JOURNAL OF RADIATION PROTECTION, VOL.36 NO.3 SEPTEMBER 2011 119 ▣ PAPER ▣ MITOCHONDRIAL DNA DELETION AND IMPAIRMENT OF MITOCHONDRIAL BIOGENESIS ARE MEDIATED BY REACTIVE OXYGEN SPECIES IN IONIZING RADIATION‐INDUCED PREMATURE SENESCENCE Hyeon Soo Eom *,† , Uhee Jung * , Sung-Kee Jo * , and Young Sang Kim † * Radiation Biotechnology Research Division, Korea Atomic Energy Research Institute, Korea † Department of Biochemistry, College of Natural Sciences, Chungnam National University, Korea Received August 23, 2011 / 1st Revised September 22, 2011 / Accepted for Publication September 23, 2011 Mitochondrial DNA (mtDNA) deletion is a well‐known marker for oxidative stress and aging, and contributes to harmful effects in cultured cells and animal tissues. mtDNA biogenesis genes (NRF‐1, TFAM) are essential for the maintenance of mtDNA, as well as the transcription and replication of mitochondrial genomes. Considering that oxidative stress is known to affect mitochondrial biogenesis, we hypothesized that ionizing radiation (IR)‐induced reactive oxygen species (ROS) causes mtDNA deletion by modulating the mitochondrial biogenesis, thereby leading to cellular senescence. Therefore, we examined the effects of IR on ROS levels, cellular senescence, mitochondrial biogenesis, and mtDNA deletion in IMR‐90 human lung fibroblast cells. Young IMR‐90 cells at population doubling (PD) 39 were irradiated at 4 or 8 Gy. Old cells at PD55, and H2O2‐treated young cells at PD 39, were compared as a positive control. The IR increased the intracellular ROS level, senescence‐associated β‐galactosidase (SA‐β‐gal) activity, and mtDNA common deletion (4977 bp), and it decreased the mRNA expression of NRF‐1 and TFAM in IMR‐90 cells. Similar results were also observed in old cells (PD 55) and H2O2‐treated young cells. To confirm that a increase in ROS level is essential for mtDNA deletion and changes of mitochondrial biogenesis in irradiated cells, the effects of N‐acetylcysteine (NAC) were examined. In irradiated and H2O2–treated cells, 5 mM NAC significantly attenuated the increases of ROS, mtDNA deletion, and SA‐β‐gal activity, and recovered from decreased expressions of NRF‐1 and TFAM mRNA. These results suggest that ROS is a key cause of IR‐induced mtDNA deletion, and the suppression of the mitochondrial biogenesis gene may mediate this process. Keywords: Reactive oxygen species, Mitochondrial DNA deletion, Mitochondrial biogenesis, Nuclear Respiratory Factor‐1, Mitochondrial transcription factor A, Ionizing radiation 1. INTRODUCTION 1) Many reactive oxygen species (ROS) are formed by several external and endogenous factors. ROS have an affect on lipids, proteins, and DNA, and can cause dam- age to cells and tissues. The accumulation of damage from oxidative stress leads to a change of biological outcome, including inflammation, cancer, and diabetes [1]. Namely, the immoderate generation of ROS, or a deficiency in ROS scavenging, causes body impairment Corresponding author : Uhee Jung, uhjung@kaeri.re.kr Radiation Biotechnology Division, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute 1566 Sinjeong‐dong, Jeongeupsi, Jeonbuk 580‐185 South Korea [2]. Also, a recent study reported that ROS are an im- portant cause of cellular senescence and aging [3]. The immoderate generation of ROS by external stress and the accumulation of generated ROS induce aging by degree. Human mitochondria consist of 16569 bp double‐ stranded DNA of a circular form [4]. Since mitochon- dria lack histone protein, and are involved electron transport complexes that generate ROS, mtDNA is more easily injured than nuclear DNA [5]. Increased ROS from various types of stress can mediate mitochondrial DNA damage and the alteration of mitochondrial gene expression [6,7]. Also, mtDNA common deletion, which is one of the occurring events in aged cells, causes an accumulation of ROS [8,9]. ROS can affect the steady