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Induction of apoptosis in mouse thymocytes by cadmium
Toxicology Letters 115 (2000) 99 – 105 www.elsevier.com/locate/toxlet
Induction of apoptosis in mouse thymocytes by cadmium Hidekazu Fujimaki *, Masami Ishido, Keiko Nohara En6ironmental Health Sciences Di6ision, National Institute for En6ironmental Studies 16 -2, Onogawa, Tsukuba, Ibaraki 305 -0053 Japan Received 20 September 1999; received in revised form 7 January 2000; accepted 10 January 2000
Abstract In the thymus apoptosis is an important process in T cell maturation and differentiation. Cadmium (Cd) is an ubiquitous toxic metal that is capable of modulating immune responses. To investigate the induction of apoptosis and immunomodulation by environmental chemicals, we cultured mouse thymocytes with Cd and/or dexamethasone (DEX). DNA fragmentation was analyzed by gel electrophoresis, ELISA and flow cytometry. Treatment with either Cd or DEX induced DNA fragmentation in the thymocytes. Exposure to 10 mM Cd killed thymocytes by apoptosis rather than necrosis. However, no synergistic or additive effect was observed in the induction of apoptosis when DEX was added to the Cd. These results suggest that Cd may modulate the function of the thymus by the induction of apoptosis through mechanisms that differ from those used by DEX. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Apoptosis; Thymocyte; Cadmium
1. Introduction Apoptosis is a significant process in T cell development and differentiation. Modulation of apoptosis results in dysfunction of T cell dependent immune responses. Environmental chemicals such as tributyltin, arsenide, indium, and methylcholanthrene have been shown to induce apoptosis in rodent thymocytes (Raffray and Cohen, 1993; Bustamante et al., 1997; Lutz et al., 1998). Exposure to cadmium (Cd) results in modulation of antibody production, inhibition of B-cell * Corresponding author.
activation, enhancement of mortality in mice challenged with streptococci, and decreased mortality of influenza-infected mice (Koller et al., 1976; Gardner et al., 1977; Fujimaki et al., 1982; Chaumard et al., 1983; Ohsawa et al., 1986; Daum et al., 1993). Injection of Cd induces thymic atrophy in mice (Yamada et al., 1981). However, it is uncertain whether apoptosis is a key mechanism in Cd-induced thymic atrophy. Cd at low concentrations (4–10 mM) kills the human T cell line CEM-C12 by apoptosis (Azzouzi et al., 1994), and treatment with 10 mM Cd induces DNA fragmentation in cultured LLC-PK1 cells (Ishido et al., 1995). In the T-cell line CCRF-CEM and the lymphoblastoid cell line Molt-3, apoptosis has been detected with exposure to 10 mM Cd (Tsan-
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garis and Tzortzatou-Stathopoulou, 1998). These in vitro studies suggest that exposure to 10 mM Cd may activate apoptotic mechanisms that affect the intracellular enzymatic pathways. It is therefore possible to hypothesize that the process of apoptosis in the thymus is influenced by exposure to Cd. The aims of our study were to clarify whether in vitro treatment with Cd could induce apoptosis in mouse thymocytes and to compare the effects of Cd with those of dexamethasone (DEX), an agent with well-documented immunomodulatory properties.
2. Materials and methods
2.1. Cell culture Thymocytes were isolated from male BALB/c mice (1–3 months old) purchased from Clea
Fig. 1. Induction of DNA fragmentation in mouse thymocytes. Mouse thymocytes (2 × 107) were exposed to control (lane 2), 1 mM DEX (lane 3), or 10 mM Cd (lane 4) for 7 h. The treated cells were then lysed in 5 mM Tris buffer, pH 7.4, containing 0.5% Triton X-100 and 20 mM EDTA. The DNA fragments formed were extracted with phenol-chloroform and electrophoresed on a 1.2% agarose gel. Lane 1 shows a size marker. One experiment representative of two is shown.
Fig. 2. Apoptotic features were detected by ELISA for histone-associated DNA fragments. After the thymocytes had been treated with Cd or DEX, the cells were centrifuged, and the supernatants collected. The Cell pellet fractions were incubated with lysis buffer for 30 min at room temperature, then centrifuged and the supernatants collected. Culture supernatants or cell lysates were added to streptavidin-coated microtiter plates in the presence of biotin-labeled anti-histone and peroxidase conjugated anti-DNA antibodies and incubated for 2 h at room temperature. After the plates had been washed 3 times, substrate solution (2,2%-Azino-di[2-ethylbenzthiazolinsulfonat]) was added and the plates were incubated for 10 – 20 min. Absorbance was measured at 405 and 490 nm with a microplate reader. Data represent the means and standard errors of three experiments.
Japan, Inc., Tokyo, Japan. Single cell suspension of the thymocytes was passed through stainlesssteel mesh and a cell count was performed in RPMI-1640 with 10% FCS, 2 mM glutamine, 5 mM HEPES, 0.1 mM nonessential amino acids, and 100 U/ml penicillin- 100 mg/ml streptomycin. Thymocytes with various concentrations of cadmium chloride (Cd) (Wako Pure Chemicals, Osaka, Japan) and 1 mM DEX as a positive control were cultured in RPMI-1640 without FCS for either 3 or 7 h and then prepared for the following investigations.
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2.2. DNA extraction and agarose gel electrophoresis
2.3. ELISA for in 6itro determination of nuclease clea6es
DNA fragmentation was investigated by gel electrophoresis (Ishido et al., 1995). Briefly, cellular DNA from thymocytes that had been cultured for 7 h with 10 mM Cd or 1 mM DEX was isolated in 5 mM Tris–HCl, pH 7.4, containing 20 mM EDTA and 0.5% Triton X-100, at 4°C for 20 min. After this solution had been centrifuged at 28 000× g for 20 min, the supernatants were extracted with phenol-chloroform and precipitated in ethanol. Half of each sample was treated with 20 mg/ml RNase A and electrophoresed on 1.2% agarose gel. After electrophoresis, the gel was stained with ethidium bromide and visualized under UV light.
To further confirm the induction of apoptosis, ELISA for the qualitative and quantitative determination of cytoplasmic histone-associated-DNAfragments was performed with commercial kits (Boehringer Mannheim GmbH, Mannheim, Germany). After the thymocytes had been cultured with Cd and DEX for 7 h, the cells were centrifuged, and the supernatants collected. The cell pellet fractions were incubated with lysis buffer for 30 min at room temperature. They were then centrifuged and the supernatants were collected. Culture supernatants or cell lysate were added to streptavidin-coated microtiter plate in the presence of biotin-labeled anti-histone and peroxidase
Fig. 3. Apoptotic cells discriminated by the TUNEL technique in thymocytes exposed to various doses of Cd. Thymocytes were cultured in the absence or presence of 0.1, 1.0 and 10 mM Cd for 7 h. With the TUNEL method, 3%-OH DNA ends generated by DNA fragmentation are nick end labeled with biotinylated dUTP, introduced by terminal deoxytransferase (TdT) and then stained with avidin-conjugated FITC to allow specific staining. (a) Control, (b) 0.1 mM Cd, (c) 1.0 mM Cd and (d) 10 mM Cd. One representative experiment of four separate thymocyte preparations is shown.
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nm with a microplate reader (Bio-Rad, Nippon Bio-Rad Laboratory KK, Japan).
2.4. Flow cytometry assay
Fig. 4. Induction of apoptotic cells discriminated by the TUNEL technique in thymocytes exposed to Cd or DEX. Thymocytes were cultured with 1.0 mM DEX or 10 mM Cd for 7 h. With the TUNEL method, 3%-OH DNA ends generated by DNA fragmentation are nick end labeled with biotinylated dUTP, introduced by terminal deoxytransferase (TdT) and then stained with avidin-conjugated FITC to allow specific staining. (a) Control, (b) 1.0 mM DEX and (c) 10 mM Cd. One experiment representative of three is shown.
conjugated anti-DNA antibodies and incubated for 2 h at room temperature. After the plates had been washed three times, substrate solution (2,2%Azino-di[2-ethylbenzthiazolin-sulfonat]) was added, and the plates were incubated for 10 – 20 min. Absorbance was measured at 405 and 490
Flow cytometry analysis was done with a MEBSTAIN apoptosis kit (MBL, Nagoya, Japan). In brief, thymocytes that had been cultured with Cd and DEX for 7 h were washed twice with PBS containing 0.2% BSA and then fixed with 4% paraformaldehyde at 4°C for 30 min. After the cells had been washed twice again, they were incubated with proteinase K solution for 30 min, and with TdT reaction reagent for 1 h at 37°C. After the cell pellets had been washed, 10 ml of blocking reagent were added and the cells were kept at room temperature for 10 min. Thirty microliters of avidin-FITC reagent were then added and the cells were incubated in the dark at room temperature for 30 min. After washing twice, the cell pellets were suspended in 500 ml of PBS containing 0.2% BSA. The FITC labeled cells were analysed by flow cytometry. To compare the induction of apoptosis and necrosis in cells that had been treated with 10 mM Cd and/or 1 mM DEX for either 3 or 7 h, the ability to bind annexin V and exclude propidium iodide was determined with an apoptosis detection kit (R&D Systems, Minneapolis, MN). Necrotic cells stain with both annexin V FITC and propidium iodide, while cells undergoing apoptosis stain only with the annexin V FITC. Briefly, thymocytes were suspended in binding buffer at a concentration of 1×106 cells/ml. Ten microliters of FITC-conjugated annexin V and 10 ml of propidium iodide were added to the cells (1× 105/100 ml), and they were incubated in the dark for 15 min at room temperature. Following the incubation, 400 ml of binding buffer were added to the cell suspension, which was then analysed with a flow cytometer.
2.5. Statistical analysis Data were one representative experiment out of two to four and were analysed using Student’s t-test.
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3. Results To investigate whether in vitro Cd exposure may induce apoptosis in mouse thymocytes, thymocytes were cultured with Cd or DEX as a positive control for 7 h. Treatment of the thymocytes with 10 mM Cd or 1 mM DEX resulted in DNA ladder formation on gel electrophoresis, indicating degradation of nuclear DNA by both the Cd and the DEX (Fig. 1). To further confirm the apoptosis induced by Cd, apoptotic features were detected by ELISA for histone-associated DNA fragments. The absorbance of the cell lysate from cultured thymocytes exposed to 10 mM Cd or 1 mM DEX was significantly increased compared with that of the control (Fig. 2). There was no increase in absorbance in the culture supernatants of each group of thymocytes. These results suggest that Cd treatment may induce the formation of histone-associated DNA fragments in thymocytes.
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For the evaluation of apoptosis in thymocytes by the TUNEL method, thymocytes were cultured in the absence or presence of 0.1 mM, 1.0 mM and 10 mM Cd for 7 h. A marked increase in the number of labeled thymocytes was detected with 10 mM Cd (Fig. 3). Concentrations of 0.1 mM and 1 mM Cd did not induce a significant increase in the numbers of labeled cells with nuclear DNA fragments. The number of labeled thymocytes with nuclear DNA fragments was found to have been markedly increased by the presence of either 1 mM DEX (b) or 10 mM Cd (c) compared with the control (a) (Fig. 4). The effect of DEX was more pronounced than that of Cd. To compare the modulating activity of Cd with that of DEX, the percentages of necrotic and apoptotic cells induced by each were measured. These percentages were also measured for a mixture of Cd and DEX. Flow cytometric analysis with FITC-conjugated annexin V and propidium iodide showed that over 3 h of incubation, expo-
Fig. 5. Induction of apoptosis and necrosis in thymocytes exposed to Cd and/or DEX. Flow cytometric analysis with FITC-conjugated annexin V and propidium iodide was performed after incubation for 3 or 7 h with 10 mM Cd and/or 1 mM DEX. Each column shows the mean percentage of the control. (a) At 3 h the control values for apoptosis and necrosis were 15.1 9 0.9% and 8.7 90.6% (mean9SE of two different experiments), respectively. (b) At 7 h the control values for apoptosis and necrosis were 18.0 9 0.6% and 10.49 0.6% (mean9SE of three different experiments), respectively.
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sure to 1 mM DEX markedly increased the percentage of apoptotic cells, but exposure to 10 mM Cd did not (Fig. 5a). However, exposure to 10 mM Cd over a 7 h incubation significantly increased the percentage of apoptotic cells compared with that of necrotic cells (Fig. 5b). Thymocytes exposed to control, Cd, DEX and Cd + DEX showed apoptotic values of 18.0 9 0.6, 41.3 90.1, 55.4 9 0.8 and 57.5 91.0 (mean 9 SE of three experiments), respectively. The mixture of Cd and DEX did not show synergic or additive induction of apoptosis in thymocytes compared with Cd or DEX alone. Cd exposure resulted in a slow induction of apoptosis compared with DEX.
4. Discussion In this study, we investigated the in vitro effects of Cd on the induction of apoptosis in mouse thymocytes. Cd exposure is known to induce thymic atrophy as well as immunosuppression in mice (Koller et al., 1976; Gardner et al., 1977; Yamada et al., 1981; Fujimaki et al., 1982; Chaumard et al., 1983; Ohsawa et al., 1986; Daum et al., 1993; Schulte et al., 1994). However, it has remained unclear whether thymic atrophy results from the induction of apoptosis by Cd. In our study, DNA fragmentation, one of the hallmarks of apoptosis, in Cd-exposed thymocytes was characterized by gel electrophoresis, ELISA and flow cytometric analysis. Our results indicated that in vitro Cd exposure induced apoptotic features in mouse thymocytes. Although the induction of apoptosis by Cd was slower than that induced by DEX, the magnitude of the induction was similar. Zucker et al. (1994) studied the effects of tributyltin (TBT) and DEX on the induction of apoptosis in rat thymocytes and showed that simultaneous exposure to TBT and DEX resulted in a decreased induction of apoptosis. They suggested that TBT and DEX, are mutual antagonists and initiate endonuclease-mediated apoptotic cell death through different mechanisms. In our present study, simultaneous exposure to DEX did not affect the extent of
Cd-induced apoptosis. The mechanisms by which glucocorticoids induce apoptosis have been discussed. Endogenous endonuclease activity during apoptosis of glucocorticoid-treated thymocytes is known to be inhibited by zinc (Nieto and LopezRivas, 1989). Expression of c-myc can trigger apoptosis, but apoptosis induced by glucocorticoids can proceed in the absence of c-myc protein (Bissonnette et al., 1994). Our previous papers indicate that zinc can suppress the DNA laddering formation of apoptosis elicited by Cd (Ishido et al., 1995, 1999). We have also shown that Cd induces apoptosis in the absence of transcriptional activation of c-myc (Ishido et al., 1998). In the present study, there was a slight difference in the time-dependent induction of apoptosis between Cd and DEX (Fig. 5). Taken together, these findings suggest that Cd and DEX exposure may induce apoptosis in mouse thymocytes through a mechanism independent of c-myc function and zinc may play a role. Glucocorticoids induce thymocyte apoptosis by activating a calcium-dependent endonuclease (Cohen and Duke, 1984). However, Lohmann and Beyersmann (1993) observed a significant increase in DNA fragmentation in the presence of 10 mM Cd in a Ca2 + -free system. Additional data are required to determine whether the induction of apoptosis by Cd is Ca2 + and zinc-dependent or not. In conclusion, the induction of mouse thymocyte death by in vitro Cd exposure may be caused by the induction of apoptosis rather than by necrosis. Further studies on the relationship between thymic atrophy and the induction of apoptosis in the thymocytes of mice exposed to Cd are needed.
Acknowledgements This work was supported partly by a grant-in-aid for scientific research (B) from the Ministry of Education, Science, Sports and Culture of Japan and by special coordination funds for promoting Science and Technology supplied by the Science and Technology Agency of Japan.
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References Azzouzi, B.E., Tsangaris, G.T., Pellegrini, O., Manuel, Y., Benveniste, J., Thomas, Y., 1994. Cadmium induces apoptosis in a human T cell line. Toxicology 88, 127–139. Bissonnette, R.P., Shi, Y., Mahboubi, A., Glynn, J.M., Green, D.R., 1994. C-myc and apoptosis. In: by David Tomei, L., Cope, F.O. (Eds.), Apoptosis II: The Molecular Basis of Apoptosis in Disease. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp. 327–356. Bustamante, J., Dock, L., Vahter, M., Fowler, B., Orrenius, S., 1997. The semiconductor elements arsenic and indium induce apoptosis in rat thymocytes. Toxicology 118, 129– 136. Chaumard, C., Quero, A.M., Bouley, G., Girard, F., Boudene, C.L., German, A., 1983. Influence of inhaled cadmium microparticles on mouse influenza pneumonia. Environ. Res. 31, 428 – 439. Cohen, J.J., Duke, R.C., 1984. Glucocorticoid activation of a calcium-dependent endonuclease in thymocyte nuclei leads to cell death. J. Immunol. 132, 38–42. Daum, J.R., Shepherd, D.M., Noelle, R.J., 1993. Immunotoxicology of cadmium and mercury on B-lymphocytes-1. Effects on lymphocyte function. Int. J. Immunophamac. 15, 383 – 394. Fujimaki, H., Murakami, M., Kubota, K., 1982. In vitro evaluation of cadmium-induced augmentation of the antibody response. Toxicol. Appl. Pharmacol. 62, 288–293. Gardner, D.E., Miller, F.J., Illing, J.W., Kirtz, J.M., 1977. Alterations in bacterial defense mechanisms of the lung induced by inhalation of cadmium. Bull. Eur. Physiopathol. Resp. 13, 157–174. Ishido, M., Homma, S.T., Leung, P.S., Tohyama, C., 1995. Cadmium-induced DNA fragmentation is inhibitable by zinc in porcine kidney LLC-PK1 cells. Life Sci. 56, 351– 356. Ishido, M., Tohyama, C., Suzuki, T., 1998. C-myc is not involved in cadmium-elicited apoptotic pathway in porcine kidney LLC-PK1 cells. Life Sci. 63, 1195–1204.
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Ishido, M., Suzuki, T., Adachi, T., Kunimoto, M., 1999. Zinc stimulates DNA synthesis during its antiapototic action independently with increments of an antiapoptotic protein, bcl-2, in porcine kidney LLC-PK1 cells. J. Pharmacol. Exp. Ther. 290, 923 – 928. Koller, L.D., Exon, J.H., Roan, J.G., 1976. Humoral antibody response in mice after a single dose exposure to lead or cadmium. Proc. Soc. Exp. Biol. Med. 151, 339 – 342. Lohmann, R.D., Beyersmann, D., 1993. Cadmium and zinc mediated changes of the Ca2 + -dependent endonuclease in apoptosis. Biochem. Biophys. Res. Commun. 190, 1097 – 1103. Lutz, C.T., Browne, G., Petzold, C.R., 1998. Methylcholanthrene causes increased thymocyte apoptosis. Toxicology 128, 151 – 167. Nieto, M.A., Lopez-Rivas, A., 1989. IL-2 protects T lymphocytes from glucocorticoid-induced DNA fragmentation and cell death. J. Immunol. 143, 4166 – 4170. Ohsawa, M., Masuko-Sato, K., Takahashi, K., Otsuka, F., 1986. Strain differences in cadmium-mediated suppression of lymphocyte proliferation in mice. Toxic. Appl. Pharmacol. 84, 379 – 388. Raffray, M., Cohen, G.M., 1993. Thymocyte apoptosis as a mechanism for tributyltin-induced thymic atrophy in vivo. Arch. Toxicol. 67, 231 – 236. Schulte, S., Mengel, K., Gatke, U., Friedberg, K.D., 1994. No influence of cadmium on the production of specific antibodies in mice. Toxicology 93, 263 – 268. Tsangaris, G.Th., Tzortzatou-Stathopoulou, F., 1998. Cadmium induces apoptosis differentially on immune system cell lines. Toxicology 128, 143 – 150. Yamada, Y., Shimizu, F., Kawamura, R., Kubota, K., 1981. Thymic atrophy in mice induced by cadmium administration. Toxicol. Lett. 8, 49 – 55. Zucker, R.M., Elstein, K.H., Thomas, D.J., Rogers, J.M., 1994. Tributyltin and dexamethasone induce apoptosis in rat thymocytes by mutually antagonistic mechanisms. Toxicol. Appl. Pharmacol. 127, 163 – 170.
Induction of apoptosis in mouse thymocytes by cadmium Hidekazu Fujimaki *, Masami Ishido, Keiko Nohara En6ironmental Health Sciences Di6ision, National Institute for En6ironmental Studies 16 -2, Onogawa, Tsukuba, Ibaraki 305 -0053 Japan Received 20 September 1999; received in revised form 7 January 2000; accepted 10 January 2000
Abstract In the thymus apoptosis is an important process in T cell maturation and differentiation. Cadmium (Cd) is an ubiquitous toxic metal that is capable of modulating immune responses. To investigate the induction of apoptosis and immunomodulation by environmental chemicals, we cultured mouse thymocytes with Cd and/or dexamethasone (DEX). DNA fragmentation was analyzed by gel electrophoresis, ELISA and flow cytometry. Treatment with either Cd or DEX induced DNA fragmentation in the thymocytes. Exposure to 10 mM Cd killed thymocytes by apoptosis rather than necrosis. However, no synergistic or additive effect was observed in the induction of apoptosis when DEX was added to the Cd. These results suggest that Cd may modulate the function of the thymus by the induction of apoptosis through mechanisms that differ from those used by DEX. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Apoptosis; Thymocyte; Cadmium
1. Introduction Apoptosis is a significant process in T cell development and differentiation. Modulation of apoptosis results in dysfunction of T cell dependent immune responses. Environmental chemicals such as tributyltin, arsenide, indium, and methylcholanthrene have been shown to induce apoptosis in rodent thymocytes (Raffray and Cohen, 1993; Bustamante et al., 1997; Lutz et al., 1998). Exposure to cadmium (Cd) results in modulation of antibody production, inhibition of B-cell * Corresponding author.
activation, enhancement of mortality in mice challenged with streptococci, and decreased mortality of influenza-infected mice (Koller et al., 1976; Gardner et al., 1977; Fujimaki et al., 1982; Chaumard et al., 1983; Ohsawa et al., 1986; Daum et al., 1993). Injection of Cd induces thymic atrophy in mice (Yamada et al., 1981). However, it is uncertain whether apoptosis is a key mechanism in Cd-induced thymic atrophy. Cd at low concentrations (4–10 mM) kills the human T cell line CEM-C12 by apoptosis (Azzouzi et al., 1994), and treatment with 10 mM Cd induces DNA fragmentation in cultured LLC-PK1 cells (Ishido et al., 1995). In the T-cell line CCRF-CEM and the lymphoblastoid cell line Molt-3, apoptosis has been detected with exposure to 10 mM Cd (Tsan-
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garis and Tzortzatou-Stathopoulou, 1998). These in vitro studies suggest that exposure to 10 mM Cd may activate apoptotic mechanisms that affect the intracellular enzymatic pathways. It is therefore possible to hypothesize that the process of apoptosis in the thymus is influenced by exposure to Cd. The aims of our study were to clarify whether in vitro treatment with Cd could induce apoptosis in mouse thymocytes and to compare the effects of Cd with those of dexamethasone (DEX), an agent with well-documented immunomodulatory properties.
2. Materials and methods
2.1. Cell culture Thymocytes were isolated from male BALB/c mice (1–3 months old) purchased from Clea
Fig. 1. Induction of DNA fragmentation in mouse thymocytes. Mouse thymocytes (2 × 107) were exposed to control (lane 2), 1 mM DEX (lane 3), or 10 mM Cd (lane 4) for 7 h. The treated cells were then lysed in 5 mM Tris buffer, pH 7.4, containing 0.5% Triton X-100 and 20 mM EDTA. The DNA fragments formed were extracted with phenol-chloroform and electrophoresed on a 1.2% agarose gel. Lane 1 shows a size marker. One experiment representative of two is shown.
Fig. 2. Apoptotic features were detected by ELISA for histone-associated DNA fragments. After the thymocytes had been treated with Cd or DEX, the cells were centrifuged, and the supernatants collected. The Cell pellet fractions were incubated with lysis buffer for 30 min at room temperature, then centrifuged and the supernatants collected. Culture supernatants or cell lysates were added to streptavidin-coated microtiter plates in the presence of biotin-labeled anti-histone and peroxidase conjugated anti-DNA antibodies and incubated for 2 h at room temperature. After the plates had been washed 3 times, substrate solution (2,2%-Azino-di[2-ethylbenzthiazolinsulfonat]) was added and the plates were incubated for 10 – 20 min. Absorbance was measured at 405 and 490 nm with a microplate reader. Data represent the means and standard errors of three experiments.
Japan, Inc., Tokyo, Japan. Single cell suspension of the thymocytes was passed through stainlesssteel mesh and a cell count was performed in RPMI-1640 with 10% FCS, 2 mM glutamine, 5 mM HEPES, 0.1 mM nonessential amino acids, and 100 U/ml penicillin- 100 mg/ml streptomycin. Thymocytes with various concentrations of cadmium chloride (Cd) (Wako Pure Chemicals, Osaka, Japan) and 1 mM DEX as a positive control were cultured in RPMI-1640 without FCS for either 3 or 7 h and then prepared for the following investigations.
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2.2. DNA extraction and agarose gel electrophoresis
2.3. ELISA for in 6itro determination of nuclease clea6es
DNA fragmentation was investigated by gel electrophoresis (Ishido et al., 1995). Briefly, cellular DNA from thymocytes that had been cultured for 7 h with 10 mM Cd or 1 mM DEX was isolated in 5 mM Tris–HCl, pH 7.4, containing 20 mM EDTA and 0.5% Triton X-100, at 4°C for 20 min. After this solution had been centrifuged at 28 000× g for 20 min, the supernatants were extracted with phenol-chloroform and precipitated in ethanol. Half of each sample was treated with 20 mg/ml RNase A and electrophoresed on 1.2% agarose gel. After electrophoresis, the gel was stained with ethidium bromide and visualized under UV light.
To further confirm the induction of apoptosis, ELISA for the qualitative and quantitative determination of cytoplasmic histone-associated-DNAfragments was performed with commercial kits (Boehringer Mannheim GmbH, Mannheim, Germany). After the thymocytes had been cultured with Cd and DEX for 7 h, the cells were centrifuged, and the supernatants collected. The cell pellet fractions were incubated with lysis buffer for 30 min at room temperature. They were then centrifuged and the supernatants were collected. Culture supernatants or cell lysate were added to streptavidin-coated microtiter plate in the presence of biotin-labeled anti-histone and peroxidase
Fig. 3. Apoptotic cells discriminated by the TUNEL technique in thymocytes exposed to various doses of Cd. Thymocytes were cultured in the absence or presence of 0.1, 1.0 and 10 mM Cd for 7 h. With the TUNEL method, 3%-OH DNA ends generated by DNA fragmentation are nick end labeled with biotinylated dUTP, introduced by terminal deoxytransferase (TdT) and then stained with avidin-conjugated FITC to allow specific staining. (a) Control, (b) 0.1 mM Cd, (c) 1.0 mM Cd and (d) 10 mM Cd. One representative experiment of four separate thymocyte preparations is shown.
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nm with a microplate reader (Bio-Rad, Nippon Bio-Rad Laboratory KK, Japan).
2.4. Flow cytometry assay
Fig. 4. Induction of apoptotic cells discriminated by the TUNEL technique in thymocytes exposed to Cd or DEX. Thymocytes were cultured with 1.0 mM DEX or 10 mM Cd for 7 h. With the TUNEL method, 3%-OH DNA ends generated by DNA fragmentation are nick end labeled with biotinylated dUTP, introduced by terminal deoxytransferase (TdT) and then stained with avidin-conjugated FITC to allow specific staining. (a) Control, (b) 1.0 mM DEX and (c) 10 mM Cd. One experiment representative of three is shown.
conjugated anti-DNA antibodies and incubated for 2 h at room temperature. After the plates had been washed three times, substrate solution (2,2%Azino-di[2-ethylbenzthiazolin-sulfonat]) was added, and the plates were incubated for 10 – 20 min. Absorbance was measured at 405 and 490
Flow cytometry analysis was done with a MEBSTAIN apoptosis kit (MBL, Nagoya, Japan). In brief, thymocytes that had been cultured with Cd and DEX for 7 h were washed twice with PBS containing 0.2% BSA and then fixed with 4% paraformaldehyde at 4°C for 30 min. After the cells had been washed twice again, they were incubated with proteinase K solution for 30 min, and with TdT reaction reagent for 1 h at 37°C. After the cell pellets had been washed, 10 ml of blocking reagent were added and the cells were kept at room temperature for 10 min. Thirty microliters of avidin-FITC reagent were then added and the cells were incubated in the dark at room temperature for 30 min. After washing twice, the cell pellets were suspended in 500 ml of PBS containing 0.2% BSA. The FITC labeled cells were analysed by flow cytometry. To compare the induction of apoptosis and necrosis in cells that had been treated with 10 mM Cd and/or 1 mM DEX for either 3 or 7 h, the ability to bind annexin V and exclude propidium iodide was determined with an apoptosis detection kit (R&D Systems, Minneapolis, MN). Necrotic cells stain with both annexin V FITC and propidium iodide, while cells undergoing apoptosis stain only with the annexin V FITC. Briefly, thymocytes were suspended in binding buffer at a concentration of 1×106 cells/ml. Ten microliters of FITC-conjugated annexin V and 10 ml of propidium iodide were added to the cells (1× 105/100 ml), and they were incubated in the dark for 15 min at room temperature. Following the incubation, 400 ml of binding buffer were added to the cell suspension, which was then analysed with a flow cytometer.
2.5. Statistical analysis Data were one representative experiment out of two to four and were analysed using Student’s t-test.
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3. Results To investigate whether in vitro Cd exposure may induce apoptosis in mouse thymocytes, thymocytes were cultured with Cd or DEX as a positive control for 7 h. Treatment of the thymocytes with 10 mM Cd or 1 mM DEX resulted in DNA ladder formation on gel electrophoresis, indicating degradation of nuclear DNA by both the Cd and the DEX (Fig. 1). To further confirm the apoptosis induced by Cd, apoptotic features were detected by ELISA for histone-associated DNA fragments. The absorbance of the cell lysate from cultured thymocytes exposed to 10 mM Cd or 1 mM DEX was significantly increased compared with that of the control (Fig. 2). There was no increase in absorbance in the culture supernatants of each group of thymocytes. These results suggest that Cd treatment may induce the formation of histone-associated DNA fragments in thymocytes.
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For the evaluation of apoptosis in thymocytes by the TUNEL method, thymocytes were cultured in the absence or presence of 0.1 mM, 1.0 mM and 10 mM Cd for 7 h. A marked increase in the number of labeled thymocytes was detected with 10 mM Cd (Fig. 3). Concentrations of 0.1 mM and 1 mM Cd did not induce a significant increase in the numbers of labeled cells with nuclear DNA fragments. The number of labeled thymocytes with nuclear DNA fragments was found to have been markedly increased by the presence of either 1 mM DEX (b) or 10 mM Cd (c) compared with the control (a) (Fig. 4). The effect of DEX was more pronounced than that of Cd. To compare the modulating activity of Cd with that of DEX, the percentages of necrotic and apoptotic cells induced by each were measured. These percentages were also measured for a mixture of Cd and DEX. Flow cytometric analysis with FITC-conjugated annexin V and propidium iodide showed that over 3 h of incubation, expo-
Fig. 5. Induction of apoptosis and necrosis in thymocytes exposed to Cd and/or DEX. Flow cytometric analysis with FITC-conjugated annexin V and propidium iodide was performed after incubation for 3 or 7 h with 10 mM Cd and/or 1 mM DEX. Each column shows the mean percentage of the control. (a) At 3 h the control values for apoptosis and necrosis were 15.1 9 0.9% and 8.7 90.6% (mean9SE of two different experiments), respectively. (b) At 7 h the control values for apoptosis and necrosis were 18.0 9 0.6% and 10.49 0.6% (mean9SE of three different experiments), respectively.
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sure to 1 mM DEX markedly increased the percentage of apoptotic cells, but exposure to 10 mM Cd did not (Fig. 5a). However, exposure to 10 mM Cd over a 7 h incubation significantly increased the percentage of apoptotic cells compared with that of necrotic cells (Fig. 5b). Thymocytes exposed to control, Cd, DEX and Cd + DEX showed apoptotic values of 18.0 9 0.6, 41.3 90.1, 55.4 9 0.8 and 57.5 91.0 (mean 9 SE of three experiments), respectively. The mixture of Cd and DEX did not show synergic or additive induction of apoptosis in thymocytes compared with Cd or DEX alone. Cd exposure resulted in a slow induction of apoptosis compared with DEX.
4. Discussion In this study, we investigated the in vitro effects of Cd on the induction of apoptosis in mouse thymocytes. Cd exposure is known to induce thymic atrophy as well as immunosuppression in mice (Koller et al., 1976; Gardner et al., 1977; Yamada et al., 1981; Fujimaki et al., 1982; Chaumard et al., 1983; Ohsawa et al., 1986; Daum et al., 1993; Schulte et al., 1994). However, it has remained unclear whether thymic atrophy results from the induction of apoptosis by Cd. In our study, DNA fragmentation, one of the hallmarks of apoptosis, in Cd-exposed thymocytes was characterized by gel electrophoresis, ELISA and flow cytometric analysis. Our results indicated that in vitro Cd exposure induced apoptotic features in mouse thymocytes. Although the induction of apoptosis by Cd was slower than that induced by DEX, the magnitude of the induction was similar. Zucker et al. (1994) studied the effects of tributyltin (TBT) and DEX on the induction of apoptosis in rat thymocytes and showed that simultaneous exposure to TBT and DEX resulted in a decreased induction of apoptosis. They suggested that TBT and DEX, are mutual antagonists and initiate endonuclease-mediated apoptotic cell death through different mechanisms. In our present study, simultaneous exposure to DEX did not affect the extent of
Cd-induced apoptosis. The mechanisms by which glucocorticoids induce apoptosis have been discussed. Endogenous endonuclease activity during apoptosis of glucocorticoid-treated thymocytes is known to be inhibited by zinc (Nieto and LopezRivas, 1989). Expression of c-myc can trigger apoptosis, but apoptosis induced by glucocorticoids can proceed in the absence of c-myc protein (Bissonnette et al., 1994). Our previous papers indicate that zinc can suppress the DNA laddering formation of apoptosis elicited by Cd (Ishido et al., 1995, 1999). We have also shown that Cd induces apoptosis in the absence of transcriptional activation of c-myc (Ishido et al., 1998). In the present study, there was a slight difference in the time-dependent induction of apoptosis between Cd and DEX (Fig. 5). Taken together, these findings suggest that Cd and DEX exposure may induce apoptosis in mouse thymocytes through a mechanism independent of c-myc function and zinc may play a role. Glucocorticoids induce thymocyte apoptosis by activating a calcium-dependent endonuclease (Cohen and Duke, 1984). However, Lohmann and Beyersmann (1993) observed a significant increase in DNA fragmentation in the presence of 10 mM Cd in a Ca2 + -free system. Additional data are required to determine whether the induction of apoptosis by Cd is Ca2 + and zinc-dependent or not. In conclusion, the induction of mouse thymocyte death by in vitro Cd exposure may be caused by the induction of apoptosis rather than by necrosis. Further studies on the relationship between thymic atrophy and the induction of apoptosis in the thymocytes of mice exposed to Cd are needed.
Acknowledgements This work was supported partly by a grant-in-aid for scientific research (B) from the Ministry of Education, Science, Sports and Culture of Japan and by special coordination funds for promoting Science and Technology supplied by the Science and Technology Agency of Japan.
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