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

Soo Hah Sang, Janna M. Mundt, Hyung M. Kim, Rhoda A. Sumbad, Ken W Turteltaub, Paul Henderson

Research output: Contribution to journalArticle

43 Scopus citations

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 [14C]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 17β-estradiol resulted in a reduction in the rate of [14C]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. 17β-Estradiol-induced 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.

Original languageEnglish (US)
Pages (from-to)11203-11208
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume104
Issue number27
DOIs
StatePublished - Jul 3 2007
Externally publishedYes

Keywords

  • Breast cancer
  • DNA repair
  • Nucleoside metabolism
  • Oxidative stress

ASJC Scopus subject areas

  • Genetics
  • General

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