The Journal of Toxicological Sciences (J. Toxicol. Sci.) 935 Vol.35, No.6, 935-939, 2010 Toxicogenomics/proteomics Report DNA microarray analysis of the liver of mice treated with cobalt chloride Kanako Matsumoto1, Hitomi Fujishiro1, Masahiko Satoh2 and Seiichiro Himeno1 1Laboratory of Molecular Nutrition and Toxicology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Yamashiro-cho, Tokushima 770-8514, Japan 2Laboratory of Pharmaceutical Health Sciences, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya, Aichi 464-8650, Japan (Received August 25, 2010; Accepted September 3, 2010) ABSTRACT — To investigate the in vivo effects of cobalt chloride on gene expression at early time points, DNA microarray analysis was performed on the liver of mice injected subcutaneously with cobalt chloride. The liver tissue samples were taken 0.5, 1, and 3 hr after injection. Of the 14 genes up-regu- lated at 0.5 hr after injection, 7 are related to immunological responses, and 4 of the 7 were found to be involved in the activation of interferon. Key words: Cobalt, DNA microarray, Liver, Mouse INTRODUCTION vivo, especially at early time points, have not been clari- fied. In this study, we used DNA microarray to examine The human body contains about 1-2 mg of cobalt, a the gene expression profiles in the liver of mice injected trace element essential for human health as a compo- with CoCl2. nent of vitamin B12. On the other hand, excessive expo- sure to cobalt has been reported to induce asthma, pneu- MATERIALS AND METHODS monia, hematological abnormalities, cardiomyopathy, and carcinogenicity in humans (Kesteloot et al., 1968; Heath, Treatment of animals 1956). LD50 of CoCl2 in various animals fell in a range Five-week-old male ICR mice were purchased from between 150 and 500 mg/kg. A high concentration of Japan SLC Co. (Shizuoka, Japan) and housed in plastic cobalt can induce cell death by apoptosis and necrosis in cages at 23-24°C with a 12-hr light and dark cycle. The many types of cells. The mechanisms involved in cobalt- mice were given free access to a commercial diet and tap induced apoptosis include DNA fragmentation, activa- water. All animal experiments were performed according tion of caspases, increased production of reactive oxygen to protocols approved by the Animal Care and Use Com- species, augmented phosphorylation of mitogen activated mittee of Tokushima Bunri University. Mice were subcu- protein kinases, and elevated levels of p53 in cells (Zou taneously injected with CoCl2 at a dose of 300 µmol/kg, et al., 2001, 2002; Yang et al., 2004). It is well-known and sacrificed 0.5, 1, and 3 hr after CoCl2 treatment. Con- that CoCl2 can mimic hypoxic conditions and, conse- trol mice were given saline. Tissue samples of liver were quently, activate hypoxia inducible factor-1 (Kalpana collected under anesthesia, immediately immersed in liq- et al., 2008). In addition, CoCl2 induces heme oxygenase uid nitrogen, and stored at -80°C. 1, which is a rate-limiting enzyme in the catabolism of heme and protects against hepatic ischemia/reperfusion Preparation of RNAs for DNA microarray analysis injury (Badr et al., 1999; Tom et al., 1996; Glanemann et Total RNA was extracted and purified from the fro- al., 2005). Thus, cobalt plays multi-functional roles in the zen liver tissue samples using the SV Total RNA Isolation body at nutritional, physiological, pharmacological, and System (Promega, Madison, WI, USA). Pooled total RNA toxicological levels. Although cobalt is involved in a vari- (5 µg) was applied to OpArrayTM Mouse V4.0 (Operon ety of biological events in the body, the effects of CoCl2 Technologies, Alameda, CA, USA), in which 28,800 administration on the whole profiles of gene expression in genes are registered. We used the Low RNA Fluorescent Correspondence: Seiichiro Himeno (E-mail: [email protected]) Vol. 35 No. 6 936 K. Matsumoto et al. Linear Amp Kit PLUS (Agilent Technologies Inc., San- ly, after CoCl2 administration. The genes showing ratios ta Clara, CA, USA) to synthesize double-stranded (ds) (CoCl2-treated to control) of 2.5 or higher are listed as up- cDNA from the total RNA and then to synthesize cRNA regulated genes, and those with ratios of 0.4 or less were from the ds cDNA as a template. Total RNA was reverse listed as down-regulated genes. Administration of CoCl2 transcribed with the T7 Promoter primer. After second- up-regulated the expression of 14, 27, and 32 genes, and strand synthesis, we used the ds cDNA as a template for down-regulated the expression of 5, 19, and 25 genes in Cyanine (Cy) 3/Cy5-labeled cRNA synthesis. cRNA from the liver at 0.5, 1, and 3 hr, respectively. Of the 14 genes the control group was labeled with Cy3 and that from the enhanced at 0.5 hr after CoCl2 treatment, 7 (Isg15, Ifi27, CoCl2-injected group was labeled with Cy5. The OpAr- Pbef1, Oas1g, Igtp, Gbp9, Psmb9) are related to immuno- ray slide was hybridized with 20-25 µg of Cy3- and logical responses. Especially, Isg15, Ifi27, Igtp, and Gbp9 Cy5-labeled cRNA competitively and washed with the are involved in the activation of interferon (Broering OpArray Hybridization Buffer Kit (Operon). A fluores- et al., 2010; Asselah et al., 2008; Taylor et al., 2000). The cent image of the OpArray slide was then recorded with expression of these four genes remained high up to 3 hr CRBIO (Hitachi Software Engineering, Tokyo, Japan). after CoCl2 treatment. In addition, the expression of glu- The digitized image data were processed with DNA- tathione S-transferase α2 (Gsta2) was up-regulated at all SIS Array software (Hitachi Software Engineering). The the time points from 0.5 to 3 hr after CoCl2 treatment. ratios of the intensity of Cy5 to that of Cy3 were calcu- On the other hand, there were no persistent changes in lated. Information on each gene on the slide was obtained the expression of down-regulated genes. The expression from the National Center for Biotechnology Information of metallothionein I (Mt1) was low at early time points (NCBI) database (Honda et al., 2010). after CoCl2 treatment (0.33 at 0.5 hr and 0.27 at 1 hr), although its expression should be examined further at lat- RESULTS AND DISCUSSION er time points since cobalt is known to induce the synthe- sis of metallothionein (Kalpana et al., 2008). Tables 1, 2, and 3 show the gene expression profiles In a preliminary study, we found that administration of the liver of mice examined 0.5, 1, and 3 hr, respective- of CoCl2 to mice induced the expression of a few genes Table 1. Changes in gene expression in the liver of mice treated with cobalt chloride. (0.5 hr after administration) Name of gene Definition Accession No. Ratio Up-regulated gene ( > 2.5) Isg15 ISG15 ubiquitin-like modifier NM_015783 5.92 Gsta2 glutathione S-transferase, alpha 2 NM_008182 3.43 Ifi27 interferon, alpha-inducible protein 27 NM_029803 3.30 Pbef1 pre-B-cell colony-enhancing factor 1 NM_021524 3.30 Oas1g 2’-5’ oligoadenylate synthetase 1G NM_011852 3.29 Bst2 Mus musculus bone marrow stromal cell antigen 2 NM_198095 3.27 Sfrs12ip1 SFRS12-interacting protein 1 NM_026075 3.05 Fbxo39 F-box protein 39 XM_282966 2.95 Rcc2 regulator of chromosome condensation 2 NM_173867 2.73 Igtp interferon gamma induced GTPase NM_018738 2.69 Dnajb9 DnaJ (Hsp40) homolog, subfamily B, member 9 NM_013760 2.67 Gbp9 guanylate-binding protein 9 NM_172777 2.65 Lyplal1 lysophospholipase-like 1 NM_146106 2.57 Psmb9 proteosome (prosome, macropain) subunit, beta type 9 NM_013585 2.52 Down-regulated gene ( < 0.4) Mt1 metallothionein 1 NM_013602 0.33 Caskin1 CASK interacting protein 1 NM_027937 0.33 Tmem12 transmembrane protein 12 NM_178669 0.36 Rbm21 RNA binding motif protein 21 NM_197993 0.39 Dusp6 dual specificity phosphatase 6 NM_026268 0.39 Vol. 35 No. 6 937 Hepatic gene expression in mice after cobalt chloride injection Table 2. Changes in gene expression in the liver of mice treated with cobalt chloride. (1 hr after administration) Name of gene Definition Accession No. Ratio Up-regulated gene ( > 2.5) Kcnab1 potassium voltage-gated channel, shaker-related subfamily, NM_010597 6.84 beta member 1 Txnl4 thioredoxin-like 4 transcript variant 2 NM_178604 5.35 Oas1g 2’-5’ oligoadenylate synthetase 1G NM_011852 4.95 Gsta2 glutathione S-transferase, alpha 2 NM_008182 4.13 Fam108b family with sequence similarity 108, member B NM_146096 4.09 Akap13 anchor protein 13, transcript variant 1 XM_133543 3.82 Fez2 fasciculation and elongation protein zeta 2 NM_199448 3.73 Por P450 (cytochrome) oxidoreductase NM_008898 3.73 Isg15 ISG15 ubiquitin-like modifier NM_015783 3.64 Oxnad1 oxidoreductase NAD-binding domain containing 1 NM_145460 3.42 Klk5 kallikrein related-peptidase 5 NM_026806 3.18 Tmem51 transmembrane protein 51 NM_145402 3.17 Cml4 camello-like 4 NM_023455 3.17 Fbxo39 F-box protein 39 XM_282966 3.12 Ifi27 interferon, alpha-inducible protein 27 NM_029803 3.11 Pno1 partner of NOB1 homolog NM_025443 3.08 Samd8 sterile alpha motif domain containing 8 NM_026283 3.08 Zbp1 Z-DNA binding protein 1 NM_021394 3.03 Gbp9 guanylate-binding protein 9 NM_172777 2.85 Mtfr1 mitochondrial fission regulator 1 NM_026182 2.85 Ifi27l1 interferon, alpha-inducible protein 27 like 1 NM_194067 2.84 Nubpl nucleotide binding protein-like NM_029760 2.77 Mum1 melanoma associated antigen NM_023431 2.64 Ogfr opioid growth factor receptor NM_031373 2.59 Ift52 intraflagellar transport 52 homolog NM_172150 2.57 Papola poly (A) polymerase alpha NM_011112 2.55 Acd adrenocortical dysplasia NM_008739 2.52 Down-regulated