Analysis of 8-Hydroxydeoxyguanosine 5-Monophosphate (8-OH-dGMP) as a Reliable Marker of Cellular Oxidative DNA Damage After -Irradiation Nan Mei, 1 Kazuyoshi Tamae, 1 Naoki Kunugita, 2 Takeshi Hirano, 1 and Hiroshi Kasai 1 * 1 Department of Environmental Oncology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Kitakyushu, Japan 2 Department of Health Information Science, School of Health Sciences, University of Occupational and Environmental Health, Kitakyushu, Japan In order to improve 8-hydroxyguanine (8-OH-Gua) detection in DNA, we digested isolated DNA with nuclease P1 and analyzed for 8-hydroxydeox- yguanosine 5'-monophosphate (8-OH-dGMP) us- ing a high-performance liquid chromatography system equipped with an electrochemical detector (HPLC-ECD). The amount of 8-OH-Gua in the DNA was expressed as the ratio of 8-OH-dGMP to de- oxycytidine monophosphate (dCMP). Using this analysis, the background level of 8-OH-Gua in DNA from human lung carcinoma cells (A549) was several-fold lower than that obtained by a previous method. A549 cells were exposed to 20 – 60 Gy of -radiation and an increase in 8-OH- Gua concentration was observed with increasing -ray dose (0.3 residues per 10 7 dCMP per Gy). Moreover, by an immunohistochemical procedure using a commercial FITC-kit, 8-OH-Gua was clearly detected in A549 cells and the fluorescence in- tensity of cells with oxidative DNA damage in- creased with the doses of -irradiation. Using an endonuclease nicking assay, we also found that -rays decreased 8-OH-Gua repair activity. The results indicate that 8-OH-dGMP is a useful and sensitive marker for estimating oxidative damage in DNA. Environ. Mol. Mutagen. 41:332–338, 2003. © 2003 Wiley-Liss, Inc. Key words: DNA damage; -irradiation; 8-hydroxyguanine; 8-OH-dGMP; repair activity INTRODUCTION Oxygen radicals are generated by environmental factors, such as ionizing radiation and chemical carcinogens, and also by endogenous processes, including energy metabolism in mitochondria [Kasai and Hirano, 2000]. A fraction of oxygen radicals may escape the cellular defenses and cause permanent or transient damage to proteins, lipids, and nu- cleic acids. Reactive oxygen species (ROS), such as hydro- gen peroxide, superoxide anions (O 2 - ), singlet oxygen, and hydroxyl radicals (OH  ), can damage cellular DNA and proteins. Among these ROS, the hydroxyl radical is gener- ally assumed to be the critical reactive species that directly attacks DNA [Breen and Murphy, 1995]. The types of DNA damage caused by ROS include strand breaks and base modifications. 8-Hydroxyguanine (8-OH-Gua), also called 7,8-dihydro-8-oxoguanine (8-oxo-Gua), is a typical form of ROS-induced DNA damage [Kasai and Nishimura, 1984]. It was reported to be a key biomarker relevant to carcinogen- esis [Kasai, 1997; Shigenaga and Ames, 1991], and to cause mainly G 3 T transversions [Grollman and Moriya, 1993; Cunningham, 1997]. About 65% of the cellular DNA damage produced by ionizing radiation is caused by hydroxyl radicals, which are formed from the radiolysis of molecular water [Ward, 1988]. The indirect effects of ionizing radiation give rise to strand breaks and base lesions as the main classes of DNA damage. However, the quantitative aspects of lesion forma- tion remain to be established, mostly due to the lack of sensitive and reliable measurement methods [Douki et al., 1996]. 8-OH-Gua was first identified in 1984 as a type of ionizing radiation-induced DNA base damage. X-irradiated DNA samples were analyzed by high-performance liquid Grant sponsors: the Japan Society for the Promotion of Science (to NM), the Radiation Effects Association of Japan (to NM). and the Ministry of Education, Culture, Sports, Science and Technology of Japan (to HK). Current address for N. Mei: Division of Genetic and Reproductive Toxi- cology, National Center for Toxicological Research, Jefferson AR 72079 USA. *Correspondence to: Hiroshi Kasai, Department of Environmental Oncol- ogy, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Kitakyushu, 807-8555 Japan. E-mail: h-kasai@ med.uoeh-u.ac.jp Received 11 December 2002; provisionally accepted 26 January 2003; and in final form 22 February 2003 DOI 10.1002/em.10165 Environmental and Molecular Mutagenesis 41:332–338 (2003) © 2003 Wiley-Liss, Inc.