J. Radiat. Res., 50, 241–252 (2009) Regular Paper Microarray Analysis of Differentially Expressed Genes in the Kidneys and Testes of Mice after Long-term Irradiation with Low-dose-rate γ-rays Keiko TAKI1, Bing WANG1, Tetsuo NAKAJIMA1, Jianyu WU1, Tetsuya ONO2, Yoshihiko UEHARA2, Tsuneya MATSUMOTO3, Yoichi OGHISO3, Kimio TANAKA3, Kazuaki ICHINOHE3, Shingo NAKAMURA3, Satoshi TANAKA3, Junji MAGAE4, Ayana KAKIMOTO1 and Mitsuru NENOI1* Kidney/Testis/Low-dose-rate radiation/Microarray/Mitochondrial oxidative phosphorylation. Measuring global gene expression using cDNA or oligonucleotide microarrays is an effective approach to understanding the complex mechanisms of the effects of radiation. However, few studies have been carried out that investigate gene expression in vivo after prolonged exposure to low-dose-rate radia- tion. In this study, C57BL/6J mice were continuously irradiated with γ-rays for 485 days at dose-rates of 0.032–13 μGy/min. Gene expression profiles in the kidney and testis from irradiated and unirradiated mice were analyzed, and differentially expressed genes were identified. A combination of pathway analysis and hierarchical clustering of differentially expressed genes revealed that expression of genes involved in mitochondrial oxidative phosphorylation was elevated in the kidney after irradiation at the dose-rates of 0.65 μGy/min and 13 μGy/min. Expression of cell cycle-associated genes was not profoundly modulated in the kidney, in contrast to the response to acute irradiation, suggesting a threshold in the dose-rate for modulation of the expression of cell cycle-related genes in vivo following exposure to radiation. We demonstrated that changes to the gene expression profile in the testis were largely different from those in the kidney. The Gene Ontology categories “DNA metabolism”, “response to DNA damage” and “DNA replication” overlapped significantly with the clusters of genes whose expression decreased with an increase in the dose-rate to the testis. These observations provide a fundamental insight into the organ- specific responses to low-dose-rate radiation. radiation on human health. A number of these studies have INTRODUCTION suggested there were changes in the immune system1) DNA repair rate2) and chromosome aberration frequency.3) However, There is increasing concern about the biological effects of the effects of low-dose-rate radiation are still controversial low-dose-rate radiation. Epidemiological studies in naturally because many confounding factors, including lifestyle, smok- high-background-radiation areas (HBRAs) have provided ing habits, and dietary habits, may have affected the results. opportunities to directly observe the effects of low-dose-rate Animal studies under controlled conditions are therefore important in order to compensate for the uncertainties of epidemiological studies. Tanaka et al.4) have investigated the *Corresponding author: Phone: +81-43-206-3082, life shortening of mice after long-term (about 400 days) Fax: +81-43-255-6497, irradiation with γ-rays at extremely low-dose-rates in the range E-mail: [email protected] μ 1Radiation Effect Mechanisms Research Group, National Institute of of 0.038-16 Gy/min, and reported that the lifespan of female Radiological Sciences, 9-1, Anagawa-4-chome, Inage-ku, Chiba 263-8555 mice irradiated with 0.83 μGy/min and mice of both sexes Japan; 2Graduate School of Medicine, Tohoku University, 2-1 Seiryou- irradiated with 16 μGy/min were significantly shortened. 3 machi, Aoba-ku, Sendai-shi 980-8575 Japan; Institute for Environmental In order to generalize animal data to humans, a study of Sciences, 1-7, Ienomae, Obuchi, Rokkasho-mura, Kamikita-gun, Aomori the mechanism of radiation effects is required. Determina- 039-3212 Japan; 4Nuclear Technology Research Laboratory, Radiation safety Research Center, Central Research Institute of Electric Power tion of global gene expression patterns using cDNA or Industry, 2-11-1, Iwadokita, Komae-shi, Tokyo 277-0861 Japan. oligonucleotide microarrays is an effective approach to doi:10.1269/jrr.09011 understanding the complex mechanisms of radiation effects. J. Radiat. Res., Vol. 50, No. 3 (2009); http://jrr.jstage.jst.go.jp 242 K. Taki et al. Alteration of gene expression profiles in vivo after whole for 485 days. The kidney and testis have been classified as body irradiation of mice has been investigated by Amundson relatively radio-sensitive organs,13) and the likelihood of et al.5) where they reported that only a few genes were com- detecting effects of such low-dose-rate radiation was thought monly regulated in the thymus, spleen and liver following to be high in these organs. Our study is expected to provide exposure to 0.2–2 Gy of γ–rays. On the basis of this obser- insight into the mechanisms underlying the biological vation, they concluded that gene regulation was highly response to low-dose-rate radiation and normal tissue injuries. tissue-specific following γ-irradiation. Markedly different gene expression responses between the kidney and the brain MATERIALS AND METHODS were also reported by Zhao et al.6) who observed altered expression of genes related to transcription/translation and Mice and irradiation oxidation/reduction in the kidney after 10 Gy of whole-body Male specific pathogen free (SPF) C57BL/6J mice, 7 to 8 irradiation.6) It has been also reported that genes associated weeks of age, were obtained from an animal breeding facil- with inflammation, proliferation, metabolism, migration/ ity (CLEA Japan, Tokyo). Mice were not littermate. We used invasion, and cytoskeleton/microtubules were modulated in male mice because the data from female mice are considered the kidney 10 – 30 weeks after irradiation with 16 Gy of γ– to be perturbed by an estradiol cycle. Irradiation with 137Cs rays.7) These studies provided basic insight into the patho- γ-rays was carried out in a facility at the Institute for genesis of late injuries of the kidney. In the brain, differential Environmental Sciences for 485 days (22 h/day) at dose- modulation of genes associated with ribosomal proteins, rates of 0.032 μGy/min, 0.65 μGy/min, and 13 μGy/min. electron transport, and intracellular signaling involving G The total dose after irradiation at these dose-rates was 21 proteins, TGF-β, and Wnt were observed after irradiation mGy, 420 mGy, and 8000 mGy, respectively. Dosimetry and with 20 Gy of X-rays.8) DNA replication/repair, prolifera- maintenance of the mice were carried out in the same tion/apoptosis, cell cycle, and RNA processing genes were manner as described in Tanaka et al..4) We examined 12 reported to be altered in bone marrow following whole-body mice (3 per each dose-rate condition, including the control). exposure of mice to 6.5 Gy of γ–rays.9) Other results indicate The number of mice per dose-rate condition (n = 3) was that the response of the liver to the internal exposure to α– chosen because this is the minimum sample size needed for particles is characterized by up-regulation of genes related statistical analysis. Each mouse was subjected to dissection, to transcription and down-regulation of genes associated during which it was observed that the gross appearance was with signal transduction.10) The irradiated liver is also normal without organ hypertrophy, neoplasia, and/or hair suggested to be in an inflammatory state characterized by loss. All experiments were conducted according to Japanese up-regulation of positive acute phase proteins.10) Although legal regulations and followed the Guidelines for Animal the mouse strains and radiation doses used were not Experiments of the Institute for Environmental Sciences. identical, these studies suggest that the genes regulated by radiation differ between tissues. Preparation of RNA and hybridization The dose-rate effect has been assessed in vitro by Sokolov Mice were euthanized immediately after the termination et al.11) using human normal fibroblasts irradiated with 1 Gy of irradiation, and the kidney and testis were removed and of γ-rays at the dose-rate of 1 Gy/min or 0.045 Gy/min. They stored at –80°C in RNAlater (Ambion; Aplied Biosystems, observed that approximately one-third of genes were differ- Foster City, CA). Total RNA was extracted using an RNeasy entially expressed between these dose-rate conditions. Mini Kit and RNase-Free DNase Set (Qiagen, Valencia, Amundson et al.12) also investigated the dose-rate effect CA). The quality of total RNA samples was assessed using using human myeloid leukemia cells irradiated with γ–rays an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa in the dose-rate range of 0.0028–2.9 Gy/min. It was reported Clara, CA). From each sample, 150 ng of the total RNA was that there exist two classes of low-dose-rate responders; one labeled using an Illumina RNA Amplification Kit (Ambion; was genes induced in a dose-rate dependent fashion, while Aplied Biosystems, Foster City, CA). A total of 1.5 μg of the other was genes with dose-rate independent induction. biotin-labeled cRNA was hybridized for 18 h to the microarray Apoptosis-related genes were predominantly included in the (Sentrix Mouse-6 v1.0 Expression BeadChips, Illumina, San dose-rate dependent gene group, and the majority of genes Diego, CA). RNA samples from each mouse were not in the dose-rate independent gene group were involved in pooled but instead hybridized to the array separately. The cell cycle regulation. hybridized, biotinylated cRNA was detected with Streptavidin- In contrast to the accumulating data on global gene Cy3 (GE, Fairfield, CT) and quantitated using a BeadStation expression in vitro after exposure to low-dose-rate radiation, 500GX-WG Systems scanner (Illumina). Sentrix Mouse-6 few studies have been carried out in vivo. In this study, we v1.0 Expression BeadChips are constructed from small analyzed alteration of gene expression profiles in the kidney beads coated with 46,000 species of oligonucleotide probes.