DETECTION OF ALKYLATED DNA BASES IN HUMAN TISSUES

人体组织中烷基化 DNA 碱基的检测

• 批准号: 3551201
• 负责人: RUGGERO MONTESANO
• 金额: $ 9.22万
• 依托单位: INTERNATIONAL AGENCY FOR RES ON CANCER
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-16 至 1990-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3551201
• 关键词: DNA; alkylating agents; alkylation; alkyltransferase; antibody; antibody formation; cancer risk; environment related neoplasm /cancer; environmental contamination; enzyme mechanism; esophagus neoplasm; high performance liquid chromatography; human tissue; immunofluorescence technique; laboratory rabbit; monoclonal antibody; neoplasm /cancer diagnosis; neoplasm /cancer epidemiology; neoplasm /cancer immunodiagnosis; nitrosamines; nucleobase; radioimmunoassay; scintillation counter; spectrometry; stomach neoplasms

Humans are exposed to nitrosamines either directly or as a result of their formation in vivo; however, epidemiological studies indicating that a particular type of human cancer is causally associated with exposure to nitrosamines are not available. This difficulty in relating the measurement of environmental nitrosamines to an observed increased rate of a cancer may be overcome by the development of methods that permit the measurement of past exposure at the individual level. Nitrosamines are enzymatically converted into metabolites which bind covalently to DNA and various DNA adducts have been identified. The present studies have the specific aims of 1) developing immunological methods using monoclonal and/or polyclonal antibodies to quantitate certain of these DNA adducts in human tissues 2) assessing the value of this approach as a method for determining human exposure to environmental alkylating agents. Antibodies against 7-methyldeoxyguanosine (7-medG), O4-methyldeoxythymidine (O4-medT) and phosphotriesters will be developed and characterized for their sensitivity and specificity. These antibodies and those already available against O6-methyldeoxyguanosine (O6-medG) and O6-ethyldeoxyguanosine (O6-etdG) will be used in immunoassays to detect these adducts in DNA from human surgical tissue specimens of oesophagus or stomach obtained from populations at differential risk of cancer at either or both of these sites and for which some evidence of exposure to environmental nitrosamines exists. DNA is extracted from the tissues, hydrolyzed, fractionated chromatographically and the isolated DNA adducts are quantitated by immunoassay using the appropriate antibodies. Using this methodology with the antibody for O6-medG and a 2 mg sample of DNA levels of modification approaching one adduct in 10-8 of the corresponding normal nucleoside can be detected. In preliminary studies of oesophageal and stomach tissue samples from one of the proposed study populations from Linxian County in People's Rep. of China significant levels of O6-medG have already been detected. The capacity of the same tissues to repair O6-alkyldG and O4-medT will also be examined in conjunction with determination of adduct levels. In parallel, experimental studies in rats are planned to examine the presence of DNA adducts in peripheral blood cells following nitrosamine exposure. These experiments are designed to assess the potential of this approach as an alternative method of assessing human exposure to environmental nitrosamines.

人类直接或直接接触亚硝胺 体内形成；但是，流行病学研究表明 特定类型的人类癌症与暴露有关 硝基胺不可用。 这个困难在关联 测量环境亚硝胺，观察到的速率提高 可以通过开发允许的方法来克服癌症 测量过去在个人层面上的暴露。 亚硝胺是 酶促转化为代谢产物，它们与DNA和 已经确定了各种DNA加合物。 目前的研究具有 1）使用单克隆的特定目标开发免疫学方法 和/或多克隆抗体，以量化这些DNA加合物中的某些抗体 人体组织2）评估这种方法的价值作为一种方法 确定人类接触环境烷基化剂。 抗体 针对7-甲基氧鸟苷（7-MEDG），O4-甲脱氧胸苷（O4-MEDT） 和磷酸化的人将被开发和表征 灵敏度和特异性。 这些抗体和已经可用的抗体 反对O6-甲基氧鸟苷（O6-MEDG）和O6-乙基氧鸟苷 （O6-ETDG）将在免疫测定中使用，以检测来自DNA中的这些加合物 从中获得的食管或胃的手术组织标本 在这两个地点，这两个地点的癌症风险差异的种群 并为此证明了暴露于环境硝基胺的证据 存在。 从组织中提取DNA，水解，分级 色谱和分离的DNA加合物通过 使用适当的抗体进行免疫测定。 将此方法与 O6-MEDG的抗体和2 mg的DNA修饰水平样品 在相应的正常核苷的10-8中接近一个加合物可以 被检测到。 在对食道和胃组织的初步研究中 来自Linxian县拟议的研究人群的样本 中国人民众议员的大量O6 Medg已经存在 检测到。 相同组织修复O6-烷基的能力和 O4-MEDT也将与加合物的确定一起检查 水平。 同时，计划在大鼠中进行实验研究以检查 亚硝胺后，外周血细胞中的DNA加合物的存在 接触。 这些实验旨在评估这一潜力 方法是评估人类接触的另一种方法 环境亚硝胺。