CHEMICAL CARCINOGENS AND DNA INTERACTIONS IN TISSUE CULT

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The primary objective of this proposal is to develop a highly sensitive liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) system for the analysis of methyldeoxyribonucleosides formed in carcinogen-treated DNA. This system will allow the detection and quantitation of methylated nucleosides generated from non-radioisotopically labeled carcinogens at levels comparable to those which induce biological effects in vivo. The plan is to utilize the specificity of MS/MS to detect methylated deoxyribonucleosides isolated from calf thymus DNA treated with methyl methanesulfonate (MeMS) or N-methyl-N-nitrosourea (MeNU). New scan procedures for tandem mass spectrometers will be employed. The MS/MS methodology will also be used in the development of a method for the rapid and direct detection of methylated deoxyribonucleosides in DNA without the requirement for prior separation of the nucleosides by LC. The sensitivity of detection will be further enhanced by adapting recent advances in ionization methodology, specifically secondary ion mass spectrometry, fast atom bombardment and laser desorption. These methods will be implemented in both the LC/MS and LC/MS/MS procedures. Emphasis will be placed on characterizing conditions, especially these of sample matrix, which maximize the utility of these new procedures. The optimized mass spectrometric system will then be used to measure the methylation products in DNA prepared by exposure of Chinese hamster V79 cells in tissue culture to MeMS and MeNU at the levels required to induce mutation and cytotoxicity. The success of this project will provide a unique approach adaptable to the analysis of the DNA adducts of other chemical carcinogens or antitumor agents. This will help to elucidate their mechanisms of action at the molecular level.