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

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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.