776 RADIATION RESEARCH 170, 776–783 (2008) 0033-7587/ 08 $15.00  2008 by Radiation Research Society. All rights of reproduction in any form reserved. The Nucleotide Pool, a Target for Low-Dose -Ray-Induced Oxidative Stress Traimate Sangsuwan and Siamak Haghdoost 1 Department of Genetics, Microbiology and Toxicology, Radiobiology Group, Stockholm University, S 106 91, Stockholm, Sweden Sangsuwan, T. and Haghdoost, S. The Nucleotide Pool, a Target for Low-Dose -Ray-Induced Oxidative Stress. Radiat. Res. 170, 776–783 (2008). Oxidative stress occurs when the generation of reactive ox- ygen species (ROS) exceeds the cellular antioxidant capacity. The excess ROS react with and modify cellular components. Nucleic acid modifications are of principal interest because they may cause mutations. 8-Oxo-7,8-dihydro-2-deoxyguano- sine (8-oxo-dG) is a mutagenic lesion that can be formed by ROS in DNA as well as in the nucleotide pool. 8-Oxo-dG is removed from the DNA by base excision repair and from the nucleotide pool by the nucleotide sanitization enzyme hMTH1. hMTH1 hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, which is re- leased to the extracellular environment and can serve as a marker of oxidative stress. The aim of this work was to es- tablish the dose–response relationship for radiation-induced extracellular 8-oxo-dG and hMTH1 in the mGy range of  rays in three cellular model systems: human whole blood, hu- man fibroblasts and stimulated lymphocytes. Extracellular 8-oxo-dG was analyzed with the use of an ELISA and hMTH1 by Western blotting. Our results demonstrate that low-dose ionizing radiation induces a stress response that leads to the formation of extracellular 8-oxo-dG and induction of hMTH1 in all three cellular model systems tested. This suggests that the nucleotide pool is an important target for radiation-in- duced stress response.  2008 by Radiation Research Society INTRODUCTION The present knowledge on cellular responses to ionizing radiation in the mGy range is limited, and the dose–re- sponse relationships for the genotoxic action for this dose range are not known. The use of novel markers could help to fill the gaps in our knowledge about the cellular response to low-dose radiation. Low-LET ionizing radiation causes different types of damage to cellular DNA through two known mechanisms of action: the direct action that is responsible for about 30% of the effect and is caused by direct ionization of the target 1 Address for correspondence: Siamak Haghdoost, Department of Ge- netics, Microbiology and Toxicology, Stockholm University, S-106 91 Stockholm, Sweden; e-mail: Siamak.haghdoost@gmt.su.se. and the indirect action that accounts for about 70% of the effect (1). The indirect effect is caused by radiolysis of intracellular water and the subsequent formation of reactive oxygen species, ROS (e.g., OH, O 2 ). ROS are also formed endogenously in living cells as a result of cell me- tabolism. The majority of the endogenous ROS is derived from the mitochondrial electron transport chain (2). ROS can induce mutations through direct oxidation of DNA or through oxidation of deoxyribonucleotides that are then incorporated into the DNA (3). A high level of intra- cellular production of ROS can lead to a depletion of ri- bonucleotide reductase, an enzyme that catalyzes the re- duction of ribonucleotide to 2-deoxyribonucleotide that in turn may cause an imbalance of the dNTP pool (4). The fidelity of DNA repair and synthesis is dependent on a bal- anced dNTP pool. It has been suggested that halogenated pyrimidines, e.g. 5-fluoro-2-deoxyuridine (FrdU) (5) as well as exposure to UV radiation (6), induce nucleotide pool imbalance that may be involved in cell death. Modi- fications of the nucleotide pool composition by FrdU treat- ment may also influence various cellular processes such as cell cycle regulation, proliferation and differentiation (5). One of the frequently studied mutagenic base lesions is 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxo-dG). This le- sion is produced when one hydroxyl radical reacts with 2-dG at the C8 position (7, 8). When present in DNA, 8- oxo-dG can cause a G:C to T:A transversion (9). Free rad- icals can react with dGTP in the nucleotide pool and give rise to 8-oxo-dGTP, which can be incorporated into DNA and induce an A:T to C:G transversion (10). hMTH1 in- hibits incorporation of 8-oxo-dGTP into DNA by hydro- lyzing it to 8-oxo-dGMP that can be transported from the intracellular to the extracellular milieu as 8-oxo-dG by the action of 8-oxo-dGMPase (11). The absence of mutT (bac- terial homologue of hMTH1) activity in E. coli leads to 1000 times more spontaneous A:T to C:G transversions compared to the wild type (12). Further, a higher cancer frequency in mice defective in MTH1 has been reported (13). Extracellular 8-oxo-dG (in urine, blood serum, cell culture medium) as well as the 8-oxo-dG content of the DNA has been used as a marker of oxidative stress (14). According to our previously published data, the yield of radiation-induced extracellular 8-oxo-dG was 35 times