releasing DNA, adducts are also present in human tissues. Therefore our specific aims in this proposal are: 1) fully characterize the adducts formed upon reaction of alpha-acetoxy-N-nitrosopyrrolidine (alpha- acetoxyNPYR) and alpha-acetoxyNPIP with DNA in vitro; 2) identify the DNA adducts formed upon treatment of rats with NPYR or NPIP and determine their persistence in liver and esophagus; 3) develop DNA-based biomarkers of NPYR and NPIP formation and metabolic activation in humans; and 4) investigate the formation of crotonaldehyde-derived paraldol releasing adducts in rats and humans. DNA adducts will be characterized by nuclear magnetic resonance spectrometry, mass spectrometry, ultraviolet spectroscopy and by independent synthesis. Persistence studies will be carried out with radiolabelled carcinogens and with the use of specific methods for adduct detection. Mass spectrometric methods will be developed for quantitation of DNA adducts in human tissues. The results obtained in this program will test the overall hypothesis that the formation and persistence of specific DNA adducts are important in organospecific carcinogenesis by cyclic nitrosamines and that these adducts are present in human DNA as a result of endogenous formation of nitrosamines and related electrophiles. The insights gained here will be important in developing rational strategies for cancer prevention. This grant proposal will replace the corresponding portion of the Principal Investigator's Outstanding Investigator Grant, as the latter program is being phased out by the National Cancer Institute.
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