DEVELOPMENT OF A 14C-POSTLABELING PROCEDURE FOR TRACE DETECTION OF DNA ADDUCTS

用于 DNA 加合物痕量检测的 14C 后标记程序的开发

• 批准号: 7602408
• 负责人: Karen A Brown
• 金额: $ 2.55万
• 依托单位: UNIVERSITY OF CALIF-LAWRNC LVRMR NAT LAB
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7602408
• 关键词: Acetylation; Animals; Biological Assay; Carcinogens; Computer Retrieval of Information on Scientific Projects Database; Cultured Cells; DNA; DNA Adducts; Deoxyguanosine; Detection; Development; Exposure to; Food; Funding; Future; Grant; High Pressure Liquid Chromatography; Institution; Label; Lesion; Measures; Nitrates; Nitrosamines; Nucleotides; Procedures; Radiolabeled; Reaction; Research; Research Personnel; Resources; Sampling; Scintillation Counting; Source; Standards of Weights and Measures; Structure; Tobacco smoke; Translating; United States National Institutes of Health; acetic anhydride; adduct; cytotoxic; exposed human population; nitrate; radiotracer; tool

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Original Description: The aim of this project is to develop a specific 14C-postlabelling assay to detect very low levels of a particular type of DNA adduct, O6-methyl deoxyguanosine (O6-MedG). This adduct is one of the less abundant lesions formed by alkylating carcinogens, has been detected at levels of 1 adduct/10^8 nucleotides by 32P-postlabelling and is known to be mutagenic, recombinogenic and cytotoxic. O6-MedG is formed as a consequence of exposure to methylating agents, nitrosamines present in tobacco smoke, and nitrate treated foods. This assay will therefore provide a valuable tool for the detection of low levels of O6-MedG adducts in human populations exposed to such alkylating adducts. In order that adduct levels can be measured by AMS a procedure has been developed which successfully incorporates 14C-radiolabel into O6-MedG samples (two 14C-acetyl groups) via an acetylation reaction using 14C-acetic anhydride. The acetylation reaction has been developed using a synthesized adduct standard and the decreasing limits of detection determined by HPLC, LC-MS and scintillation counting. The limits of detection by AMS have been established for the 14C-diacetly-O6-MedG adduct standard and if this can now be translated to DNA samples, then the assay can be used to analyze 14C-labelled adduct samples isolated from cultured cells exposed to methylating agents and/or DNA from treated animals. This assay could also be applied to the detection and quantification of other adducts with similar structures in the future.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 原文描述： 该项目的目的是开发一种特定的 14C 标记后测定法，以检测极低水平的特定类型 DNA 加合物 O6-甲基脱氧鸟苷 (O6-MedG)。这种加合物是由烷基化致癌物形成的较少量的损伤之一，通过 32P 标记后检测到的水平为 1 个加合物/10^8 个核苷酸，并且已知具有诱变性、重组性和细胞毒性。 O6-MedG 是由于接触甲基化剂、烟草烟雾中的亚硝胺和硝酸盐处理食品而形成的。因此，该测定将为检测暴露于此类烷基化加合物的人群中低水平的 O6-MedG 加合物提供有价值的工具。 为了通过 AMS 测量加合物水平，我们开发了一种程序，通过使用 14C-乙酸酐的乙酰化反应，成功地将 14C-放射性标记掺入 O6-MedG 样品（两个 14C-乙酰基）中。使用合成的加合物标准品和通过 HPLC、LC-MS 和闪烁计数确定的降低的检测限开发了乙酰化反应。 AMS 的检测限已确定为 14C-二乙酰基-O6-MedG 加合物标准品，如果现在可以将其转化为 DNA 样品，则该测定可用于分析从暴露于甲基化剂和/或来自处理动物的 DNA 的培养细胞中分离出的 14C 标记加合物样品。该方法未来还可应用于其他具有类似结构的加合物的检测和定量。