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利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 原文描述： 该项目的目的是开发一种特殊的14C-后标记分析方法，以检测非常低水平的特定类型的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数量较少的损伤之一，已通过32P-后标记检测到1个加合物/108个核苷酸的水平，并已知具有诱变性、重组原性和细胞毒性。O6-MedG是由于接触甲基化试剂、烟草烟雾中存在的亚硝胺和硝酸盐处理食品而形成的。因此，本试验将为检测接触这种烷基化加合物的人群中低水平的O6-MedG加合物提供一个有价值的工具。 为了用AMS测量加合物水平，建立了一种方法，通过使用14C-乙酸酐的乙酰化反应，将14C-放射性标记成功地结合到O6-MedG样品(两个14C-乙酰基)中。用合成的加合物标准进行了乙酰化反应，并用高效液相色谱、LC-MS和闪烁计数法确定了检测下限。AMS已经为14C-双乙酰-O6-MedG加合物标准建立了检测限，如果现在可以将其转化为DNA样本，那么该分析方法可以用于分析从接触甲基化试剂的培养细胞和/或处理动物的DNA中分离出来的14C标记加合物样本。该方法还可用于其他结构相似的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.