Measurement of 7,8-dihydro-8-oxo-2′-deoxyguanosine metabolism in MCF-7 cells at low concentrations using accelerator mass spectrometry

Abstract

Growing evidence suggests that oxidative damage to cells generates mutagenic 7,8-dihydro-8-oxo-2 -deoxyguanosine (8-oxodG), which may initiate diseases related to aging and carcinogenesis. Kinetic measurement of 8-oxodG metabolism and repair in cells has been hampered by poor assay sensitivity and by difficulty characterizing the flux of oxidized nucleotides through the relevant metabolic pathways. We report here the development of a sensitive and quantitative approach to characterizing the kinetics and metabolic sources of 8-oxodG in MCF-7 human breast cancer cells by accelerator mass spectrometry. We observed that [ 14 C]8-oxodG at medium concentrations of up to 2 pmol/ml was taken up by MCF-7 cells, phosphorylated to mono-, di-, and triphosphate derivatives, and incorporated into DNA. Oxidative stress caused by exposure of the cells to 17␤-estradiol resulted in a reduction in the rate of [ 14 C]8-oxodG incorporation into DNA and an increase in the ratio of 8-oxodG monophosphate (8-oxodGMP) to 8-oxodG triphosphate (8-oxodGTP) in the nucleotide pool. 17␤-Estradiolinduced oxidative stress up-regulated the nucleotide pool cleansing enzyme MTH1 and possibly other Nudix-related pyrophosphohydrolases. These data support the conclusion that 8-oxodGTP is formed in the nucleotide pool by both 8-oxodG metabolism and endogenous reactive oxygen species. The metabolism of 8-oxodG to 8-oxodGTP, followed by incorporation into DNA is a mechanism by which the cellular presence of this oxidized nucleoside can lead to mutations.DNA repair ͉ nucleoside metabolism ͉ oxidative stress ͉ breast cancer

Probing the Active Site of the Deoxynucleotide N-Hydrolase Rcl Encoded by the Rat Gene c6orf108

“…These DNA lesions are subject to a network of DNA repair mechanisms that generally excise the modified base (28). However, in some cases the modified base can be recycled as illustrated by 8-oxo-2Ј-deoxyguanosine and 8-oxo-guanine (29). We hypothesized that modified dNMPs could be present during this cycle and that Rcl could be involved in their catabolism.…”

8-Oxo-deoxyguanosine: Reduce, reuse, recycle?

“…Indeed, there is evidence to suggest that bromodeoxycytidine can be taken up by dividing cells and, after deamination and phosphorylation, and/or vice versa, is present in the dNTP pool as a substrate for DNA synthesis (12). A further consequence of the findings of Hah et al (6) is to ascribe even greater importance to the activities of Nudix-type enzymes. These enzymes would appear to be the true gatekeepers ensuring that 8- The authors also comment on a hypothesis that has received limited discussion or experimental examination in the literature: that of further oxidation of 8-oxodG.…”

“…Indeed, significant contribution from the latter two routes probably negates the utility of measuring this lesion in urine, under any circumstances. The findings of Hah et al (6) now extend this caveat to include assessment of nuclear, and potentially mitochondrial, 8-oxodG. Such measurements would, therefore, not be uniquely reflective of cellular oxidative stress but would nonetheless represent a DNA damage burden and hence a risk/threat to the cell.…”

“…Although the data of Hah et al (6) certainly demonstrate the potential for extracellular 8-oxodG to be incorporated into cellular DNA, the references that we cite above add to the debate and give a fuller picture of the possible processes surrounding this interesting area. For researchers studying nucleic acid-derived biomarkers of oxidative stress, it is interesting to note that of 10.8 pmol of extracellular, radiolabeled 8-oxodG added to cells in culture, Ϸ8% was localized on or inside the cells.…”

“…1). In a recent issue of PNAS, Hah et al (6) used accelerator mass spectrometry, a technique with exquisite sensitivity, to experimentally examine previously difficult questions concerning the metabolic fate of 8-oxodG, using more realistic levels of substrates than were formerly feasible.…”