Measurement of 7,8-dihydro-8-oxo-2′-deoxyguanosine metabolism in MCF-7 cells at low concentrations using accelerator mass spectrometry
- Sang Soo Hah,
- Janna M. Mundt,
- Hyung M. Kim,
- Rhoda A. Sumbad,
- Kenneth W. Turteltaub, and
- Paul T. Henderson*
- Chemistry, Materials, and Life Sciences Directorate and Center for Accelerator Mass Spectrometry, Lawrence Livermore National Laboratory, 7000 East Avenue, L-452, Livermore, CA 94551
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Edited by Philip C. Hanawalt, Stanford University, Stanford, CA, and approved May 24, 2007 (received for review February 24, 2007)
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.
Footnotes
- *To whom correspondence should be addressed. E-mail: henderson48{at}llnl.gov
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Author contributions: S.S.H. and J.M.M. contributed equally to this work; S.S.H., J.M.M., K.W.T., and P.T.H. designed research; S.S.H., J.M.M., H.M.K., and R.A.S. performed research; S.S.H., J.M.M., K.W.T., and P.T.H. analyzed data; and S.S.H. and P.T.H. wrote the paper.
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The authors declare no conflict of interest.
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This article is a PNAS Direct Submission.
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This article contains supporting information online at www.pnas.org/cgi/content/full/0701733104/DC1.
- Abbreviations:
- AMS,
- accelerator mass spectrometry;
- BER,
- base excision repair;
- dG,
- 2′-deoxyguanosine;
- E2,
- 17β-estradiol;
- Fpg,
- formamidopyrimidine-DNA glycosylase;
- MTH1,
- MutT homologous 1;
- 8-oxodG,
- 7,8-dihydro-8-oxo-2′-deoxyguanosine;
- 8-oxodGMP,
- 8-oxodG monophosphate;
- 8-oxodGDP,
- 8-oxodG diphosphate;
- 8-oxodGTP,
- 8-oxodG triphosphate;
- qRT-PCR,
- quantitative RT-PCR.
- © 2007 by The National Academy of Sciences of the USA
