The nucleotide pool is a significant target for oxidative stress

doi:10.1016/j.freeradbiomed.2006.05.003

Free Radical Biology and Medicine

Volume 41, Issue 4, 15 August 2006, Pages 620-626

https://doi.org/10.1016/j.freeradbiomed.2006.05.003 Get rights and content

Abstract

Oxidative stress is considered to be one of the most important phenomena involved in the process of aging and age-related diseases. 8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) has been frequently used as a marker for oxidative stress. However, the origin of extracellular 8-oxo-dG is not well understood. The aim of this work was to investigate the nucleotide pool and the role of the human mutT homologue protein (hMTH1) in the appearance of extracellular 8-oxo-dG in a cellular model system. For this purpose we used primary human fibroblast cells, which were transfected by siRNAs homologous to hMTH1. Extracellular 8-oxo-dG in cell culture media after exposure of the cells to ionizing radiation was measured as enzyme-linked immunosorbent assay reactivity. Our results demonstrate the profound effect of both hMTH1 expression and nucleotide pool size on the cellular excretion of 8-oxo-dG, suggesting that the nucleotide pool is a significant target for the formation of extracellular 8-oxo-dG.

Section snippets

Cells and reagents

Primary human fibroblast cells VH10 were cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Paisley, UK) containing 10% fetal bovine serum (FBS; Invitrogen) and PEST (Invitrogen). The cells were grown at 37°C in 5% CO₂ in 154-cm² petri dishes for analysis of extracellular 8-oxo-dG and in 21.2-cm² petri dishes for Western blot analysis of hMTH1 protein expression.

Transfection of cells with siRNA

Two different siRNAs (ID 11691 Refseq NM_002452, ID 46023 Refseq NM_198954; Ambion, Huntingdon, UK) homologous to

Results

A representative example of Western blot analysis of radiation-induced hMTH1 in transfected and nontransfected VH10 cells is shown in Fig. 2. SiRNA transfection depresses the hMTH1 protein in unirradiated VH10 cells by approximately 60%. For control, the transfection medium was used without siRNA but with Lipofectamine in two experiments (data not shown) in order to investigate effects of transfection medium on hMTH1 protein and extracellular 8-oxo-dG concentration with and without irradiation.

Discussion

The constant endogenous production of reactive oxygen species and the resulting oxidatively generated damage are thought to significantly contribute to aging and age-related diseases [1], [35]. A major form of oxidatively generated DNA damage is 8-oxo-dG, a well-established and sensitive marker of oxidative stress under in vitro and in vivo conditions [1], [10]. The appearances of mutagenic 8-oxo-dG in DNA can be attributed to direct hydroxylation of C8 position of dG in DNA or incorporation of

Acknowledgments

This work was supported by the Swedish Cancer and Allergy Foundation, Swedish Radiation Protection Authority, and Commission of the European Union (FI6R-CT-2003-508842). We thank Björn Palmgren for statistical advice and Professor Dag Jensen and Associate Professor Klaus Erixon, Department of Genetics, Microbiology, and Toxicology, Stockholm University, for valuable comments and providing us with V79 cells.

References (43)

S. Loft et al.
Cancer risk and oxidative DNA damage in man
J. Mol. Med.
(1996)
M.D. Evans et al.
Oxidative DNA damage and disease: induction, repair and significance
Mutat. Res.
(2004)
M. Dizdaroglu
Formation of an 8-hydroxyguanine moiety in deoxyribonucleic acid on gamma-irradiation in aqueous solution
Biochemistry
(1985)
H. Kasai et al.
Hydroxylation of the C-8 position of deoxyguanosine by reducing agents in the presence of oxygen
Nucleic Acids Symp. Ser.
(1983)
H. Hayakawa et al.
Generation and elimination of 8-oxo-7,8-dihydro-2′-deoxyguanosine 5′-triphosphate, a mutagenic substrate for DNA synthesis, in human cells
Biochemistry
(1995)
Y. Nakabeppu et al.
Biological significance of the defense mechanisms against oxidative damage in nucleic acids caused by reactive oxygen species: from mitochondria to nuclei
Ann. N. Y. Acad. Sci.
(2004)
P. Svoboda et al.
Protection or sensitization by thiols or ascorbate in irradiated solutions of DNA or deoxyguanosine
Radiat. Res.
(1999)
A. Valenzuela
The biological significance of malondialdehyde determination in the assessment of tissue oxidative stress
Life Sci.
(1991)
H. Kasai
Chemistry-based studies on oxidative DNA damage: formation, repair, and mutagenesis
Free Radic. Biol. Med.
(2002)
M.K. Shigenaga et al.
Urinary 8-hydroxy-2′-deoxyguanosine as a biological marker of in vivo oxidative DNA damage
Proc. Natl. Acad. Sci. USA
(1989)

M.S. Cooke et al.

DNA repair: insights from urinary lesion analysis

Free Radic. Res.

(2002)

M. Murata et al.

Oxidative DNA damage induced by nitrotyrosine, a biomarker of inflammation

Biochem. Biophys. Res. Commun.

(2004)

Y. Hinokio et al.

Oxidative DNA damage in diabetes mellitus: its association with diabetic complications

Diabetologia

(1999)

S. Haghdoost et al.

Can 8-oxo-dG be used as a predictor for individual radiosensitivity?

Int. J. Radiat. Oncol. Biol. Phys.

(2001)

C. Tagesson et al.

Determination of urinary 8-hydroxydeoxyguanosine by automated coupled-column high performance liquid chromatography: a powerful technique for assaying in vivo oxidative DNA damage in cancer patients

Eur. J. Cancer

(1995)

D.D. Kennedy et al.

8-Oxo-dG elevated in children during leukemia treatment

Integr. Cancer Ther.

(2004)

H. Kasai et al.

Life style and urinary 8-hydroxydeoxyguanosine, a marker of oxidative DNA damage: effects of exercise, working conditions, meat intake, body mass index, and smoking

Jpn. J. Cancer Res.

(2001)

H. Prieme et al.

Effect of smoking cessation on oxidative DNA modification estimated by 8-oxo-7,8-dihydro-2′-deoxyguanosine excretion

Carcinogenesis

(1998)

S. Loft et al.

Increased urinary excretion of 8-oxo-2′-deoxyguanosine, a biomarker of oxidative DNA damage, in urban bus drivers

Mutat. Res.

(1999)

M.S. Cooke et al.

DNA repair is responsible for the presence of oxidatively damaged DNA lesions in urine

Mutat. Res.

(2005)

M.S. Cooke et al.

Progress in the analysis of urinary oxidative DNA damage

Free Radic. Biol. Med.

(2002)

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¹: Present address: Genetic Toxicology, Safety Assessment, AstraZeneca R&D Södertälje, SE-151 85 Södertälje, Sweden.

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Original ContributionThe nucleotide pool is a significant target for oxidative stress

Abstract

Section snippets

Cells and reagents

Transfection of cells with siRNA

Results

Discussion

Acknowledgments

Cancer risk and oxidative DNA damage in man

J. Mol. Med.

Oxidative DNA damage and disease: induction, repair and significance

Mutat. Res.

Formation of an 8-hydroxyguanine moiety in deoxyribonucleic acid on gamma-irradiation in aqueous solution

Biochemistry

Hydroxylation of the C-8 position of deoxyguanosine by reducing agents in the presence of oxygen

Nucleic Acids Symp. Ser.

Generation and elimination of 8-oxo-7,8-dihydro-2′-deoxyguanosine 5′-triphosphate, a mutagenic substrate for DNA synthesis, in human cells

Biochemistry

Biological significance of the defense mechanisms against oxidative damage in nucleic acids caused by reactive oxygen species: from mitochondria to nuclei

Ann. N. Y. Acad. Sci.

Protection or sensitization by thiols or ascorbate in irradiated solutions of DNA or deoxyguanosine

Radiat. Res.

The biological significance of malondialdehyde determination in the assessment of tissue oxidative stress

Life Sci.

Chemistry-based studies on oxidative DNA damage: formation, repair, and mutagenesis

Free Radic. Biol. Med.

Urinary 8-hydroxy-2′-deoxyguanosine as a biological marker of in vivo oxidative DNA damage

Proc. Natl. Acad. Sci. USA

DNA repair: insights from urinary lesion analysis

Free Radic. Res.

Oxidative DNA damage induced by nitrotyrosine, a biomarker of inflammation

Biochem. Biophys. Res. Commun.

Oxidative DNA damage in diabetes mellitus: its association with diabetic complications

Diabetologia

Can 8-oxo-dG be used as a predictor for individual radiosensitivity?

Int. J. Radiat. Oncol. Biol. Phys.

Determination of urinary 8-hydroxydeoxyguanosine by automated coupled-column high performance liquid chromatography: a powerful technique for assaying in vivo oxidative DNA damage in cancer patients

Eur. J. Cancer

8-Oxo-dG elevated in children during leukemia treatment

Integr. Cancer Ther.

Life style and urinary 8-hydroxydeoxyguanosine, a marker of oxidative DNA damage: effects of exercise, working conditions, meat intake, body mass index, and smoking

Jpn. J. Cancer Res.

Effect of smoking cessation on oxidative DNA modification estimated by 8-oxo-7,8-dihydro-2′-deoxyguanosine excretion

Carcinogenesis

Increased urinary excretion of 8-oxo-2′-deoxyguanosine, a biomarker of oxidative DNA damage, in urban bus drivers

Mutat. Res.

DNA repair is responsible for the presence of oxidatively damaged DNA lesions in urine

Mutat. Res.

Progress in the analysis of urinary oxidative DNA damage

Free Radic. Biol. Med.

Original Contribution
The nucleotide pool is a significant target for oxidative stress