CHEMICAL CARCINOGENS AND DNA INTERACTIONS IN TISSUE CULT

这项提议的主要目标是开发一种高度敏感的 LC/MS/MS(LC/MS/MS)系统 用于分析致癌物处理后形成的甲基脱氧核糖核苷 DNA该系统将允许甲基化的检测和定量 由非放射性同位素标记的致癌物产生的核苷 水平与在体内引起生物效应的水平相当。这个 计划是利用MS/MS的特异性来检测甲基化 甲基处理小牛胸腺DNA中脱氧核糖核苷的分离 甲烷磺酸盐(MEMS)或N-甲基-N-亚硝脲(菜单)。新扫描 将采用串联质谱计的程序。MS/MS 方法论也将用于开发一种快速的 直接检测DNA中的甲基化脱氧核糖核苷 核苷事先用LC分离的要求。敏感度 将通过采用最新的进展来进一步加强检测 电离方法学，特别是二次离子质谱学 原子轰击和激光解吸。这些办法将付诸实施 在LC/MS和LC/MS/MS程序中。重点将放在 刻画条件，尤其是样本矩阵的刻画条件 最大限度地发挥这些新程序的效用。最优质量 然后将使用光谱系统来测量甲基化产物 中国仓鼠V79细胞在组织培养中暴露制备的DNA 到MEMS和菜单所需的水平，以诱导突变和 细胞毒性。该项目的成功将提供一种独特的方法 适用于其他化学致癌物的DNA加合物的分析 或者抗肿瘤药物。这将有助于阐明它们的致病机制。 在分子水平上的作用。

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.