DNA Microarray Analysis of the Liver of Mice Treated with Cobalt Chloride Kanako Matsumoto1, Hitomi Fujishiro1, Masahiko Satoh2 and Seiichiro Himeno1

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-regulated 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])

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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 synthesis 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

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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 gene ( < 0.4) Dusp6 dual specificity phosphatase 6 NM_026268 0.21 Arl4a ADP-ribosylation factor-like 4A, transcript variant 1 NM_007487 0.21 Igfbp1 insulin-like growth factor binding protein 1 NM_008341 0.24 Tubb2a tubulin, beta 2a - 0.25 Mt1 Metallothionein 1 NM_013602 0.27 Tubb2a tubulin, beta 2a NM_009450 0.30 Dscr1 Down syndrome critical region homolog 1 NM_019466 0.31 Hbb-b1 hemoglobin, beta adult major chain NM_008220 0.34 Gadd45g growth arrest and DNA-damage-inducible 45 gamma NM_011817 0.34 Ocel1 occludin/ELL domain containing 1 XM_134253 0.34 Cyp2b10 cytochrome P450, family 2, subfamily b, polypeptide 10 NM_009999 0.35 Clpx caseinolytic peptidase X NM_011802 0.35 Zfp277 zinc finger protein 277 NM_172575 0.35 Adamts1 a disintegrin-like and metallopeptidase with thrombospondin NM_009621 0.36 type 1 motif Creld2 cysteine-rich with EGF-like domains 2 NM_029720 0.36 Dio1 deiodinase, iodothyronine, type I NM_007860 0.37 Txnip thioredoxin interacting protein NM_023719 0.38 Btg1 B-cell translocation gene 1, anti-proliferative NM_007569 0.38 Col3a1 procollagen, type III, alpha 1 NM_009930 0.40

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Table 3. Changes in gene expression in the liver of mice treated with cobalt chloride. (3 hr after administration) Name of gene Definition Accession No. Ratio Up-regulated gene ( > 2.5) Samd9l sterile alpha motif domain containing 9-like NM_010156 6.48 Cyfip1 cytoplasmic FMR1 interacting protein 1 NM_011370 5.67 Isg15 ISG15 ubiquitin-like modifier NM_015783 5.53 Arhgap29 Rho GTPase activating protein 29 NM_172525 4.76 Gsta2 glutathione S-transferase, alpha 2 NM_008182 4.63 Taok3 TAO kinase 3 NM_001081308 4.19 Clec2h C-type lectin domain family 2, member h NM_053165 4.06 Gpr98 G protein-coupled receptor 98 NM_054053 3.60 Rps6ka1 ribosomal protein S6 kinase polypeptide 1 NM_009097 3.55 Hspa1a heat shock protein 1A NM_010479 3.54 Mrps28 mitochondrial ribosomal protein S28 NM_025434 3.54 Ogfr opioid growth factor receptor NM_031373 3.51 Gbp9 guanylate-binding protein 9 NM_172777 3.41 Phf17 PHD finger protein 17 NM_172303 3.29 Brd7 bromodomain containing 7 NM_012047 3.28 Fam108b family with sequence similarity 108, member B NM_146096 3.12 Inca1 inhibitor of CDK, cyclin A1 interacting protein 1 NM_213729 3.11 Snap47 synaptosomal-associated protein, 47 NM_144521 3.09 Zc3h3 zinc finger CCCH type containing 3 NM_172121 3.03 Chac1 ChaC, cation transport regulator-like 1 NM_026929 3.00 Iigp2 interferon inducible GTPase 2 NM_019440 2.92 Ifi27 interferon, alpha-inducible protein 27 NM_029803 2.90 Fdps farnesyl diphosphate synthetase NM_134469 2.86 Igtp interferon gamma induced GTPase NM_018738 2.80 Hcn3 hyperpolarization-activated, cyclic nucleotide-gated K+ 3 NM_008227 2.79 Tmem14c transmembrane protein 14C NM_025387 2.77 H6pd hexose-6-phosphate dehydrogenase (glucose 1-dehydroge- NM_173371 2.77 nase) Ifi47 interferon gamma inducible protein 47 NM_008330 2.73 Smarcd2 SWI/SNF related, matrix associated, NM_031878 2.72 actin dependent regulator of chromatin, subfamily d, member 2 Ppil2 peptidylprolyl isomerase (cyclophilin)-like 2 NM_144954 2.71 Eftud1 elongation factor Tu GTP binding domain containing 1 NM_175317 2.70 Rcc2 regulator of chromosome condensation 2 NM_173867 2.70 Pacs2 phosphofurin acidic cluster sorting protein 2 NM_001081170 2.67 Ucp1 uncoupling protein 1 (mitochondrial, proton carrier) NM_009463 2.67 Ube3a ubiquitin protein ligase E3A NM_011668 2.66 Sec24c SEC24 related gene family, member C NM_172596 2.65 Bms1l BMS1-like, ribosome assembly protein NM_194339 2.62 Ctbp1 C-terminal binding protein 1 NM_013502 2.56 Lrrc35 leucine rich repeat containing 35 NM_173038 2.55 Esf1 nucleolar pre-rRNA processing protein, homolog XM_130548 2.55 Gltpd1 glycolipid transfer protein domain containing 1 NM_024472 2.54 Gstp2 glutathione S-transferase, pi 2 NM_181796 2.52 Down-regulated gene ( < 0.4) Cyp2b10 cytochrome P450, family 2, subfamily b, polypeptide 10 NM_009999 0.15 Dhrs2 dehydrogenase/reductase member 2 NM_027790 0.16 Zfp277 zinc finger protein 277 NM_172575 0.18 Ren2 renin 2 tandem duplication of Ren1 NM_031193 0.18 Hao3 hydroxyacid oxidase (glycolate oxidase) 3 NM_019545 0.19 Xlr3b X-linked lymphocyte-regulated 3B NM_011727 0.22 Ccdc21 coiled-coil domain containing 21 NM_144527 0.25 Olfr826 olfactory receptor 826 NM_146676 0.25 Slc24a4 solute carrier family 24 , member 4 NM_172152 0.25 Tfrc transferrin receptor NM_011638 0.25

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Table 3. (Continued) Name of gene Definition Accession No. Ratio Agxt alanine-glyoxylate aminotransferase NM_016702 0.26 Marcksl1 MARCKS-like 1 NM_010807 0.26 Itprip inositol 1,4,5-triphosphate receptor interacting protein NM_001001738 0.28 Macc1 metastasis associated in colon cancer 1 XM_138397 0.28 Tas2r115 taste receptor, type 2, member 115 NM_207020 0.28 Slco1a4 solute carrier organic anion transporter family, member 1a4 NM_030687 0.29 Olfr1462 olfactory receptor 1462 NM_146693 0.30 Hist2h3c2 histone cluster 2, H3c2 NM_054045 0.32 Bcl11a Immunoglobulin heavy chain V gene segment NM_016707 0.33 Olfr143 olfactory receptor 143 NM_146806 0.37 Crygd crystallin, gamma D NM_007776 0.38 Lsm6 LSM6 homolog, U6 small nuclear RNA associated NM_030145 0.39 Ptp4a1 protein tyrosine phosphatase 4a1 NM_011200 0.39 Bnip3l BCL2/adenovirus E1B interacting protein 3-like NM_009761 0.39 Acsl5 acyl-CoA synthetase long-chain family member 5 XM_484816 0.40

involved in immunological responses at early time points cobalt chloride. Toxicol. Appl. Pharmacol., 231, 354-363. such as 1 hr. The data on microarray analyses shown here Kesteloot, H., Roelandt, J., Willems, J., Claes, J.H. and Joossens, J.V. (1968): An enquiry into the role of cobalt in the heart dis- also demonstrated that the genes related to immunologi- ease of chronic beer drinkers. Circulation, 37, 854-864. cal responses were enhanced as early as 0.5 hr after CoCl2 Heath, J.C. (1956): The production of malignant tumours by cobalt administration. The results of the present study may lead in the rat. Br. J. Cancer, 10, 668-673. to a better understanding of the physiological, pharmaco- Honda, A., Komuro, H., Nagase, H., Hozumi, I., Inuzuka, T., Hara, logical and toxicological actions of cobalt compounds. H., Fujiwara, Y. and Satoh, M. (2010): Microarray analysis of the liver in metallothionein-III null mice treated with cadmium. J. Toxicol. Sci., 35, 271-273 ACKNOWLEDGMENT Taylor, G.A., Collazo, C.M., Yap, G.S., Nguyen, K., Gregorio, T.A., Taylor, L.S., Eagleson, B., Secrest, L., Southon, E.A., Reid, This work was partly supported by a Grant-in-Aid for S.W., Tessarollo, L., Bray, M., McVicar, D.W., Komschlies, Scientific Research (B-19390169) from Japan Society for K.L., Young, H.A., Biron, C.A., Sher, A. and Vande Woude, G.F. (2000): Pathogen-specific loss of host resistance in mice lack- the Promotion of Science. ing the IFN-gamma-inducible gene IGTP. Proc. Natl. Acad. Sci. USA, 97, 751-755. REFERENCES Tom, D.J., Rodgers, P.A., Shokoohi, V., Stevenson, D.K. and Dennery, P.A. (1996): Hepatic heme oxygenase is inducible in Asselah, T., Bieche, I., Narguet, S., Sabbagh, A., Laurendeau, I., neonatal rats during the early postnatal period. Pediatr. Res., 40, Ripault, M.P., Boyer, N., Martinot-Peignoux, M., Valla, D., 288-293. Vidaud, M. and Marcellin, P. (2008): Liver gene expression sig- Yang, S.J., Pyen, J., Lee, I., Lee, H., Kim, Y. and Kim, T. (2004): nature to predict response to pegylated interferon plus ribavirin Cobalt chloride-induced apoptosis and extracellular signal-regu- combination therapy in patients with chronic hepatitis C. Gut, lated protein kinase 1/2 activation in rat C6 glioma cells. J. Bio- 57, 516-524. chem. Mol. Biol., 37, 480-486. Badr, G.A., Zhang, J.Z., Tang, J., Kern, T.S. and Ismail-Beigi, F. Zou, W., Yan, M., Xu, W., Huo, H., Sun, L., Zheng, Z. and Liu, (1999): Glut1 and glut3 expression, but not capillary density, is X. (2001): Cobalt chloride induces PC12 cells apoptosis through increased by cobalt chloride in rat cerebrum and retina. Brain reactive oxygen species and accompanied by AP-1 activation. J. Res. Mol. Brain Res., 64, 24-33. Neurosci. Res., 64, 646-653. Broering, R., Zhang, X., Kottilil, S., Trippler, M., Jiang, M., Lu, M., Zou, W., Zeng, J., Zhuo, M., Xu, W., Sun, L., Wang, J. and Liu, Gerken, G. and Schlaak, J.F. (2010): The interferon stimulated X. (2002): Involvement of caspase-3 and p38 mitogen-activat- gene 15 functions as a proviral factor for the hepatitis C virus ed protein kinase in cobalt chloride-induced apoptosis in PC12 and as a regulator of the IFN response. Gut, 59, 1111-1119. cells. J. Neurosci. Res., 67, 837-843. Glanemann, M., Schirmeier, A., Lippert, S., Langrehr, J.M., Neuhaus, P. and Nussler, A.K. (2005): Cobalt-protoporphyrin induced heme oxygenase overexpression and its impact on liver regeneration. Transplant. Proc., 37, 3223-3225. Kalpana, S., Dhananjay, S., Anju, B., Lilly, G. and Sai Ram, M. (2008): Cobalt chloride attenuates hypobaric hypoxia induced vascular leakage in rat brain: molecular mechanisms of action of

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