Growing evidence suggests that oxidative damage to cells generates mutagenic 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG), which initiates diseases related to aging and carcinogenesis. Insufficient detection sensitivity has hampered discrimination between incorporation of 8-oxodG into DNA and direct oxidation of the genome. We report here the use of accelerator mass spectrometry (AMS) to measure incorporation and removal of 8-oxodG in vivo. AMS provides zeptomole (10-21 mol) sensitivity to compounds labeled with isotopes such as 14C. Human breast cancer cells were grown in the presence of either [14C]8-oxodG or [14C]dG in order to trace the amount of each nucleoside that was incorporated into DNA by nucleotide salvage. Unexpectedly, the rate of incorporation of 8-oxodG was approximately equal to that of dG, with concentrations of the incorporated nucleosides approaching that of background 8-oxodG levels in the DNA of most cell types. After exclusion of 14C-labeled compound from the media, only the 14C from 8-oxodG was removed from the cellular DNA, presumably by base excision repair (BER). The incorporation and repair data indicate that nucleotide salvage contributes significantly to levels of 8-oxodG in DNA, and that base excision repair rather than nucleotide pool cleansing is the predominant mode of repairing this type of nucleobase damage.
This work was performed in part under the auspices of the U.S. Department of Energy by University of California Lawrence Livermore National Laboratory under Contract No. W-7405-Eng-48.
See more of AMS in Low Dose Bioscience Workshop
See more of The 10th International Conference on Accelerator Mass Spectrometry (September 5-10, 2005)