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

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 原始描述： 本项目的目的是开发一种特定的14 C-后标记检测，以检测非常低水平的一种特定类型的DNA加合物，O 6-甲基脱氧鸟苷（O 6-MedG）。这种加合物是由烷化剂形成的含量较少的损伤之一，通过32 P后标记检测到的水平为1加合物/10^8个核苷酸，已知具有致突变性、重组性和细胞毒性。O 6-MedG是由于暴露于甲基化剂、烟草烟雾中存在的亚硝胺和硝酸盐处理的食物而形成的。因此，该测定将提供用于检测暴露于此类烷基化加合物的人群中的低水平O 6-MedG加合物的有价值的工具。 为了通过AMS测量加合物水平，开发了一种程序，该程序通过使用14 C-乙酸酐的乙酰化反应将14 C-放射性标记成功掺入O 6-MedG样品（两个14 C-乙酰基）中。乙酰化反应已开发使用合成的加合物标准品和通过HPLC，LC-MS和闪烁计数测定的检测限下降。 AMS的检测限已确定为14 C-二乙酰基-O 6-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的培养细胞和/或处理动物的DNA中分离的14 C标记的加合物样品。该方法也可应用于其他类似结构加合物的检测和定量。

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.