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Metallothionein expression and localization in rat bone tissue after cadmium injection
Toxicology Letters 123 (2001) 143– 150 www.elsevier.com/locate/toxlet
Metallothionein expression and localization in rat bone tissue after cadmium injection N. Oda a, C.A. Sogawa a,*, N. Sogawa a, K. Onodera a, H. Furuta a, T. Yamamoto b a
Department of Dental Pharmacology, Okayama Uni6ersity Dental School, 2 -5 -1 Shikata-cho, Okayama 700 -8525, Japan b Department of Oral Anatomy I, Okayama Uni6ersity Dental School, 2 -5 -1 Shikata-cho, Okayama 700 -8525, Japan Received 13 March 2001; received in revised form 30 May 2001; accepted 31 May 2001
Abstract We investigated the induction of metallothionein (MT) by cadmium (Cd) in the bone tissue of rats. To clarify the cell response to Cd in bone, the isoform-specific expression of MT mRNAs (MT-I and MT-II) was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Both MT-I and MT-II mRNA levels were increased within 3 h by Cd administration. MT (MT-I/MT-II) localization after single Cd injection were also confirmed by immunohistochemical studies. Notably, MT-positive cells were time-dependently increased, and the positive cells were mainly localized in osteocytes. The cell-specific induction of MT may be associated with Cd accumulation and Cd-induced bone injury in vivo. Furthermore, we also found that MT was consecutively expressed in some osteoclasts of control rats. This finding suggested a new role of osteoclasts in bone metabolism. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Metallothionein; Cadmium; Bone; Osteocyte; Osteoclast; Osteoblast
1. Introduction Metallothionein (MT), which was discovered as a cadmium (Cd) and zinc binding protein in the horse kidney, is well known as a protein that binds Cd with high affinity and is induced by Cd (Margoshes and Vallee, 1957; Ka¨gi and Vallee, 1960). Due to its high affinity for Cd, MT functions in protection against Cd toxicity (Hamer, * Corresponding author. Tel.: + 81-86-235-6662; fax: + 8186-235-6664. E-mail address: [email protected] (C.A. Sogawa).
1986; Klaassen et al., 1999). Although the liver is the most responsive organ to MT induction, MT is also induced in other organs (Waalkes and Klaassen, 1985; Kimura et al., 1974). Kimura et al. (1974) reported that most Cd was bound to MT in the kidney of Cd-treated rats, but only 20% of Cd were bound to MT in the bone. MT-like proteins were induced in bones of Cdtreated rats, but the cysteine content of MT in the bones was lower than that in the liver (Kono et al., 1981). Further, MT-like protein was induced in MC3T3-E1 cells, an osteogenic cell line (Miyahara et al., 1986). However, there were no earlier
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reports concerning MT mRNA expression and the localization of MT in bone in relation to Cd toxicity. In this study, MT expression was examined in bone cells after a single Cd injection. To investigate the mRNA expression of each isoform of MT (MT-I and MT-II), we used MT-I and MT-II mRNA-specific primers with the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Furthermore, the localization of MT (MT-I/MT-II) in bone (femur and tibia) was examined by immunohistochemical staining.
2. Materials and methods
2.1. Chemicals CdCl2 was purchased from Nacalai Tesque Inc, Kyoto, Japan. TRIZOL was purchased from Gibco BRL, Tokyo, Japan. DNase I and RNA PCR kits were purchased from Takara Shuzo Co., Ltd., Shiga, Japan. Oligonucleotide primer sets were synthesized by Amersham Pharmacia Biotech, Tokyo, Japan. Anti-MT monoclonal antibody (against MT-I and MT-II, DAKO-MT E9), biotinylated rabbit anti-mouse immunoglobulin, and ABComplex/HRP were purchased from DAKO Co, Ltd, Carpinteria, CA. 3,3%-Diaminobenzidine tetrahydrochloride was purchased from Sigma, St. Louis, MO.
2.2. Animals and treatments Male Wistar rats (5 weeks old) were obtained from Japan SLC Co, Ltd. (Shizuoka, Japan). They were fed CE-II (CLEA Japan, Inc, Osaka, Japan) and water ad libitum for at least 1 week prior to experiment. Animals were maintained under a 12-h light:12-h dark (lights on: 06:00 to 18:00 h) cycle and temperature was maintained at 24 91 °C. Rats were injected subcutaneously (s.c.) with cadmium chloride (CdCl2, 4.5 mg Cd/ kg) dissolved in saline. Control rats were injected s.c. with saline. All injections were in a volume of 2 ml/kg.
2.3. RNA isolation and re6erse transcriptase-polymerase chain reaction (RT-PCR) Total RNA was isolated from each rat bone at 0, 3 and 6 h after Cd injection using TRIZOL as earlier described (Chomczynski and Sacchi, 1987). The proximal ends of tibiae and femora from rats were homogenized with a stainless steel homogenizer in ultrapure diethyl pyrocarbonate (DEPC)treated water. TRIZOL was added to the homogenized solution, and total RNA was isolated, according to the manufacturer’s protocol. Isolated RNA was treated with DNase I. RNA yield and purity were determined by monitoring absorbance at 260 nm and A260/A280. The expression levels of MT-I and MT-II mRNA in rat bone were measured by RT-PCR. Oligonucleotide primer sets were as follows: MT-I/II consensus sense primer, 5%-ACCCCAACTGCTCCTG(C/ T)(T/G)CC-3%; MT-I-specific antisense primer, 5%AGGTGTACGGCAAGACTCTG-3%; and MT-II-specific antisense primer, 5%-ACACCATTGTGAGGACGCCC-3%. These primers were constructed according to the known sequences (Andersen et al., 1986). The sequence of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (G3PDH) was amplified to allow comparison of relative expression of each MT isoform mRNA in samples. G3PDH primers were the G3PDH control amplimer set (CLONTECH Laboratories Inc Palo Alto, CA). Aliquots of 0.12 mg of DNase-pretreated total RNA was subjected to 30 cycles of PCR for MT-I, MT-II and G3PDH. Taq DNA polymerase and each primer were added and amplified in each reaction mixture in a final volume of 50 ml. The conditions were such that the amplifications of the PCR products were linear with respect to the amount of DNase-pretreated total RNA. PCR products for MT isoforms and G3PDH were electrophoresed on 8% polyacrylamide gels. The gels were stained with 0.5 mg/ml ethidium bromide (EtBr) solution and visualized under UV illumination. In another experiments, each reaction mixture contained [a-32P] dCTP (Amersham Pharmacia Biotech, Tokyo, Japan) for quantification of amplified DNA fragments. Each PCR
N. Oda et al. / Toxicology Letters 123 (2001) 143–150
product is also electrophoresed on 8% polyacrylamide gels. The gels were dried, exposed to an imaging plate, and analyzed using a Bioimaging Analyzer (BAS2000, Fuji Film Co., Ltd., Tokyo, Japan) as photostimulated luminescence (PSL).
2.4. Immunohistochemistry We earlier reported that the location of MT in rat dental tissue was determined by immunohistochemical staining (Sogawa, et al., 2001). Rats were anesthetized with pentobarbital (40 mg/kg) and perfused via the left ventricle with 4% paraformaldehyde in 0.08 M phosphate buffer (pH 7.3) at 0, 9, 24, 48 and 72 h after Cd injection. The proximal ends of tibiae and femora were removed and fixed in the same fixative for 2 h at 4 °C. After demineralization with 10% formic acid at 4 °C for approximately 3 weeks, these samples were dehydrated through an ethanol and xylene series and embedded in paraffin. Sections 5 mm thick were cut from the blocks, deparaffinized and immersed in 3% hydrogen peroxide in distilled water for 3 min to extinguish endogenous peroxidase activity. To block non-specific antibody binding, the sections were incubated in 5% normal rabbit serum for 30 min, followed by anti-MT monoclonal antibody (1:50) for 2.5 h at room temperature. The antibody was specifically reactive with a conserved epitope common to several mammalian species of MT-I and MT-II (Jasani and Elmes, 1991). After washing with PBS-Tween 20 (0.05%) (PBS-T), the sections were incubated with biotinylated rabbit anti-mouse immunoglobulin (1:300) for 30 min at room temperature. After washing with PBS-T, they were incubated in ABComplex/HRP for 30 min at room temperature. They were then washed with PBS-T and 0.05 M Tris– HCl (pH 7.6), followed by staining with 0.05% 3,3%-diaminobenzidine tetrahydrochloride and 0.3% hydrogen peroxide in 0.05 M Tris– HCl (pH 7.6). Sections were counterstained with hematoxylin, dehydrated and mounted in Entellan. The specificity of the staining reaction was checked by omission of anti-MT monoclonal antibody from the procedure.
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3. Results As shown in Fig. 1 A and B, both MT-I and MT-II mRNA were expressed in the proximal ends of rat bones. The expression of G3PDH mRNA was not changed by Cd treatment in the rat bone. The levels of expression of MT-I and MT-II mRNA increased within 3 h, and remained at high levels until 6 h after Cd injection. The increased levels of MT-I and MT-II mRNA were not different at 3 and 6 h.
Fig. 1. RT-PCR amplification of MT-I, MT-II and G3PDH mRNA. Aliquots of 0.12 mg DNase-I pretreated total RNA from rat bone at 0, 3 and 6 h after Cd injection (4.5 mg Cd/kg, s.c.) were reverse transcribed to cDNA and amplified by PCR for 30 cycles. PCR products were electrophoresed on an 8% polyacrylamide gel, and stained with EtBr (A). The bands corresponding to MT-I (253 base pairs (bp)), MT-II (207 bp) and G3PDH (450 bp). As the amount of PCR products of MT-II was less than that of MT-I, we used two times more amount of MT-II products (8 ml) than that of MT-I (4 ml) for taking the EtBr staining photo to clarify the gene expression for visual proof. The PSL values of PCR products corresponding to MT-I, MT-II and G3PDH were measured by a Bioimaging Analyzer (B). ( ) 0 h; ( ) 3 h; ( ), 6 h after Cd injection.
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Fig. 2. Immunohistochemical localization of MT in rat bones. Sections through the trabecular bone from a control rat (A), and a Cd-treated rat (B, at 48 h after Cd injection (4.5 mg Cd/kg, s.c.)), and the cortical bone from a control rat (C), and a Cd-treated rat (D, at 48 h after Cd injection (4.5 mg Cd/kg, s.c.)). Each section was immunostained for MT. Note the MT-positive osteoclasts (arrowheads: typical MT-positive osteoclasts), and MT-positive osteocytes in Cd-treated rats (arrows in Band D, typical MT positive osteocytes). All scale bars shown equal 40 mm.
The location of MT in rat bone was determined by immunohistochemical staining. As shown in Fig. 2, some osteoclasts were positive for MT immunostaining in the trabecular (Fig. 2A and B) and cortical (Fig. 2C and D) bone of both control
and Cd-treated rats. In the control rats, no staining was observed in osteocytes or osteoblasts (Fig. 2A and C). The number of MT-positive cells increased at 48 h after Cd injection (Fig. 2B and D). Although Cd increased MT expression in
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some osteocytes, the number of MT-positive osteocytes was greater in trabecular than in cortical bone (Fig. 2 B and D). Fig. 3 shows the time course of changes in MT location. The number of MT-positive osteocytes increased in a time-dependent manner until 48 h after Cd injection (Fig. 3A– D). Furthermore, a few MT-positive osteoblasts were detected at 48 and 72 h after injection (Fig. 3C and D). Staining for MT was observed in both the cytoplasm and nucleus (Fig. 4A and B), and nucleus staining was prominent in some MT-positive cells (Fig. 4B).
4. Discussion We showed that Cd induced MT-I and MT-II mRNA expression in rat bone. The MT-I and MT-II mRNA levels of the bone increased within 3 h, and remained at high levels until 6 h. Although the MT induction in bone was similar to that in the liver, we supposed that the MT mRNA levels are lower in the bone than in the liver because each MT mRNA PCR product from rat liver could be detected with 20 cycles of PCR, but that in rat bone could not (data not shown). We also immunohistochemically investigated the localization of MT in bone after a single Cd injection (4.5 mg Cd/kg) (Fig. 2 and Fig. 3). As the MT antibody used in the present study was specifically reactive with a conserved epitope common to several mammalian species of MT-I and MT-II, we detected both MT-I and MT-II in rat bone. MT appeared first in osteocytes after Cd injection, and later appeared in osteoblasts in addition to osteocytes (Fig. 3). However, the number of MT-positive osteoblasts was very low. Epiphyseal chondrocytes showed no induction of MT until 72 h after Cd injection (data not shown). Cd is an environmental pollutant and is known to cause loss of bone mass as seen in itai-itai patients and laboratory animals (Itokawa et al., 1974; Kimura et al., 1974; Yoshiki et al., 1975; Furuta, 1978; Kajikawa et al., 1981). In Japan, itai-itai disease, characterized by osteomalacia and renal dysfunction, is attributable to chronic Cd poisoning (Nogawa and Ishizaki, 1979). MT
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has been reported to play an important role in protection against Cd toxicity (Hamer, 1986; Klaassen et al., 1999). However, the mechanism by which MT protects against Cd-induced bone injury has not been determined. MT may protect against Cd-induced nephrotoxicity (Itokawa et al., 1974; Kajikawa et al., 1981; Hiratsuka et al., 1997), and/or against the direct Cd toxicity on bone cells (Kimura et al., 1974; Yoshiki et al., 1975; Furuta, 1978; Wang and Bhattacharyya, 1993). Recent studies have shown that MT-I and MT-II gene knockout (MT-null) mice are more sensitive to acute Cd-induced lethality and hepatotoxicity (Masters et al., 1994) and to chronic Cd-induced nephrotoxicity (Liu et al., 1998). Furthermore, Habeebu et al. (2000) reported that the loss of bone mass was more marked in MT-null mice than in wild-type controls by chronic Cd exposure. These results suggested that MT may protect against Cd-induced bone injury indirectly by reducing Cd nephropathy. On the other hand, induction of MT has been shown to offer cellular protection against Cd toxicity in various cell types, including osteosarcoma cells (Angle et al., 1993; Thomas et al., 1990), osteoblast-like cells (Suzuki et al., 1990), osteogenic cell lines (Miyahara, 1986), and cultured embryonic chick bone (Kaji et al., 1988). MT may also directly reduce the osteotoxic effects of Cd. In the present study, MT was rapidly induced in mainly osteocytes of Cd-treated rat bone. The cell-specific induction of MT may be associated with Cd accumulation and Cd-induced bone injury in vivo. Furthermore, the present study showed that MT was consecutively expressed in osteoclasts in normal rat bone. Although the consecutive MT expression was highly restricted to osteoclasts, MT-negative osteoclasts were also seen (Fig. 2). Danielson et al. (1982) demonstrated the expression of MT in specific epithelial cells of different organs, and because of the secretory, absorptive, or nutritive properties of the cells shown to express MT they suggested that MT may be involved in metal storage or transport in addition to its commonly proposed detoxification role. Our results suggested that MT may play a role in metal storage and transport in the normal bone, especially in the osteoclasts. This finding suggests a new role of osteoclasts in bone metabolism.
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Fig. 3. Time course of induction of MT in rat bones. Sections through the trabecular bone from Cd-treated rats (A, 9 h; B, 24 h; C, 48 h, and D, 72 h time point after Cd injection (4.5 mg Cd/kg, s.c.)). Each section was immunostained for MT. Note the MT-positive osteocytes (arrowheads, typical MT-positive osteocytes in A – D) and a few MT-positive osteoblasts (arrows in C and D). All scale bars shown equal 40 mm. Fig. 4. Higher power images of immunohistochemical localization of MT in rat bones. Sections through the trabecular bone from a control rat (A), and a Cd-treated rat (B, at 48 h after Cd injection (4.5 mg Cd/kg, s.c.)). Each section was immunostained for MT. Note the MT-positive osteoclasts (A), and MT-positive osteocytes (B). Staining for MT was present in both the cytoplasm and nucleus of each MT positive cell. Nuclear staining was prominent in some MT-positive cells (arrows in B). All scale bars shown equal 10 mm.
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In this study, staining for MT was observed in both the cytoplasm and nucleus of rat bone cells, and nucleus staining was prominent in some cells (Fig. 4). Several reports showed that MT was localized in the cytoplasm and nucleus of some cells (Banerjee et al., 1982; Cherian, 1994; Nartey et al., 1987; Sogawa et al., 2001). In some epithelium cells, MT was present in cytoplasm in control rat, but it was present in both the nucleus and cytoplasm following Cd treatment (Banerjee et al., 1982). Cd could enter the cell nucleus where it binds with and unknown nuclear component, within 1 h of Cd administration (Bryan and Hidalgo, 1976). MT also may be transport Cd outside the nucleus to detoxify in the bone tissue following Cd treatment. On the other hand, MT was present in the nucleus of the epithelial cells only in proliferating phase of the cell cycle (Cherian, 1994). As MT can bind Zn or Cu and transport them across the nuclear membrane, it may have a significant role to supply metals to enzymes in the proliferating phase. MT may transport consecutively essential metals inside the nucleus of osteoclasts. In this study, it was of interest that Cd induced MT-I and MT-II mRNA expression in rat bone, and notably the cells showing the most rapid induction were osteocytes where MT may protect Cd-induced bone injury.
References Andersen, R.D., Burren, B.W., Taokutz, S.J., Herschman, H.R., 1986. Rat metalltionein-1 structural gene and three pseudogenes, one of which contains 5%-regulatory sequences. Mol. Cell. Biol. 6, 302 –314. Angle, C., Thomas, D.J., Swanson, S.A., 1993. Osteotoxicity of cadmium and lead in HOS TE 85 and ROS 17/2.8 cells: relation to metallothionein induction and mitochondrial binding. Biometals 6, 179 –184. Banerjee, D., Onosaka, S., Cherian, M.G., 1982. Immunohistochemical localization of metallothionein in specific epithelial cell nucleus and cytoplasm of rat liver and kidney. Toxicology 24, 95 – 105. Bryan, S.E, Hidalgo, H.A., 1976. Nuclear 115cadmium: uptake and disappearance correlated with cadmium-binding protein synthesis. Biochem. Biophys. Res. Commun. 68, 858 –866. Cherian, M.T., 1994. The significance of the nuclear and cytoplasmic localization of metallothionein in human liver
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and tumor cells. Environ. Health Perspect. 102, 131 – 135. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal. Biochem. 162, 156 – 159. Danielson, K.G., Ohi, S., Huang, P.C., 1982. Immunochemical detection of metallothionein in specific epithelial cells of rat organs. Proc. Natl. Acad. Sci. USA 79, 2301 – 2304. Furuta, H., 1978. Cadmium effects an bone and dental tissues of rats in acute and subacute poisoning. Experientia 34, 1317 – 1318. Habeebu, S.S., Liu, J., Liu, Y., Klaassen, C.D., 2000. Metallothionein-null mice are more susceptible than wild-type mice to chronic CdCl2-induced bone injury. Toxicol. Sci. 56, 211 – 219. Hamer, D.H., 1986. Metallothionein. Ann. Rev. Biochem. 55, 913 – 951. Hiratsuka, H., Katsyta, O., Toyota, N., Tsuchitani, M., Akiba, T., Marumo, F., Umemura, T., 1997. Iron deposition at mineralization fronts and osteoid formation following chronic cadmium exposure in ovariectomized rats. Toxicol. Appl. Pharmacol. 143, 348 – 356. Itokawa, Y., Abe, T., Tabei, R., Tanaka, S., 1974. Renal and skeletal lesions in experimental cadmium poisoning. Arch. Environ. Health 28, 149 – 154. Jasani, B., Elmes, M.E., 1991. Immunohistochemical detection of metallothionein. Method Enzymol. 205, 205 – 215. Ka¨ gi, J.H., Vallee, B., 1960. Metallothionein: a cadmium- and zinc-containing protein from equine renal cortex. J. Biol. Chem. 235, 3460 – 3465. Kaji, T., Takata, M., Hosino, T., Miyahara, T., Kozuka, H., Hurashige, Y., Koizumi, F., 1988. Role of zinc in protection against cadmium-induced toxicity in formation of embryonic chick bone in tissue culture. Toxicol. Lett. 44, 221 – 227. Kajikawa, K., Nakanishi, I., Kuroda, K., 1981. Morphological changes of the kidney and bone of rats in chronic cadmium poisoning. Exp. Mol. Pathol. 34, 9 – 24. Kimura, M., Otaki, N., Yashikim, S., Suzuki, M., Horiuchi, N., Suda, T., 1974. The isolation of metallothionein and its protective role in cadmium poisoning. Arch. Biochem. Biophys. 165, 340 – 348. Klaassen, C.D., Liu, J., Choudhuri, S, 1999. Metallothionein: an intracellular protein to protect against cadmium toxicity. Annu. Rev. Toxicol. 39, 267 – 294. Kono, Y., Ishikawa, T., Kondo, M., 1981. Purification and properties of cadmium binding protein in bone. Eisei Kagaku 27, 212 – 217. Liu, J., Liu, Y., Habeebu, S.S., Klaassen, C.D., 1998. Susceptibility of MT-null mice to chronic CdCl2-induced nephrotoxicity indicates that renal injury is not mediated by the CdMT complex. Toxicol. Sci. 46, 197 – 203. Margoshes, M., Vallee, B.L., 1957. A cadmium protein from equine kidney cortex. J. Am. Chem. Soc. 79, 4813 – 4814. Masters, B.A., Kelly, E.J., Quaife, C.J., Brinster, R.L., Palmiter, R.D., 1994. Targeted disruption of metallothionein I and II genes increases sensitivity to cadmium. Proc. Natl. Acad. Sci. USA 91, 584 – 588.
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Miyahara, T., Yamada, M., Ando, R., Nemoto, S., Kaji, T., Mori, M., Kozuka, H., Itoh, N., Sudo, H., 1986. The effects of cadmium on a clonal osteogenetic cell, MC3T3E1: inhibition of calcification and induction of metallothionein-like protein by cadmium. Toxicol. Lett. 32, 19 –27. Nartey, N.O., Banerjee, D., Cherian, M.G., 1987. Immunohistochemical localization of metallothionein in cell nucleus and its changes during development. Pathology 19, 233 – 238. Nogawa, K., Ishizaki, A., 1979. A comparison between cadmium in rice and renal effects among inhabitants of Jinzu Rever Basin. Environ. Res. 18, 410 –420. Sogawa, C.A., Sogawa, N., Yamamoto, T., Oda, N., Inoue, T., Onodera, K., Furuta, H., 2001. Localization of metallothionein (MT) and expression of MT isoforms induced by cadmium in rat dental pulp. Jpn. J. Pharmacol. 86, 65– 72. Suzuki, Y., Morita, I., Yamane, Y., Murota, S., 1990. Preven-
tive effect of zinc against cadmium-induced bone resorption. Toxicology 62, 27 – 34. Thomas, D., Angle, C.R., Swanson, S.A., 1990. Effect of 5-azacytidinee on metallothionein inducibility and sensitivity to lethality of cadmium in rat osteosarcoma (ROS 17/2.8) cells. Toxicology 65, 169 – 178. Yoshiki, S., Yanagisawa, T., Kimura, M., Otaki, N., Suzuki, M., Suda, T., 1975. Bone and kidney lesions in experimental cadmium intoxication. Arch. Environ. Health 30, 559 – 562. Waalkes, M.P., Klaassen, C.D., 1985. Concentration of metallothionein in major organs of rats after administration of various metals. Fundam. Appl. Toxicol. 5, 473 – 477. Wang, C., Bhattacharyya, M.H., 1993. Effect of cadmium on bone calcium and 45Ca in nonpregnant mice on a calciumdeficient diet: evidence of direct effect of cadmium on bone. Toxicol. Appl. Pharmacol. 120, 228 – 239.
Metallothionein expression and localization in rat bone tissue after cadmium injection N. Oda a, C.A. Sogawa a,*, N. Sogawa a, K. Onodera a, H. Furuta a, T. Yamamoto b a
Department of Dental Pharmacology, Okayama Uni6ersity Dental School, 2 -5 -1 Shikata-cho, Okayama 700 -8525, Japan b Department of Oral Anatomy I, Okayama Uni6ersity Dental School, 2 -5 -1 Shikata-cho, Okayama 700 -8525, Japan Received 13 March 2001; received in revised form 30 May 2001; accepted 31 May 2001
Abstract We investigated the induction of metallothionein (MT) by cadmium (Cd) in the bone tissue of rats. To clarify the cell response to Cd in bone, the isoform-specific expression of MT mRNAs (MT-I and MT-II) was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Both MT-I and MT-II mRNA levels were increased within 3 h by Cd administration. MT (MT-I/MT-II) localization after single Cd injection were also confirmed by immunohistochemical studies. Notably, MT-positive cells were time-dependently increased, and the positive cells were mainly localized in osteocytes. The cell-specific induction of MT may be associated with Cd accumulation and Cd-induced bone injury in vivo. Furthermore, we also found that MT was consecutively expressed in some osteoclasts of control rats. This finding suggested a new role of osteoclasts in bone metabolism. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Metallothionein; Cadmium; Bone; Osteocyte; Osteoclast; Osteoblast
1. Introduction Metallothionein (MT), which was discovered as a cadmium (Cd) and zinc binding protein in the horse kidney, is well known as a protein that binds Cd with high affinity and is induced by Cd (Margoshes and Vallee, 1957; Ka¨gi and Vallee, 1960). Due to its high affinity for Cd, MT functions in protection against Cd toxicity (Hamer, * Corresponding author. Tel.: + 81-86-235-6662; fax: + 8186-235-6664. E-mail address: [email protected] (C.A. Sogawa).
1986; Klaassen et al., 1999). Although the liver is the most responsive organ to MT induction, MT is also induced in other organs (Waalkes and Klaassen, 1985; Kimura et al., 1974). Kimura et al. (1974) reported that most Cd was bound to MT in the kidney of Cd-treated rats, but only 20% of Cd were bound to MT in the bone. MT-like proteins were induced in bones of Cdtreated rats, but the cysteine content of MT in the bones was lower than that in the liver (Kono et al., 1981). Further, MT-like protein was induced in MC3T3-E1 cells, an osteogenic cell line (Miyahara et al., 1986). However, there were no earlier
0378-4274/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 4 2 7 4 ( 0 1 ) 0 0 3 8 7 - 3
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reports concerning MT mRNA expression and the localization of MT in bone in relation to Cd toxicity. In this study, MT expression was examined in bone cells after a single Cd injection. To investigate the mRNA expression of each isoform of MT (MT-I and MT-II), we used MT-I and MT-II mRNA-specific primers with the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Furthermore, the localization of MT (MT-I/MT-II) in bone (femur and tibia) was examined by immunohistochemical staining.
2. Materials and methods
2.1. Chemicals CdCl2 was purchased from Nacalai Tesque Inc, Kyoto, Japan. TRIZOL was purchased from Gibco BRL, Tokyo, Japan. DNase I and RNA PCR kits were purchased from Takara Shuzo Co., Ltd., Shiga, Japan. Oligonucleotide primer sets were synthesized by Amersham Pharmacia Biotech, Tokyo, Japan. Anti-MT monoclonal antibody (against MT-I and MT-II, DAKO-MT E9), biotinylated rabbit anti-mouse immunoglobulin, and ABComplex/HRP were purchased from DAKO Co, Ltd, Carpinteria, CA. 3,3%-Diaminobenzidine tetrahydrochloride was purchased from Sigma, St. Louis, MO.
2.2. Animals and treatments Male Wistar rats (5 weeks old) were obtained from Japan SLC Co, Ltd. (Shizuoka, Japan). They were fed CE-II (CLEA Japan, Inc, Osaka, Japan) and water ad libitum for at least 1 week prior to experiment. Animals were maintained under a 12-h light:12-h dark (lights on: 06:00 to 18:00 h) cycle and temperature was maintained at 24 91 °C. Rats were injected subcutaneously (s.c.) with cadmium chloride (CdCl2, 4.5 mg Cd/ kg) dissolved in saline. Control rats were injected s.c. with saline. All injections were in a volume of 2 ml/kg.
2.3. RNA isolation and re6erse transcriptase-polymerase chain reaction (RT-PCR) Total RNA was isolated from each rat bone at 0, 3 and 6 h after Cd injection using TRIZOL as earlier described (Chomczynski and Sacchi, 1987). The proximal ends of tibiae and femora from rats were homogenized with a stainless steel homogenizer in ultrapure diethyl pyrocarbonate (DEPC)treated water. TRIZOL was added to the homogenized solution, and total RNA was isolated, according to the manufacturer’s protocol. Isolated RNA was treated with DNase I. RNA yield and purity were determined by monitoring absorbance at 260 nm and A260/A280. The expression levels of MT-I and MT-II mRNA in rat bone were measured by RT-PCR. Oligonucleotide primer sets were as follows: MT-I/II consensus sense primer, 5%-ACCCCAACTGCTCCTG(C/ T)(T/G)CC-3%; MT-I-specific antisense primer, 5%AGGTGTACGGCAAGACTCTG-3%; and MT-II-specific antisense primer, 5%-ACACCATTGTGAGGACGCCC-3%. These primers were constructed according to the known sequences (Andersen et al., 1986). The sequence of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (G3PDH) was amplified to allow comparison of relative expression of each MT isoform mRNA in samples. G3PDH primers were the G3PDH control amplimer set (CLONTECH Laboratories Inc Palo Alto, CA). Aliquots of 0.12 mg of DNase-pretreated total RNA was subjected to 30 cycles of PCR for MT-I, MT-II and G3PDH. Taq DNA polymerase and each primer were added and amplified in each reaction mixture in a final volume of 50 ml. The conditions were such that the amplifications of the PCR products were linear with respect to the amount of DNase-pretreated total RNA. PCR products for MT isoforms and G3PDH were electrophoresed on 8% polyacrylamide gels. The gels were stained with 0.5 mg/ml ethidium bromide (EtBr) solution and visualized under UV illumination. In another experiments, each reaction mixture contained [a-32P] dCTP (Amersham Pharmacia Biotech, Tokyo, Japan) for quantification of amplified DNA fragments. Each PCR
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product is also electrophoresed on 8% polyacrylamide gels. The gels were dried, exposed to an imaging plate, and analyzed using a Bioimaging Analyzer (BAS2000, Fuji Film Co., Ltd., Tokyo, Japan) as photostimulated luminescence (PSL).
2.4. Immunohistochemistry We earlier reported that the location of MT in rat dental tissue was determined by immunohistochemical staining (Sogawa, et al., 2001). Rats were anesthetized with pentobarbital (40 mg/kg) and perfused via the left ventricle with 4% paraformaldehyde in 0.08 M phosphate buffer (pH 7.3) at 0, 9, 24, 48 and 72 h after Cd injection. The proximal ends of tibiae and femora were removed and fixed in the same fixative for 2 h at 4 °C. After demineralization with 10% formic acid at 4 °C for approximately 3 weeks, these samples were dehydrated through an ethanol and xylene series and embedded in paraffin. Sections 5 mm thick were cut from the blocks, deparaffinized and immersed in 3% hydrogen peroxide in distilled water for 3 min to extinguish endogenous peroxidase activity. To block non-specific antibody binding, the sections were incubated in 5% normal rabbit serum for 30 min, followed by anti-MT monoclonal antibody (1:50) for 2.5 h at room temperature. The antibody was specifically reactive with a conserved epitope common to several mammalian species of MT-I and MT-II (Jasani and Elmes, 1991). After washing with PBS-Tween 20 (0.05%) (PBS-T), the sections were incubated with biotinylated rabbit anti-mouse immunoglobulin (1:300) for 30 min at room temperature. After washing with PBS-T, they were incubated in ABComplex/HRP for 30 min at room temperature. They were then washed with PBS-T and 0.05 M Tris– HCl (pH 7.6), followed by staining with 0.05% 3,3%-diaminobenzidine tetrahydrochloride and 0.3% hydrogen peroxide in 0.05 M Tris– HCl (pH 7.6). Sections were counterstained with hematoxylin, dehydrated and mounted in Entellan. The specificity of the staining reaction was checked by omission of anti-MT monoclonal antibody from the procedure.
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3. Results As shown in Fig. 1 A and B, both MT-I and MT-II mRNA were expressed in the proximal ends of rat bones. The expression of G3PDH mRNA was not changed by Cd treatment in the rat bone. The levels of expression of MT-I and MT-II mRNA increased within 3 h, and remained at high levels until 6 h after Cd injection. The increased levels of MT-I and MT-II mRNA were not different at 3 and 6 h.
Fig. 1. RT-PCR amplification of MT-I, MT-II and G3PDH mRNA. Aliquots of 0.12 mg DNase-I pretreated total RNA from rat bone at 0, 3 and 6 h after Cd injection (4.5 mg Cd/kg, s.c.) were reverse transcribed to cDNA and amplified by PCR for 30 cycles. PCR products were electrophoresed on an 8% polyacrylamide gel, and stained with EtBr (A). The bands corresponding to MT-I (253 base pairs (bp)), MT-II (207 bp) and G3PDH (450 bp). As the amount of PCR products of MT-II was less than that of MT-I, we used two times more amount of MT-II products (8 ml) than that of MT-I (4 ml) for taking the EtBr staining photo to clarify the gene expression for visual proof. The PSL values of PCR products corresponding to MT-I, MT-II and G3PDH were measured by a Bioimaging Analyzer (B). ( ) 0 h; ( ) 3 h; ( ), 6 h after Cd injection.
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Fig. 2. Immunohistochemical localization of MT in rat bones. Sections through the trabecular bone from a control rat (A), and a Cd-treated rat (B, at 48 h after Cd injection (4.5 mg Cd/kg, s.c.)), and the cortical bone from a control rat (C), and a Cd-treated rat (D, at 48 h after Cd injection (4.5 mg Cd/kg, s.c.)). Each section was immunostained for MT. Note the MT-positive osteoclasts (arrowheads: typical MT-positive osteoclasts), and MT-positive osteocytes in Cd-treated rats (arrows in Band D, typical MT positive osteocytes). All scale bars shown equal 40 mm.
The location of MT in rat bone was determined by immunohistochemical staining. As shown in Fig. 2, some osteoclasts were positive for MT immunostaining in the trabecular (Fig. 2A and B) and cortical (Fig. 2C and D) bone of both control
and Cd-treated rats. In the control rats, no staining was observed in osteocytes or osteoblasts (Fig. 2A and C). The number of MT-positive cells increased at 48 h after Cd injection (Fig. 2B and D). Although Cd increased MT expression in
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some osteocytes, the number of MT-positive osteocytes was greater in trabecular than in cortical bone (Fig. 2 B and D). Fig. 3 shows the time course of changes in MT location. The number of MT-positive osteocytes increased in a time-dependent manner until 48 h after Cd injection (Fig. 3A– D). Furthermore, a few MT-positive osteoblasts were detected at 48 and 72 h after injection (Fig. 3C and D). Staining for MT was observed in both the cytoplasm and nucleus (Fig. 4A and B), and nucleus staining was prominent in some MT-positive cells (Fig. 4B).
4. Discussion We showed that Cd induced MT-I and MT-II mRNA expression in rat bone. The MT-I and MT-II mRNA levels of the bone increased within 3 h, and remained at high levels until 6 h. Although the MT induction in bone was similar to that in the liver, we supposed that the MT mRNA levels are lower in the bone than in the liver because each MT mRNA PCR product from rat liver could be detected with 20 cycles of PCR, but that in rat bone could not (data not shown). We also immunohistochemically investigated the localization of MT in bone after a single Cd injection (4.5 mg Cd/kg) (Fig. 2 and Fig. 3). As the MT antibody used in the present study was specifically reactive with a conserved epitope common to several mammalian species of MT-I and MT-II, we detected both MT-I and MT-II in rat bone. MT appeared first in osteocytes after Cd injection, and later appeared in osteoblasts in addition to osteocytes (Fig. 3). However, the number of MT-positive osteoblasts was very low. Epiphyseal chondrocytes showed no induction of MT until 72 h after Cd injection (data not shown). Cd is an environmental pollutant and is known to cause loss of bone mass as seen in itai-itai patients and laboratory animals (Itokawa et al., 1974; Kimura et al., 1974; Yoshiki et al., 1975; Furuta, 1978; Kajikawa et al., 1981). In Japan, itai-itai disease, characterized by osteomalacia and renal dysfunction, is attributable to chronic Cd poisoning (Nogawa and Ishizaki, 1979). MT
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has been reported to play an important role in protection against Cd toxicity (Hamer, 1986; Klaassen et al., 1999). However, the mechanism by which MT protects against Cd-induced bone injury has not been determined. MT may protect against Cd-induced nephrotoxicity (Itokawa et al., 1974; Kajikawa et al., 1981; Hiratsuka et al., 1997), and/or against the direct Cd toxicity on bone cells (Kimura et al., 1974; Yoshiki et al., 1975; Furuta, 1978; Wang and Bhattacharyya, 1993). Recent studies have shown that MT-I and MT-II gene knockout (MT-null) mice are more sensitive to acute Cd-induced lethality and hepatotoxicity (Masters et al., 1994) and to chronic Cd-induced nephrotoxicity (Liu et al., 1998). Furthermore, Habeebu et al. (2000) reported that the loss of bone mass was more marked in MT-null mice than in wild-type controls by chronic Cd exposure. These results suggested that MT may protect against Cd-induced bone injury indirectly by reducing Cd nephropathy. On the other hand, induction of MT has been shown to offer cellular protection against Cd toxicity in various cell types, including osteosarcoma cells (Angle et al., 1993; Thomas et al., 1990), osteoblast-like cells (Suzuki et al., 1990), osteogenic cell lines (Miyahara, 1986), and cultured embryonic chick bone (Kaji et al., 1988). MT may also directly reduce the osteotoxic effects of Cd. In the present study, MT was rapidly induced in mainly osteocytes of Cd-treated rat bone. The cell-specific induction of MT may be associated with Cd accumulation and Cd-induced bone injury in vivo. Furthermore, the present study showed that MT was consecutively expressed in osteoclasts in normal rat bone. Although the consecutive MT expression was highly restricted to osteoclasts, MT-negative osteoclasts were also seen (Fig. 2). Danielson et al. (1982) demonstrated the expression of MT in specific epithelial cells of different organs, and because of the secretory, absorptive, or nutritive properties of the cells shown to express MT they suggested that MT may be involved in metal storage or transport in addition to its commonly proposed detoxification role. Our results suggested that MT may play a role in metal storage and transport in the normal bone, especially in the osteoclasts. This finding suggests a new role of osteoclasts in bone metabolism.
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Fig. 3. Time course of induction of MT in rat bones. Sections through the trabecular bone from Cd-treated rats (A, 9 h; B, 24 h; C, 48 h, and D, 72 h time point after Cd injection (4.5 mg Cd/kg, s.c.)). Each section was immunostained for MT. Note the MT-positive osteocytes (arrowheads, typical MT-positive osteocytes in A – D) and a few MT-positive osteoblasts (arrows in C and D). All scale bars shown equal 40 mm. Fig. 4. Higher power images of immunohistochemical localization of MT in rat bones. Sections through the trabecular bone from a control rat (A), and a Cd-treated rat (B, at 48 h after Cd injection (4.5 mg Cd/kg, s.c.)). Each section was immunostained for MT. Note the MT-positive osteoclasts (A), and MT-positive osteocytes (B). Staining for MT was present in both the cytoplasm and nucleus of each MT positive cell. Nuclear staining was prominent in some MT-positive cells (arrows in B). All scale bars shown equal 10 mm.
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In this study, staining for MT was observed in both the cytoplasm and nucleus of rat bone cells, and nucleus staining was prominent in some cells (Fig. 4). Several reports showed that MT was localized in the cytoplasm and nucleus of some cells (Banerjee et al., 1982; Cherian, 1994; Nartey et al., 1987; Sogawa et al., 2001). In some epithelium cells, MT was present in cytoplasm in control rat, but it was present in both the nucleus and cytoplasm following Cd treatment (Banerjee et al., 1982). Cd could enter the cell nucleus where it binds with and unknown nuclear component, within 1 h of Cd administration (Bryan and Hidalgo, 1976). MT also may be transport Cd outside the nucleus to detoxify in the bone tissue following Cd treatment. On the other hand, MT was present in the nucleus of the epithelial cells only in proliferating phase of the cell cycle (Cherian, 1994). As MT can bind Zn or Cu and transport them across the nuclear membrane, it may have a significant role to supply metals to enzymes in the proliferating phase. MT may transport consecutively essential metals inside the nucleus of osteoclasts. In this study, it was of interest that Cd induced MT-I and MT-II mRNA expression in rat bone, and notably the cells showing the most rapid induction were osteocytes where MT may protect Cd-induced bone injury.
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