The Journal of Toxicological Sciences (J. Toxicol. Sci.) 173 Vol.39, No.1, 173-177, 2014

Toxicomics Report expression differences in the duodenum of 129/Sv and DBA/2 mice compared with that of C57BL/6J mice

Shunji Imai1,2, Maki Tokumoto1,3, Yasuyuki Fujiwara1, Akiko Honda1,4, Tatsuya Hasegawa5, Yoshiyuki Seko5, Jin-Yong Lee1, Hisamitsu Nagase2 and Masahiko Satoh1

1Laboratory of Pharmaceutical Health Sciences, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650, Japan 2Laboratory of Hygienic Chemistry and Molecular Toxicology, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan 3Laboratory of Chemical Toxicology and Environmental Health, Showa Pharmaceutical University, 3-3165 Higashi-Tamagawagakuen, Machida 194-8543, Japan 4Environmental Health Division, Department of Environmental Engineering, Graduate School of Engineering, Kyoto University, C Cluster, Kyoto-Daigaku-Katsura, Nishikyo-ku, Kyoto 615-8540, Japan 5Department of Environmental Biochemistry, Yamanashi Institute of Environmental Sciences, 5597-1 Kenmarubi, Kamiyoshida, Fujiyoshida 403-0005, Japan

(Received November 1, 2013; Accepted November 11, 2013)

ABSTRACT — We compared the cadmium (Cd) concentration in the liver and kidney of different strains of mice after exposure to 50 ppm Cd for 30 days via drinking water. Cd concentration in the liver and kid- ney of C57BL/6J mice were higher than those of 129/Sv and DBA/2 mice. Since orally ingested heavy metals are absorbed in the small intestine and then widely distributed to target tissues, microarray analy- ses were performed to compare the expression levels of transport-related in the duodenum between C57BL/6J mice and 129/Sv or DBA/2 mice. The expression levels of 9 and 11 genes were elevated more than 2.0-fold and 13 and 12 genes were reduced less than 0.5-fold in 129/Sv mice and DBA/2 mice, respectively. Among these low expressed genes, 10 genes (Slc2a2, Slc5a1, Slc16a2, Slc22a13, Slc22a18, Slc25a11, Slc36a1, Slco6c1, Abca3 and Abcd1) were common between the two types of strains. These results suggest that some of those genes might be involved in Cd absorption and its toxicity.

Key words: Cadmium, DNA microarray, Duodenum, Transport-related gene

INTRODUCTION ence the expression of Cd toxicity and Cd tissue distribu- tion (Hata et al., 1980; King et al., 1998, 1999; Habeebu Cadmium (Cd) is a ubiquitous environmental pollutant et al., 2001). Therefore, we used the DNA microarray that causes toxicity in multiple organs, including the kid- analysis method, which is well established for obtaining ney and liver (Järup et al., 1998; Zalups and Ahmad, 2003; gene expression data (Hwang et al., 2011; Tokumoto et Tokumoto et al., 2011a). Cd-contaminated food is the one al., 2011b, 2013; Lee et al., 2013), to compare the expres- of major sources of Cd exposure. Previous studies dem- sion pattern of transport-related genes in the duodenum of onstrated that several intestinal transporters, such as diva- C57BL/6J, 129/Sv and DBA/2 mice following compari- lent metal transporter 1, calcium transporter 1, Zrt-, Irt-re- son of Cd tissue accumulation, with the aim of identify- lated 8 (ZIP8) and ZIP14, play an important role ing new candidate genes involved in Cd absorption and in intestinal Cd absorption (Park et al., 2002; Ryu et al., its toxicity. 2004; Min et al., 2008; Dalton et al., 2005; He et al., 2006; Girijashanker et al., 2008; Liu et al., 2008). However, it MATERIALS AND METHODS is possible that there are some unknown Cd transporters that correlate with Cd body burden. Animals and treatment Mouse strain differences have been reported to influ- Seven-week-old female C57BL/6J, 129/Sv and

Correspondence: Masahiko Satoh (E-mail: [email protected])

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DBA/2 mice were purchased from Japan SLC RNA was extracted and purified from mouse duodenum (Shizuoka, Japan). Mice were housed in cages in an ani- using the SV Total RNA Isolation System (Promega, mal room at a controlled temperature of 23 ± 1°C with Madison, WI, USA). Purified total RNA (100 μg) from relative humidity, and a 12-hr light/dark cycle accord- the intestine of 5 mice was reverse transcribed by Super- ing to the vivarium of the laboratory animal facility of script II reverse transcriptase with Oligo (dT)12-18 prim- Gifu Pharmaceutical University. Eight-week-old mice er and dNTPmix, which contains Aminoallyl-dUTP (Life were fed standard laboratory chow ad libitum, and sep- Technologies, Carlsbad, CA, USA). Complementary DNA arated into the control group (n = 5) and Cd-exposed (cDNA) from C57BL/6J mice was labeled with fluores- group (n = 5). Mice in the Cd-treated group were pro- cent dye Cyanine (Cy) 3-labeled nucleotide, and cDNAs vided CdCl2 (50 ppm Cd) as drinking water for 30 days. from 129/Sv and DBA/2 mice were labeled with Cy5-la- After Cd exposure, mice were sacrificed by ether anesthe- beled nucleotide (GE Healthcare, Buckingham, UK). The sia, and the liver and kidney were removed from each Ace Gene Oligo Chip 30K DNA microarray slide (DNA mouse for analysis of Cd concentration. Separately, non- Chip Research Inc., Kanagawa, Japan), which contains treated 8-week-old C57BL/6J, 129/Sv and DBA/2 mice 30,000 spots of mouse oligo DNA, was used for the tar- (n = 5, each group) were sacrificed by ether anesthesia get gene search. The mixture of the labeled cDNAs from and the duodenum was removed from each mouse for the C57BL/6J and 129/Sv mice was hybridized with the DNA microarray analysis. DNA probes on the array for 16 hr at 42°C using a Luci- dea SlidePro Hybridizer (GE Healthcare). The mixture of Determination of Cd concentration the labeled cDNAs from the C57BL/6J and DBA/2 mice The tissues were digested with nitric acid and hydro- was also hybridized with the DNA probes on the array. gen peroxide, and inorganic residues were dissolved A fluorescent image of slide was recorded with CRBIO in ultrapure water. Metal analysis was carried out using (Hitachi Software Engineering, Tokyo, Japan). The dig- inductively coupled plasma-mass spectrometry (ICP-MS; itized image data were processed with DNASIS Array Agilent Technologies, Santa Clara, CA, USA). software (Hitachi Software Engineering). After global normalization, the data were filtered to exclude genes DNA microarray analysis with low expression levels. The ratio of the intensity of RNA samples for microarray analysis were prepared Cy5 to that of Cy3 was then calculated based on these sig- as reported previously (Tokumoto et al., 2011b). Total nals. Information on each gene on the slide was obtained

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Fig. 1. Cd concentrations in the liver and kidney of C57BL/6J, 129/Sv and DBA/2 mice after exposure to Cd for 30 days. Values are means ± S.D. for 5 mice. *Significantly different from C57BL/6J mice (p < 0.05).

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Table 1. Comparison of gene expression patterns between C57BL/6J and 129/Sv mice. Gene symbol Accession No. Ratio to C57BL/6J mice Low expression genes (< 0.5-fold) 1. Slc2a2 (facilitated glucose transporter, Glut2) NM_031197 0.18 2. Slc5a1 (/glucose , Sglt1) NM_019810 0.34 3. Slc16a2 (monocarboxylic acid transporter, Mct8) NM_009197 0.28 4. Slc22a13 (organic cation transporter, Orctl3) NM_133980 0.13 5. Slc22a18 (organic cation transporter, Orctl2) NM_008767 0.26 6. Slc24a2 (sodium/potassium/calcium exchanger) NM_172426 0.34 7. Slc25a11 ( oxoglutarate carrier) NM_024211 0.12 8. Slc26a3 () NM_021353 0.35 9. Slc36a1 (proton/amino acid symporter, Pat1) NM_153139 0.48 10. Slc39a6 (metal transporter, Zip6) NM_139143 0.35 11. Slco6c1 (organic anion transporter) NM_028942 0.13 12. Abca3 (ABC1) NM_013855 0.31 13. Abcd1 (ALD) NM_007435 0.41

High expression genes (> 2.0-fold) 1. Slc2a8 (facilitated , Glut8) NM_019488 2.08 2. Slc4a1 (anion exchanger, Ae1) NM_011403 2.12 3. Slc6a4 (neurotransmitter transporter, serotonin) NM_010484 5.21 4. Slc6a15 (neurotransmitter transporter) NM_175328 2.70 5. Slc9a1 (sodium/hydrogen exchanger) NM_016981 2.69 6. Slc12a8 (potassium/chloride transporters) NM_134251 4.42 7. Slc14a2 () NM_030683 3.20 8. Slc22a6 (organic anion transporter, Oat1) NM_008766 5.15 9. Abcg4 (WHITE) NM_138955 2.71 The transport-related genes in the duodenum of 129/Sv mice were compared to those of C57BL/6J mice. The genes for which the levels of expression changed less than 0.5-fold and more than 2-fold are listed here.

from the National Center for Biotechnology Information Cd concentrations in the tissues of 129/Sv and DBA/2 (NCBI) database. mice were significantly lower than those in C57BL/6J mice. These results suggest that the absorption efficien- Statistical analysis cies of Cd from the intestine in 129/Sv and DBA/2 mice Data of Cd concentration was represented as mean and are lower than that of C57BL/6J mice. standard deviation (SD). The statistical significance of the We next compared the gene expression patterns in the data was analyzed by Student’s t-test. p < 0.05 was con- duodenum of untreated C57BL/6J, 129/Sv and DBA/2 sidered significant. mice. DNA microarray data demonstrated that 396 and 441 genes showed a more than 2.0-fold increase in 129/Sv RESULTS AND DISCUSSION and DBA/2 mice, respectively, while 778 and 770 showed a less than 0.5-fold decrease in 129/Sv and DBA/2 mice, Figure 1 shows the Cd concentrations in the liver and respectively (data not shown). Among the upregulated kidney of C57BL/6J, 129/Sv and DBA/2 mice after expo- genes, 9 and 11 transport-related genes, which encode the sure to Cd (50 ppm) for 30 days via drinking water. Intake solute carrier (Slc) or ATP-binding cassette (ABC) trans- of water during experiment term was not affected by Cd. porters, were highly expressed in 129/Sv and DBA/2

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Table 2. Comparison of gene expression patterns between C57BL/6J and DBA/2 mice. Gene symbol Accession No. Ratio to C57BL/6J mice Low expression genes (< 0.5-fold) 1. Slc2a2 (facilitated glucose transporter, Glut2) NM_031197 0.25 2. Slc5a1 (sodium/glucose cotransporter, Sglt1) NM_019810 0.47 3. Slc11a1 (proton-coupled divalent metal ion transporters, Nramp1) NM_013612 0.48 4. Slc16a2 (monocarboxylic acid transporter, Mct8) NM_009197 0.43 5. Slc22a13 (organic cation transporter, Orctl3) NM_133980 0.20 6. Slc22a18 (organic cation transporter, Orctl2) NM_008767 0.17 7. Slc25a11 (mitochondrial carrier oxoglutarate carrier) NM_024211 0.20 8. Slc34a1 (sodium/phosphate cotransporter) NM_011392 0.49 9. Slc36a1 (proton/amino acid symporter, Pat1) NM_153139 0.28 10. Slco6c1 (organic anion transporter) NM_028942 0.14 11. Abca3 (ABC1) NM_013855 0.22 12. Abcd1 (ALD) NM_007435 0.39

High expression genes (> 2.0-fold) 1. Slc2a9 (facilitated glucose transporter, Glut9) NM_145559 2.29 2. Slc6a4 (neurotransmitter transporter, serotonin) NM_010484 4.94 3. Slc6a15 (neurotransmitter transporter) NM_175328 2.72 4. Slc7a2 (cationic amino acid transporter, y+ system) NM_007514 2.07 5. Slc9a1 (sodium/hydrogen exchanger) NM_016981 2.01 6. Slc12a8 (potassium/chloride transporters) NM_134251 3.56 7. Slc14a2 (urea transporter) NM_030683 2.72 8. Slc20a1 (phosphate transporter) NM_015747 2.36 9. Slc22a6 (organic anion transporter, Oat1) NM_008766 6.19 10. Slc25a37 (mitochondrial iron transporter, Mfm1) NM_026331 2.03 11. Abcg4 (WHITE) NM_138955 2.72 The transport-related genes in the duodenum of DBA/2 mice were compared to those of C57BL/6J mice. The genes for which the levels of expression changed less than 0.5-fold and more than 2-fold are listed here. mice, respectively (Tables 1 and 2). Conversely, 13 and these genes may be key determinants of individual dif- 12 transport-related genes were expressed at low levels in ferences in Cd toxicity and may also be involved in the 129/Sv and DBA/2 mice, respectively (Tables 1 and 2). transport of not only Cd but also other metals and chem- Among these low expressed transport-related genes, 10 icals. Although further characterization and comparison genes (Slc2a2, Slc5a1, Slc16a2, Slc22a13, Slc22a18, are required, the present study provides initial clues for Slc25a11, Slc36a1, Slco6c1, Abca3 and Abcd1) were com- the identification of novel metal- and/or chemical-trans- mon between 129/Sv and DBA/2 mice. A previous study porters in the small intestine. showed that Slc5a1 gene expression was downregulated by Cd (Kothinti et al., 2010). To our knowledge, the rela- ACKNOWLEDGMENTS tionship between Cd and the other remaining genes has not yet been reported. This work was partly supported by the Study of the The present study suggests that some of these trans- Health Effects of Heavy Metals Organized by the Minis- port-related genes might be involved in Cd absorption try of the Environment, Japan (to M.S.). and tissue accumulation. In addition, it is possible that

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REFERENCES (2010): Cadmium down-regulation of kidney Sp1 binding to mouse SGLT1 and SGLT2 gene promoters: possible reaction Dalton, T.P., He, L., Wang, B., Miller, M.L., Jin, L., Stringer, K.F., of cadmium with the zinc finger domain of Sp1. Toxicol. Appl. Chang, X., Baxter, C.S. and Nebert, D.W. (2005): Identification Pharmacol., 244, 254-262. of mouse SLC39A8 as the transporter responsible for cadmium- Liu, Z., Li, H., Soleimani, M., Girijashanker, K., Reed, J.M., He, 2+ 2+ induced toxicity in the testis. Proc. Natl. Acad. Sci. USA, 102, L., Dalton, T.P. and Nebert, D.W. (2008) Cd versus Zn uptake by the ZIP8 HCO -dependent symporter: kinetics, electrogenici- 3401-3406. 3 Girijashanker, K., He, L., Soleimani, M., Reed, J.M., Li, H., Liu, ty and trafficking. Biochem. Biophys. Res. Commun., 365, 814- Z., Wang, B., Dalton, T.P. and Nebert, D.W. (2008): Slc39a14 820. gene encodes ZIP14, a metal/bicarbonate symporter: similarities Lee, J.Y., Tokumoto, M., Fujiwara, Y. and Satoh, M. (2013): Gene to the ZIP8 transporter. Mol. Pharmacol., 73, 1413-1423. expression analysis using DNA microarray in HK-2 human Habeebu, S.S., Liu, Y., Park, J.D. and Klaassen, C.D. (2001): Strain proximal tubular cells treated with cadmium. J. Toxicol. Sci., 38, differences in the toxicity of cadmium to trigeminal ganglia in 959-962. mice. Toxicol. Appl. Pharmacol., 177, 200-207. Min, K.S., Ueda, H., Kihara, T. and Tanaka, K. (2008): Increased Hata, A., Tsunoo, H., Nakajima, H., Shintaku, K. and Kimura, M. hepatic accumulation of ingested Cd is associated with upregu- (1980): Acute cadmium intoxication in inbred mice: a study on lation of several intestinal transporters in mice fed diets deficient strain differences. Chem. Biol. Interact., 32, 29-39. in essential metals. Toxicol. Sci., 106, 284-289. He, L., Girijashanker, K., Dalton, T.P., Reed, J., Li, H., Soleimani, Park, J.D., Cherrington, N.J. and Klaassen, C.D. (2002): Intestinal M. and Nebert, D.W. (2006): ZIP8, member of the solute-car- absorption of cadmium is associated with divalent metal trans- rier-39 (SLC39) metal-transporter family: characterization of porter 1 in rats. Toxicol. Sci., 68, 288-294. transporter properties. Mol. Pharmacol., 70, 171-180. Ryu, D.Y., Lee, S.J., Park, D.W., Choi, B.S., Klaassen, C.D. and Hwang, G.W., Lee, J.Y., Ryoke, K., Matsuyama, F., Kim, J.M., Park J.D. (2004): Dietary iron regulates intestinal cadmium Takahashi, T. and Naganuma, A. (2011): Gene expression pro- absorption through iron transporters in rats. Toxicol. Lett., 152, filing using DNA microarray analysis of the cerebellum of mice 19-25. treated with methylmercury. J. Toxicol. Sci., 36, 389-391. Tokumoto, M., Fujiwara, Y., Shimada, A., Hasegawa, T., Seko, Y., Järup, L., Berglund, M., Elinder, C.G., Nordberg, G. and Vahter, M. Nagase, H. and Satoh, M. (2011a): Cadmium toxicity is caused (1998): Health effects of cadmium exposure-- a review of the lit- by accumulation of p53 through the down-regulation of Ube2d erature and a risk estimate. Scand. J. Work Environ. Health, 24, family genes in vitro and in vivo. J. Toxicol. Sci., 36, 191-200. 1-51. Tokumoto, M., Lee, J.Y., Fujiwara, Y. and Satoh, M. (2013): DNA King, L.M., Anderson, M.B., Sikka, S.C. and George, W.J. (1998): microarray expression analysis of mouse kidney following cad- Murine strain differences and the effects of zinc on cadmium mium exposure for 12 months. J. Toxicol. Sci., 38, 799-802. concentrations in tissues after acute cadmium exposure. Arch. Tokumoto, M., Ohtsu, T., Honda, A., Fujiwara, Y., Nagase, H. and Toxicol., 72, 650-655. Satoh, M. (2011b): DNA microarray analysis of normal rat kid- King, L.M., Banks, W.A. and George, W.J. (1999): Differences in ney epithelial cells treated with cadmium. J. Toxicol. Sci., 36, cadmium transport to the testis, epididymis, and brain in cad- 127-129. mium-sensitive and -resistant murine strains 129/J and A/J. J. Zalups, R.K. and Ahmad, S. (2003): Molecular handling of cadmi- Pharmacol. Exp. Ther., 289, 825-830. um in transporting epithelia. Toxicol. Appl. Pharmacol., 186, Kothinti, R.K., Blodgett, A.B., Petering, D.H. and Tabatabai, N.M. 163-188.

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