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Induction of metallothionein in the Reuber H-35 rat hepatoma cell
ELSEVIER
Toxicology 104 (1995) 99-104
Induction of metallothionein in the Reuber H-35 rat hepatoma cell M.S. Yang*, M.K. Tang, R.N.S. Wong Department
of Biofogy, Hong Kong Baptist University, Kowloon Tong, Hong Kong
Received 12 January 1995; accepted 2 May 1995
Abstract The feasibility of using the Reuber H-35 rat hepatoma cell (RH-35 cells) as model for studying metallothionein induction was examined. The RH-35 cells were treated with Cd, a toxic metal which is known to induce metallothionein. The LCu, after a 3-h treatment was 70 pM. The value was significantly higher (P < 0.05) if the cells were pre-treated with a sublethal dose of CdCl, (5 PM) for 2 days, indicating that pre-treatment with a low dose of Cd can protect against a subsequent higher dose of the same metal. Both the mRNA and the gene product metallothionein can be identified in the cells 2 days after treatment with 5 pM Cd. In addition to Cd, Zn and Cu were also able to induce the expression of metallothionein to various degrees. The results indicate that the MT gene is present in RH-35 cells and is responsive to treatment with various metals. Thus, this cell line can be used as a model to study metallothionein induction.
Keywords: Metallothionein
induction; Reuber H-35 rat hepatoma cell; Cadmium
1. Introduction Metallothionein (MT), a cytosolic, low molecular weight, cysteine-rich metal-binding protein, is normally undetectable in cells, but its level increases upon exposure of cells or tissues to elevated levels of toxic metal such as cadmium, Abbreviations:
PBS, phosphate-buffered saline; DEPC, di-
ethyl pyrccarbonate; SSC, standard saline citrate; LC&, concentration of metal that causes 50% cell death as detected by the trypan-blue exclusion test; MEM, minimum essential medium; Tris, tris-(hydroxymethyl)amino methane; Cd, cadmium; Zn, zinc; Cu, copper; Ni, nickel. l Corresponding author, Tel.: 852-2339-7058; Fax: 8522336-1400.
mercury and silver (Fowler et al., 1987). The protein is believed to play an important role in metal detoxification since exposure to low dose of Cd can protect against a subsequent higher dose of the same toxic metal (Goering and Klaassen, 1984). It is believed that the Cd-induced metallothionein can chelate the subsequently added metal, thus preventing it from interacting with cytoplasmic organelles (Goering and Klaassen, 1983). Besides toxic metals, Zn and Cu are found capable of inducing MT (Fowler et al., 1987). Both Zn and Cu are important cofactors for a wide variety of enzymatic reactions. Thus, MT may also play a role in regulating the level of these endogenous trace metals (Bremner, 1987).
0300-483)(/95/W9.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0300-483X(95)03149-A
100
MS. Yang et al. / Toxicology 104 (199s) 99-104
Metals are not the only elements that can induce MT synthesis. Physiological stimuli, such as starvation, stress and hyperoxia; hormones, such as catecholamines, glucocorticoid, glucagon and cytokines; and chemicals, including turpentine, alcohol, carbon tetrachloride, can also stimulate MT production (Bremner, 1987). However, the mechanism(s) of MT induction are not well understood. We have been investigating the use of a simple cell culture model to study the mechanism(s) of metallothionein induction. The Reuber H-35 rat hepatoma cell (RH-35 cells) is a clonal cell line that can easily be maintained in culture. The cell line was found to express numerous functions of the hepatocytes of adult liver (Reuber et al., 1961). It has been used as an in vitro model in studies concerning the insulin and glucocorticoidscontrolled liver function (Esau and Koontz, 1985; K.itagawa and Sugimoto, 1985; Orlowski et al., 1990). The cell line has retained the ability to induce drug-metabolizing enzymes (Frey et al., 1986; Wiebel et al., 1986). In our laboratory, the RH-35 cells have been used as a model to study the molecular basis of enzyme induction (Fong et al., 1991). In the present study, metallothionein was identified in this cell line and the gene is found to be inducible by various metals. 2. Materials and methods 2.1. Determination of Cd cytotoxicity in the RH-35 ceils The Reuber H-35 rat hepatoma cells were maintained at 37°C in MEM (Gibco, Gathersburg, TX) supplemented with 10% fetal bovine serum (FBS) (Flow Laboratories, North Ryde, NSW, Australia) and antibiotics (amphotericin B and pencillinstreptomycin) in a humidified atmosphere of 95% air and 5% CO*. Five sets of plates, in triplicate, were prepared. Upon confluence, MEM containing different doses (0, 10,50,70, 100 and 150 PM) of CdC12 was added to different plates. Then, 3 h after addition of CdC&, the medium were removed and the cells were washed twice with PBS. The cells were dislodged from the plates by treatment with 0.1 ml of 0.05% trypsin followed by addition of 0.5 ml of 0.6% trypan blue solution. The tryp-
sinized cells were transferred to a hemocytometer for counting. Five different areas on the hemocytometer were selected randomly and cells were counted under the microscope. Both the trypan blue stained (dead) cells and the unstained (live) cells were counted. The number of cells in each count ranged between 60-100. The percent of cell death was calculated by dividing the total number of stained cells by the total number of cells (both stained and unstained) counted. LCso for Cd, together with the upper and lower 95% confidence limits, were calculated by the probit analysis (Klaassen and Eaton, 1991) using the SAS/SPSS statistical software package. 2.2. Protection against CdC12by pretreatment with sublethal dose of the same metal To examine the ability of CdC12 to protect against a subsequent dose of the same toxic metal, 5 PM (final concentration) of CdC12 was added to the incubation medium and the cells were allowed to grow for 2 days. After incubation, the medium was removed and the cells washed with PBS. They were then challenged with different doses of CdCl, as described previously. The percentage of cell death, the LCw and the upper and lower 95% confidence limits were also calculated. 2.3. Isolation of metallothionein from the RH-35 cells After pre-treatment with 5 PM CdCl* for 2 days, the cells were washed with 0.02 M Tris-HCl, pH 7.4, harvested and sonicated. The broken cells were centrifuged at 100 000 x g (Hitachi 7OP-72 preparative ultracentrifuge) for 60 min at 4°C. The supematant was collected and 5 ~1 of CdCl, (1 mg/rnl) was added to every 950 ~1 sample as an internal standard to facilitate subsequent detection. The samples were then heated in boiling water for 1 min. The precipitated protein was removed by centrifugation at 10 000 x g for 5 min. The supernatant (3 ml) was applied onto a Sephadex G-75 column (26 mm x 40 cm) together with blue dextran as marker for void volume measurement. The proteins were eluted with the same buffer at a flow rate of 20 ml/h at room temperature. Five-ml fractions were collected. The optical density at 254 nm for each fraction was determined and the concen-
M.S. Yang et al. /Toxicology 104 (1995) W-104
tration of cadmium was determined by flame atomic absorption spectroscopy (Varian, Spectra AA-IO). 2.4. Determination for MT induction
of doses of Zn, Ni and Cu used
The RH-35 cells were exposed to different concentrations of either ZnC&, CuC12 or NiCl* for 3 h and the percent of dead cells was measured using the trypan blue exclusion test. The LC,, was calculated by the probit analysis using the SASISPSS package. A sublethal dose was selected to test the MT inducibility (Table 2). 2.5. Extraction of total RNA All glasswares and disposables were treated with DEPC and sterilized by autoclaving to prevent the possible contamination of RNAse. Total RNA from at least 10’ treated cells was extracted using the guanidine thiocyanate method (Chomczynski and Sacchi, 1987). Cells were washed twice with ice-cold DEPCYPBS before the addition of the denaturing solution (4 M guanidine thiocyanate, 25 mM sodium citrate, pH 7, 0.5% N-lauryl sarcosine, 0.1 M &mercaptoethanol). About l/10 vol of 2 M sodium acetate, pH 4.0 was added, the solution was then extracted with phenol/chloroform/ isoamyl alcohol mixture (25:24:1 v/v). The aqueous phase containing the extracted RNA was precipitated with an equal volume of isopropanol and the mixture was allowed to stand at -20°C overnight. The precipitated RNA was pelleted by centrifugation at 10 000 x g at 4°C. The RNA, after washing with 70% ethanol, was dried and dissolved in DEPC/water. The intactness of the RNA was confirmed by denaturing agarose gel electrophoresis (Sambrook et al., 1989). 2.6. Preparation of MT-cDNA A plasmid, pBXA containing the mouse MTIcDNA, was kindly provided by Dr. R.D. Palmiter, University of Washington, Seattle, WA. After transformation into E. coli RR1 followed by amplification of the transformants in a liquid LuriaBertani medium. A large scale plasmid preparation was performed according to Sambrook et al. (1989). The MTIcDNA was released from the vector by BumHI (Promega, Madison, WI) restriction
101
digestion and the fragments were separated by electrophoresis on a 7% polyacrylamide gel. The 300bp MTI-cDNA was recovered by electroelution using the electro-eluter (Bio-Rad, San Diego, CA). For northern blot hybridization, the MTIcDNA was labeled with 32P-dCTP using the oligolabeling kit from Pharmacia (Milwaukee, WI). 2.7. Northern blot analysis Northern blot analysis was carried out as follow. Equal amounts of RNA from different samples were subjected to electrophoresis on a 1.5% agarose gel containing formaldehyde. The separated RNAs were then transferred to a nylon membrane by capillary blotting in 20 x SSC. After prehybridizing at 65°C for 2 h, the membrane was subsequently hybridized with the heat-denatured radiolabelled MTcDNA. Hybridization was performed at 65’C overnight with the MTIcDNA probe in a solution containing 6 x SSC, 5 x Denhardt’s solution, 0.5% SDS and denatured salmon sperm DNA (20 &ml). After hybridization, the excess probe was removed by washing the membrane twice at 65°C with 2 x SSC and 2 x SSC, 0.1% SDS for an interval of lo-15 min for each wash. The washed membrane was air dried and wrapped in plastic wrap for autoradiography. 3. Results and discussion 3.1. Protection against Cd-induced cell death after pre-treatment of ceils with a low dose of Cd Fig. 1 shows that increasing cell death occurred with increasing doses of Cd in the medium. The LC% for Cd was calculated to be 70 PM with the upper and lower 95% confidence levels of 90 and 60 PM respectively (Table 1). Pre-treatment of the RH-35 cells with a low dose of Cd (5 PM) for 2 days reduced the toxicity of a subsequent dose of the same metal. The LC, increased significantly (P < 0.05) from 70 PM to 220 PM (Table 1) upon Cd pre-treatment. 3.2. Isolation of a cadmium binding-protein from the RH-35 cells after 2 days pre-treatment CdCl*
with 5 PM
Fig. 2 shows that, 2 days after Cd pre-treatment,
MS.
102
Yang et al. I Toxicology 104 (1995) 99-104
70 60
0
No
0
Cd pretreatment
Cd
pre-treatment
P
50 5 d = 8 B 2 8 b 0
s 40
0.0
00 10
0
30
20 Fraction
20
30
40
0.8
08
-
2. 0.6 -
10
50
number
0.6
MT
F
6
0 10’
102
Cd concentration
(log
uM)
Fig. 1. Percentage of cell death of the RH-35 cells after exposure to different doses of CdC& for 3 h. The values are mean f SD. of triplicates. Cd pretreatment was carried out by exposing the cells to 5 PM CdC12 for 2 days prior to Cd challenge.
a fraction of protein with a molecular weight intermediate between the high and low molecular weight proteins, can be identified in the RH-35 cells upon Sephadex G-75 chromatography. This fraction of protein has low optical density at 254 nm and high Cd content. The position on the chromatogram (V&V, = 2.5) is characteristic of that of metallothionein previously reported (Winge et al., 1979). It is not present in control cells without Cd pre-treatment, indicating that it is synthesized in response to Cd. The induction of MT by Cd and its protection against metal-induced toxicity has
0
10
20
Fraction
30
40
50
number
Fig. 2. Sephadex G-75 chromatogram of the heat stable proteins from, (I) control and, (2) Cd-treated RH-35 cells. 0 represents the 254 nm readings and W represents the Cd level &/ml) in each fraction.
9.5kb_ 7.5kb_ 4&b_ 2.4kb_ 1.4kb_
MT Table I Lt.&s and the 95% confidence limits of CdClz for the RH-35 cells in culture before and after pretreatment with a low dose of cadmium Pretreatment (2 days)
None 5 CM CdC12
0.24kb_
95% Confidence limits Lower limits
Upper limits
66 150
90 330
Fig. 3. Northern blot analysis of MT from control RH-35 cells and cells treated with Cd (5 PM), Zn (75 PM), Cu (50 PM) and Ni (396 PM). The size of MT-mRNA is approximately 0.3 kb as calculated from molecular weight markers on the left.
MS. Yanget al. /Toxicology 104 (199s) 99-104 Table 2 LCa of Zn, Ni and Cu for the RH-35 cells Metal
LCss (CcM)
Induction dosage (FM)
ZnClz NQ cuq
1220 6210 170
75 390 50
been confirmed in other cell culture models. A wildtype Chinese hamster ovary (CHO) cell line which did not produce MT was extremely sensitive to Cd. Upon conversion to a MT-inducible phenotype in the presence of 5-azacytidine, the cell line became resistant to Cd (Grady et al., 1987). Similar Cd resistance was also conferred in a mouse hepatoma cell line upon transformation to the MT-producing phenotype (Durnam and Pahniter, 1987). In addition to the protein, the mRNA for MT was also identified in the RH-35 cells. Fig. 3 shows the presence of MT-mRNA in cells after a 2-day treatment with 5 PM CdC12. 3.3. Induction of MT-mRNA by other metals Table 2 shows the LC+ of Zn, Ni and Cu for the RH-35 cells. The results show that while Cu is more toxic than Zn and Ni, it is ten times less toxic than Cd. Fig. 3 shows that Zn and Cu, at concentrations below the LCso, were able to induce MT gene expression. There is little MT induction after treatment with Ni (Fig. 3). Similar to a number of mammalian cell lines studied (Rudd and Hers&man, 1979; Cherian, 1980; Dumam and Pahniter, 1984), the RH-35 cells possess the MT gene which is responsive to metal induction. Besides Cd, Zn and Cu are also good inducers. The results show that the RH-35 cells can be used as a model for studying MT gene induction. Recently, the metal-induced metallothionein has been found to reduce the side-effects associated with metal-containing antitumor drugs (e.g. cisplatin) treatment (Basu and Laze, 1990). The finding has raised considerable interest in the search for appropriate MT inducers to be used as pre-treatment regime prior to administration of the drug (Imura et al., 1989, Basu and Lazo, 1990). In addition to studying the mechanism(s) of MT induction, the RH-35 rat hepatoma cell can also be used as a model for preliminary screening for such agents.
103
References Basu,A. and Laxo, J.S. (1990) A hypothesis regarding the protective role of metallothioneins against the toxicity of DNA interactive anticancer drugs. Toxicol. Lett. 50, 123-135. Bremner, I. (1987) Nutritional and physiological significance of metallothionein. Experientia Suppl. 52, 8l- 107. Cherian, M.G. (1980) The synthesis of metallothionein and cellular adaptation to metal toxicity in primary rat kidney epithelial cell cultures. Toxicology 17, 225-231. Chomcxynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal. B&hem. 162, l56- 159. Dumam, D.M. and Palmiter, R.D. (1984) Induction of metallothionein-I mRNA in cultured cells by heavy metals and iodoacetate: Evidence of gratuitous inducers. Mol. Cell. Biol. 4, 484-49 I. Dumam, D.M. and Pahniter, R.D. (1987) Analysis of the detoxification of heavy metal ions by mouse metallothionein. Experientia Suppl. 52, 457-463. Esau, B.D. and Koontx, J.W. (1985) Dexamethasone permits the release of an inhibitor of pyruvate dehydrogenase from Reuber H-35 hepatoma cells in response to insulin. Endocrinology 117. 698-703. Fong, W.F., Pong, H.N.H., Yang, M.S. and Wong, P.C.L. (1993). Independent actions of asparagine and high levels of free Ca*+ in the induction of omithine decarboxylase. Cell Calcium 14, 45-51. Fowler, B.A., Hildebrand C.E., Kojima, Y. and Webb, M. (1987). Nomenclature of metallothionein. Experientia Suppl. 52, 19-22. Frey, A.B., Rosenfeld, M.G., Dolan, W.J., Adesnik, M. and Dreibich, G. (1984) Induction of cytochrome P-450 isoenxyme in rat hepatomaderived cell cutlures. J. Cell Physiol. 120, 169-180. Goering, P.L. and Klaassen, C.D. (1983) Altered subcellular distribution of cadmium following cadmium pretreatment: possible mechanism of tolerance to cadmium-induced lethality. Toxicol. Appl. Pharmacol. 73, 195-203. Goering, P.L. and Klaassen, C.D. (1984) Tolerance to cadmium-induced hepatotoxicity following cadmium pretreatment. Toxicol. Appl. Pharmacol. 74, 308-313. Grady, D.L., Moyxis, R.K. and Hildebrand, C.E. (1987) Molecular and cellular mechanisms of cadmium resistance in cultured cells. Experientia Suppl. 52, 447-455. Imura, N., Naganuma, A. and Satoh, M. (1989) Metallothionein as a resistance factor for antitumor drugs. Gan To Kagaku Ryoho. 16, 599-604. Kitagawa, Y. and Sugimoto, E. (1985) Interaction between glucocorticoids, 8-bromoadenosine 3’,5’-monophosphate and insulin in regulation of synthesis of carbamoyl-phosphate synthetase I in Reuber hepatoma H-35. Eur. J. B&hem. 150,249-254. Klaassen CD. and Eaton, D.L. (1991) Principles of toxicology. In: M.O. Amdur, J. Doull and C.D. Klaassen (Eds), Toxicology - The Basic Science of Poisons. 4th Edn., Pergamon Press, Oxford, pp. 18-23. Orlowski, C.C., Ooi, G.T. and Rechler, M.M. (1990) Dex-
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MS.
Yang et al. I Toxicology 104 (1995) 99-104
amethasone stimulates transcription of the insulin-like growth factor-binding protein-I gene in H4-II-E rat hepatoma cells. Mol. Endocrinol. 4, 1592-1599. Reuber, M.D. (1961) A transplantable bile-secreting hepatocellular carcinoma in the rat. J. Natl. Cancer Inst. 4, 891-897. Rudd, C.J. and Herschman, H.R. (1979) Metallothionein in a human cell line: The response of HeLa cells to cadmium and zinc. Toxicol. Appl. Pharmacol. 47, 273-278. Sambrook J., Fritsch, E.F. and Maniatis, T. (1989) In: C. Nolan (Ed), Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 7-15. Wiebel, F.J., Kiefer, F. and Murdia, U.S. (1990) Phenobarbital induces cytochrome P-450- and cytochrome P-448dependent monoxygenases in rat hepatoma cells. Chem.Biol. Interact. 52, 151-162. Winge, D.R., Premakumar, R. and Pajagopalan, K.V. (1975) Metal induced formation of metallothionein in rat liver. Arch. B&hem. Biophy. 170, 242-252.
Toxicology 104 (1995) 99-104
Induction of metallothionein in the Reuber H-35 rat hepatoma cell M.S. Yang*, M.K. Tang, R.N.S. Wong Department
of Biofogy, Hong Kong Baptist University, Kowloon Tong, Hong Kong
Received 12 January 1995; accepted 2 May 1995
Abstract The feasibility of using the Reuber H-35 rat hepatoma cell (RH-35 cells) as model for studying metallothionein induction was examined. The RH-35 cells were treated with Cd, a toxic metal which is known to induce metallothionein. The LCu, after a 3-h treatment was 70 pM. The value was significantly higher (P < 0.05) if the cells were pre-treated with a sublethal dose of CdCl, (5 PM) for 2 days, indicating that pre-treatment with a low dose of Cd can protect against a subsequent higher dose of the same metal. Both the mRNA and the gene product metallothionein can be identified in the cells 2 days after treatment with 5 pM Cd. In addition to Cd, Zn and Cu were also able to induce the expression of metallothionein to various degrees. The results indicate that the MT gene is present in RH-35 cells and is responsive to treatment with various metals. Thus, this cell line can be used as a model to study metallothionein induction.
Keywords: Metallothionein
induction; Reuber H-35 rat hepatoma cell; Cadmium
1. Introduction Metallothionein (MT), a cytosolic, low molecular weight, cysteine-rich metal-binding protein, is normally undetectable in cells, but its level increases upon exposure of cells or tissues to elevated levels of toxic metal such as cadmium, Abbreviations:
PBS, phosphate-buffered saline; DEPC, di-
ethyl pyrccarbonate; SSC, standard saline citrate; LC&, concentration of metal that causes 50% cell death as detected by the trypan-blue exclusion test; MEM, minimum essential medium; Tris, tris-(hydroxymethyl)amino methane; Cd, cadmium; Zn, zinc; Cu, copper; Ni, nickel. l Corresponding author, Tel.: 852-2339-7058; Fax: 8522336-1400.
mercury and silver (Fowler et al., 1987). The protein is believed to play an important role in metal detoxification since exposure to low dose of Cd can protect against a subsequent higher dose of the same toxic metal (Goering and Klaassen, 1984). It is believed that the Cd-induced metallothionein can chelate the subsequently added metal, thus preventing it from interacting with cytoplasmic organelles (Goering and Klaassen, 1983). Besides toxic metals, Zn and Cu are found capable of inducing MT (Fowler et al., 1987). Both Zn and Cu are important cofactors for a wide variety of enzymatic reactions. Thus, MT may also play a role in regulating the level of these endogenous trace metals (Bremner, 1987).
0300-483)(/95/W9.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0300-483X(95)03149-A
100
MS. Yang et al. / Toxicology 104 (199s) 99-104
Metals are not the only elements that can induce MT synthesis. Physiological stimuli, such as starvation, stress and hyperoxia; hormones, such as catecholamines, glucocorticoid, glucagon and cytokines; and chemicals, including turpentine, alcohol, carbon tetrachloride, can also stimulate MT production (Bremner, 1987). However, the mechanism(s) of MT induction are not well understood. We have been investigating the use of a simple cell culture model to study the mechanism(s) of metallothionein induction. The Reuber H-35 rat hepatoma cell (RH-35 cells) is a clonal cell line that can easily be maintained in culture. The cell line was found to express numerous functions of the hepatocytes of adult liver (Reuber et al., 1961). It has been used as an in vitro model in studies concerning the insulin and glucocorticoidscontrolled liver function (Esau and Koontz, 1985; K.itagawa and Sugimoto, 1985; Orlowski et al., 1990). The cell line has retained the ability to induce drug-metabolizing enzymes (Frey et al., 1986; Wiebel et al., 1986). In our laboratory, the RH-35 cells have been used as a model to study the molecular basis of enzyme induction (Fong et al., 1991). In the present study, metallothionein was identified in this cell line and the gene is found to be inducible by various metals. 2. Materials and methods 2.1. Determination of Cd cytotoxicity in the RH-35 ceils The Reuber H-35 rat hepatoma cells were maintained at 37°C in MEM (Gibco, Gathersburg, TX) supplemented with 10% fetal bovine serum (FBS) (Flow Laboratories, North Ryde, NSW, Australia) and antibiotics (amphotericin B and pencillinstreptomycin) in a humidified atmosphere of 95% air and 5% CO*. Five sets of plates, in triplicate, were prepared. Upon confluence, MEM containing different doses (0, 10,50,70, 100 and 150 PM) of CdC12 was added to different plates. Then, 3 h after addition of CdC&, the medium were removed and the cells were washed twice with PBS. The cells were dislodged from the plates by treatment with 0.1 ml of 0.05% trypsin followed by addition of 0.5 ml of 0.6% trypan blue solution. The tryp-
sinized cells were transferred to a hemocytometer for counting. Five different areas on the hemocytometer were selected randomly and cells were counted under the microscope. Both the trypan blue stained (dead) cells and the unstained (live) cells were counted. The number of cells in each count ranged between 60-100. The percent of cell death was calculated by dividing the total number of stained cells by the total number of cells (both stained and unstained) counted. LCso for Cd, together with the upper and lower 95% confidence limits, were calculated by the probit analysis (Klaassen and Eaton, 1991) using the SAS/SPSS statistical software package. 2.2. Protection against CdC12by pretreatment with sublethal dose of the same metal To examine the ability of CdC12 to protect against a subsequent dose of the same toxic metal, 5 PM (final concentration) of CdC12 was added to the incubation medium and the cells were allowed to grow for 2 days. After incubation, the medium was removed and the cells washed with PBS. They were then challenged with different doses of CdCl, as described previously. The percentage of cell death, the LCw and the upper and lower 95% confidence limits were also calculated. 2.3. Isolation of metallothionein from the RH-35 cells After pre-treatment with 5 PM CdCl* for 2 days, the cells were washed with 0.02 M Tris-HCl, pH 7.4, harvested and sonicated. The broken cells were centrifuged at 100 000 x g (Hitachi 7OP-72 preparative ultracentrifuge) for 60 min at 4°C. The supematant was collected and 5 ~1 of CdCl, (1 mg/rnl) was added to every 950 ~1 sample as an internal standard to facilitate subsequent detection. The samples were then heated in boiling water for 1 min. The precipitated protein was removed by centrifugation at 10 000 x g for 5 min. The supernatant (3 ml) was applied onto a Sephadex G-75 column (26 mm x 40 cm) together with blue dextran as marker for void volume measurement. The proteins were eluted with the same buffer at a flow rate of 20 ml/h at room temperature. Five-ml fractions were collected. The optical density at 254 nm for each fraction was determined and the concen-
M.S. Yang et al. /Toxicology 104 (1995) W-104
tration of cadmium was determined by flame atomic absorption spectroscopy (Varian, Spectra AA-IO). 2.4. Determination for MT induction
of doses of Zn, Ni and Cu used
The RH-35 cells were exposed to different concentrations of either ZnC&, CuC12 or NiCl* for 3 h and the percent of dead cells was measured using the trypan blue exclusion test. The LC,, was calculated by the probit analysis using the SASISPSS package. A sublethal dose was selected to test the MT inducibility (Table 2). 2.5. Extraction of total RNA All glasswares and disposables were treated with DEPC and sterilized by autoclaving to prevent the possible contamination of RNAse. Total RNA from at least 10’ treated cells was extracted using the guanidine thiocyanate method (Chomczynski and Sacchi, 1987). Cells were washed twice with ice-cold DEPCYPBS before the addition of the denaturing solution (4 M guanidine thiocyanate, 25 mM sodium citrate, pH 7, 0.5% N-lauryl sarcosine, 0.1 M &mercaptoethanol). About l/10 vol of 2 M sodium acetate, pH 4.0 was added, the solution was then extracted with phenol/chloroform/ isoamyl alcohol mixture (25:24:1 v/v). The aqueous phase containing the extracted RNA was precipitated with an equal volume of isopropanol and the mixture was allowed to stand at -20°C overnight. The precipitated RNA was pelleted by centrifugation at 10 000 x g at 4°C. The RNA, after washing with 70% ethanol, was dried and dissolved in DEPC/water. The intactness of the RNA was confirmed by denaturing agarose gel electrophoresis (Sambrook et al., 1989). 2.6. Preparation of MT-cDNA A plasmid, pBXA containing the mouse MTIcDNA, was kindly provided by Dr. R.D. Palmiter, University of Washington, Seattle, WA. After transformation into E. coli RR1 followed by amplification of the transformants in a liquid LuriaBertani medium. A large scale plasmid preparation was performed according to Sambrook et al. (1989). The MTIcDNA was released from the vector by BumHI (Promega, Madison, WI) restriction
101
digestion and the fragments were separated by electrophoresis on a 7% polyacrylamide gel. The 300bp MTI-cDNA was recovered by electroelution using the electro-eluter (Bio-Rad, San Diego, CA). For northern blot hybridization, the MTIcDNA was labeled with 32P-dCTP using the oligolabeling kit from Pharmacia (Milwaukee, WI). 2.7. Northern blot analysis Northern blot analysis was carried out as follow. Equal amounts of RNA from different samples were subjected to electrophoresis on a 1.5% agarose gel containing formaldehyde. The separated RNAs were then transferred to a nylon membrane by capillary blotting in 20 x SSC. After prehybridizing at 65°C for 2 h, the membrane was subsequently hybridized with the heat-denatured radiolabelled MTcDNA. Hybridization was performed at 65’C overnight with the MTIcDNA probe in a solution containing 6 x SSC, 5 x Denhardt’s solution, 0.5% SDS and denatured salmon sperm DNA (20 &ml). After hybridization, the excess probe was removed by washing the membrane twice at 65°C with 2 x SSC and 2 x SSC, 0.1% SDS for an interval of lo-15 min for each wash. The washed membrane was air dried and wrapped in plastic wrap for autoradiography. 3. Results and discussion 3.1. Protection against Cd-induced cell death after pre-treatment of ceils with a low dose of Cd Fig. 1 shows that increasing cell death occurred with increasing doses of Cd in the medium. The LC% for Cd was calculated to be 70 PM with the upper and lower 95% confidence levels of 90 and 60 PM respectively (Table 1). Pre-treatment of the RH-35 cells with a low dose of Cd (5 PM) for 2 days reduced the toxicity of a subsequent dose of the same metal. The LC, increased significantly (P < 0.05) from 70 PM to 220 PM (Table 1) upon Cd pre-treatment. 3.2. Isolation of a cadmium binding-protein from the RH-35 cells after 2 days pre-treatment CdCl*
with 5 PM
Fig. 2 shows that, 2 days after Cd pre-treatment,
MS.
102
Yang et al. I Toxicology 104 (1995) 99-104
70 60
0
No
0
Cd pretreatment
Cd
pre-treatment
P
50 5 d = 8 B 2 8 b 0
s 40
0.0
00 10
0
30
20 Fraction
20
30
40
0.8
08
-
2. 0.6 -
10
50
number
0.6
MT
F
6
0 10’
102
Cd concentration
(log
uM)
Fig. 1. Percentage of cell death of the RH-35 cells after exposure to different doses of CdC& for 3 h. The values are mean f SD. of triplicates. Cd pretreatment was carried out by exposing the cells to 5 PM CdC12 for 2 days prior to Cd challenge.
a fraction of protein with a molecular weight intermediate between the high and low molecular weight proteins, can be identified in the RH-35 cells upon Sephadex G-75 chromatography. This fraction of protein has low optical density at 254 nm and high Cd content. The position on the chromatogram (V&V, = 2.5) is characteristic of that of metallothionein previously reported (Winge et al., 1979). It is not present in control cells without Cd pre-treatment, indicating that it is synthesized in response to Cd. The induction of MT by Cd and its protection against metal-induced toxicity has
0
10
20
Fraction
30
40
50
number
Fig. 2. Sephadex G-75 chromatogram of the heat stable proteins from, (I) control and, (2) Cd-treated RH-35 cells. 0 represents the 254 nm readings and W represents the Cd level &/ml) in each fraction.
9.5kb_ 7.5kb_ 4&b_ 2.4kb_ 1.4kb_
MT Table I Lt.&s and the 95% confidence limits of CdClz for the RH-35 cells in culture before and after pretreatment with a low dose of cadmium Pretreatment (2 days)
None 5 CM CdC12
0.24kb_
95% Confidence limits Lower limits
Upper limits
66 150
90 330
Fig. 3. Northern blot analysis of MT from control RH-35 cells and cells treated with Cd (5 PM), Zn (75 PM), Cu (50 PM) and Ni (396 PM). The size of MT-mRNA is approximately 0.3 kb as calculated from molecular weight markers on the left.
MS. Yanget al. /Toxicology 104 (199s) 99-104 Table 2 LCa of Zn, Ni and Cu for the RH-35 cells Metal
LCss (CcM)
Induction dosage (FM)
ZnClz NQ cuq
1220 6210 170
75 390 50
been confirmed in other cell culture models. A wildtype Chinese hamster ovary (CHO) cell line which did not produce MT was extremely sensitive to Cd. Upon conversion to a MT-inducible phenotype in the presence of 5-azacytidine, the cell line became resistant to Cd (Grady et al., 1987). Similar Cd resistance was also conferred in a mouse hepatoma cell line upon transformation to the MT-producing phenotype (Durnam and Pahniter, 1987). In addition to the protein, the mRNA for MT was also identified in the RH-35 cells. Fig. 3 shows the presence of MT-mRNA in cells after a 2-day treatment with 5 PM CdC12. 3.3. Induction of MT-mRNA by other metals Table 2 shows the LC+ of Zn, Ni and Cu for the RH-35 cells. The results show that while Cu is more toxic than Zn and Ni, it is ten times less toxic than Cd. Fig. 3 shows that Zn and Cu, at concentrations below the LCso, were able to induce MT gene expression. There is little MT induction after treatment with Ni (Fig. 3). Similar to a number of mammalian cell lines studied (Rudd and Hers&man, 1979; Cherian, 1980; Dumam and Pahniter, 1984), the RH-35 cells possess the MT gene which is responsive to metal induction. Besides Cd, Zn and Cu are also good inducers. The results show that the RH-35 cells can be used as a model for studying MT gene induction. Recently, the metal-induced metallothionein has been found to reduce the side-effects associated with metal-containing antitumor drugs (e.g. cisplatin) treatment (Basu and Laze, 1990). The finding has raised considerable interest in the search for appropriate MT inducers to be used as pre-treatment regime prior to administration of the drug (Imura et al., 1989, Basu and Lazo, 1990). In addition to studying the mechanism(s) of MT induction, the RH-35 rat hepatoma cell can also be used as a model for preliminary screening for such agents.
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