Susceptibility of Mouse Splenic Cells to Oxidative DNA Damage by X-Ray Irradiation

Abstract

Reactive oxygen species (ROS) formed in cells by mitochondrial respiration, phagocytosis, ionizing radiation and chemical oxidizing agents have been shown to be a major cause of oxidative damage to lipids, proteins, DNA, etc. Hydroxyl radicals are suggested to play an important role in the damage caused by ionizing radiation.1-3) Many kinds of oxidative damage are induced in the body by X-ray irradiation. DNA lesions, such as modified bases, abasic sites, and DNA strand breaks, are formed by X-ray irradiation. For example, is formed in DNA by ROS, such as hydroxyl radicals and singlet oxygen, and is recognized as a biomarker of oxidative DNA damage. If 8-oxodG residues are not removed before DNA replication, they cause G-C→T-A transversions. 4)A variety of antioxidant mechanisms in the body protect tissues against damage caused by ROS. 5) In general, susceptibility of mitotic cells to oxidative stress is higher than that of postmitotic cells. If antioxidant defenses are insufficient, ROS could cause damage, including DNA lesions, to cells.Two patterns of cell death, necrosis and apoptosis, are known.6,7) Necrosis results from metabolic collapse in cells, when cells can no longer maintain their homeostasis, while apoptosis is known as programmed cell death, and plays a central role in cell death that occurs during normal development in animals. In this study, the difference in susceptibility of splenic cells as undifferentiated cells and fibroblasts as differentiated cells to X-ray irradiation was examined based on changes in survival rates and 8-oxodG contents. In addition, in order to confirm the process of cell death, we examined whether apoptosis occurs in splenic cells and fibroblasts after X-ray irradiation. MATERIALS AND METHODSAnimals Male BDF1 mice were obtained at 9 or 10 weeks of age from SLC Japan Inc. (Shizuoka, Japan) and housed 4-5 mice per cage under specific-pathogen-free conditions. The animals were fed a commercial laboratory diet, CRF-1 (Oriental Yeast Inc., Tokyo, Japan), and water ad libitum. The animals were maintained at 21Ϯ2°C in a photoperiod-controlled (12 h/d) room.Cell Culture Mice were killed by cervical dislocation under anesthesia and their spleens were quickly isolated and put in phosphate-buffered saline (PBS, pH 7.2). Splenic cells were obtained by leaching the spleens on a #100 gauge wire mesh in a-medium (ICN Biomedicals Inc., Aurora, OH, U.S.A.), and suspended in a-medium supplemented with 30% fetal bovine serum (FBS; Moregate, Melbourne, Australia), 1% deionized bovine serum albumin, 100 mM mercaptethanol, 2 units of human urine erythropoietin (Sigma, St. Louis, MO, U.S.A.) and 10% (v/v) pokeweed-mitogenstimulated murine spleen cell conditioned medium (PWM-SCCM; Stemcell Technologies Inc., Vancouver, BC, Canada). The cell suspension was mixed by pipetting and passed through another mesh. The suspensions (3 ml) containing 5ϫ10 6 splenic cells were incubated at 37°C in 35 mm plastic dishes under a humidified atmosphere of 5% CO 2 and 95% air. Human embryonic fibroblasts, TIG-7...

Pathophysiological significance of senescence marker protein‐30

“…Therefore, to evaluate the extent of senescence, a convenient biomarker is required. Some modified substances, such as 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodG) or a hormone like DHEA‐S, have been reported to be useful for this purpose 1,2 . However, these markers are not directly encoded by the genome.…”

“…Some modified substances, such as 8-oxo-7,8-dihydro-2′deoxyguanosine (8-oxodG) or a hormone like DHEA-S, have been reported to be useful for this purpose. 1,2 However, these markers are not directly encoded by the genome. Furthermore, these substances are disadvantageous for modifying a gene to establish a model of aging.…”

Increased Levels of 8-Hydroxy-2′-Deoxyguanosine in Drosophila Larval DNA after Irradiation with 364-nm Laser Light but not with X-rays

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“…There are reports showing low doses of radiation exposure encountered in diagnostic x-ray radiation may induce malicious conditions. Tahara and Kaneko (2004) reported the survival rate in splenic cells in BDF1 mice and fetal human lung fibroblasts TIG-7 irradiated with x-rays at dose of 1, 2, 5 or 10 Gy. The survival rate of both irradiated cells decreased in a dose-dependent manner.…”