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Increased radiation-induced apoptosis in mouse thymus in the absence of metallothionein
Toxicology 134 (1999) 39 – 49
Increased radiation-induced apoptosis in mouse thymus in the absence of metallothionein Diana Xi Deng, Lu Cai, Subrata Chakrabarti, M. George Cherian * Department of Pathology, Uni6ersity of Western Ontario, London, Ontario, Canada, N6A 5C1 Received 28 November 1998; accepted 14 February 1999
Abstract Metallothionein (MT) has been shown to protect cells from free radical induced DNA damage after exposure to copper, hydrogen peroxide and also radiation. In order to study the role of MT in radiation induced apoptosis, age-matched male control mice (C57BL/6J), MT-I overexpressing (MT-I*) and MT-null transgenic mice were exposed to whole body cobalt 60 g-irradiation at 0, 5, or 10 Gy, and their thymus were removed 24 h later. The basal levels of MT and zinc concentrations in the thymus were measured by 109Cadmium-heme assay and atomic absorption spectrophotometry, respectively. The MT expression after radiation was determined by immunohistochemical staining using a polyclonal antibody to MT. The extent of apoptosis in thymocytes was determined by histology (H&E stain). DNA was isolated from the thymus, and DNA fragmentation was determined by agarose gel electrophoresis. The results showed that the basal level of MT protein in MT-I* thymus was 2.4-fold higher than control mice, and that MT was inducible in both MT-I* and control C57BL6 thymus after radiation exposure. Minimal MT protein was detected in MT-null mice thymus before or after radiation, while, a significantly higher number of apoptotic cells and DNA fragmentation were found in MT-null thymus after whole body irradiation. These data demonstrated a protective role for MT in radiation-induced apoptosis in mouse thymus. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: DNA fragmentation; Apoptosis; Metallothionein; Thymus
1. Introduction Metallothioneins (MTs) are a group of low* Corresponding author. Tel.: +1-519-6612030; fax: +1519-6613370. E-mail address: [email protected] (M.G. Cherian)
molecular-weight (6000–7000 Daltons), cysteine rich (30%) intracellular proteins with high affinity for transitional metals. Four major isoforms of MT have been identified in mammalian tissues, and MT-I and MT-II are the two major isoforms (Kagi, 1993). MT is constitutively expressed in various species and tissues, including mouse thy-
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mus (Olafson, 1985). Diverse factors including metals, glucocorticoids, cytokines, and ionizing radiation have been shown to induce MT synthesis (Cherian, 1995). The distribution of MT and the induction in MT levels may vary depending on species and organs (Koropatnick et al., 1989; Cherian, 1995). Several physiologic roles have been proposed for MT, including detoxification of potentially toxic metals, regulation of essential trace metals such as zinc (Zn) and copper, and participation in the cellular antioxidant defense system. Apoptosis or programmed cell death is distinct from necrotic cell death both in morphology and in mechanism (Wyllie et al., 1980; Lowe et al., 1993). Apoptosis plays a key role in a number of immune functions, including antiself or nonsense clone deletions. Abnormal programmed cell death may be a key mechanism of cell loss in several diseases such as autoimmune disease, AIDS, cancer, neurodegenerative disorders. Mild oxidative stress is a well-recognized inducer of apoptosis (Slater et al., 1995; Simonian and Coyle, 1996). Reactive oxygen intermediates generated by exposure to hydrogen peroxide, redox-cycling quinones, thiol-alkylating agents, or nitric oxide can act as second messengers for a variety of signal transduction processes, including programmed cell death (Suzuki et al., 1997). There are reports which suggest a protective role for MT in cellular injury induced by ionizing radiation. For example, induction of MT conferred increased resistance against ionizing radiation in several cell lines (Bakka et al. 1982; Mello-Filho et al., 1988). Mice orally given MTinducing metals such as Zn showed protection against g-irradiation compared to controls (Matsubara et al., 1986). MT can also protect against radiation-induced DNA damage. Zn/CdMT was shown to be an effective radioprotector against damages to the DNA sugar – phosphate backbone (Greenstock et al., 1987). High levels of MT induced by pretreatment with metals in both in vitro and in vivo can protect rabbit lymphocytes from ionizing radiation-induced chromosome aberrations (Cai and Cherian, 1996). However, the role of MT in apoptosis
remains unclear. Apoptosis was enhanced more in MT-null cells than controls when exposed to several anti-cancer agents (Kondo et al., 1997). On the other hand, MT bound to cadmium has been shown to promote apoptosis in human kidney 293 cells (Hamada et al., 1996). Since ionizing radiation can induce both MT synthesis (Shiraishi et al., 1989) and apoptosis (Lowe et al. 1993), present study was undertaken to evaluate the role of MT in radiation induced apoptosis in the thymus. Thymus is one of the most radiosensitive organs both in vivo and in vitro (Okada, 1970). In addition, apoptosis can be induced readily in the thymus by a variety of modulators such as glucocorticoids and ionizing radiation. The present study was undertaken to investigate the role of MT in radiation induced apoptosis in the thymus which is a sensitive organ to radiation injury. The transgenic mice with genetically altered MT genes used in this experiment were an excellent model to study the role of MT in radiation induced apoptosis in the thymus. The MT-I* mice have previously been shown to express MT mRNA and MT protein in a manner similar to control mice with regard to cell specificity and inducibility (Palmiter et al., 1993; Iszard et al., 1995). The MT-null mice used in this experiment transcribe the MT genes, but do not translate MT protein (Masters et al., 1994). In this study, therefore, C57BL/6 mice were used as controls because both transgenes have a partial C57BL/6J background, and the effect of radiation-induced apoptosis was investigated in the thymus in these strains. 2. Materials and methods
2.1. Animals and treatments The two strains of transgenic mice: MT-I* and MT-null, were purchased from Jackson Laboratories (Bar Harbor, ME) and were housed at the animal care facility, University of Western Ontario, London, Ontario in accordance with guidelines established by the Canadian Council on Animal Care (1984). Litters from the inbred transgenic mice were used for
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subsequent experiments. All C57BL/6J mice were purchased from Charles River Laboratories (Montreal, Quebec). Normal thymus from adult male MT-I*, MT-null and control C57BL/6J mice were collected for analysis of the basal levels of MT and Zn concentrations in the thymus. Four age-matched (2 months) male mice per experimental group were exposed to whole body cobalt 60 g-irradiation at a dose rate of 0.65 Gy/min. In previous studies (Liu et al., 1996; Weil et al., 1996), 4 or 5 Gy dose of radiation was used, and induction of apoptosis in the thymus was shown at various time intervals until 24 h after exposure. Therefore, a radiation dose of 5 Gy was used in present study to investigate the influence of different MT levels on radiation-induced apoptosis in the thymus. At 24 h after a whole-body radiation, MT synthesis was induced in thymus and this time was selected to study apoptosis. In addition, we wanted to examine these effects at a higher dose of radiation. Therefore, 10 Gy dose of radiation was also used in this study.
2.2. Estimation of MT Whole thymus was homogenized in 1 ml of a 0.25 M sucrose solution. The homogenate was centrifuged at 10,000×g for 10 min at 4°C. The supernatant was stored at − 80°C for analysis of MT protein. Quantification of MT was performed by a 109cadmium-heme saturation method as described previously (Eaton and Cherian, 1991). Briefly, an aliquot of the resulting supernatant fraction, diluted with 30 mM Tris – HCl buffer (pH 8.0), was incubated with 10 ppm 109Cd solution with known specific activity to saturate the metal-binding sites of MT. Excess Cd was removed by addition of rat hemolysate to the assay tubes followed by heat treatment in a boiling water bath, which caused precipitation of cadmium–hemoglobin and other proteins, except MT which is heat stable. The denatured proteins were removed by centrifugation at 10,000 rpm for 2 min. Hemolysate treatment/heat denaturation/ centrifugation steps were repeated three times. The Cd concentrations in the final supernatant were calculated from the radioactivity of the 109Cd which were measured by a g counter (1272 Clin-
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igamma, LKB Wallac; Turku, Finland), and were converted to MT concentration on the basis of 7 g atoms of cadmium/MT. The total MT concentrations in the thymus were expressed as mg/g wet tissue.
2.3. Estimation of Zn For metal analysis, the whole thymus was digested in 1ml nitric acid overnight at room temperature, heated at 95°C for 1 h and the total volumes were adjusted to 2 ml with distilled water. The Zn concentrations in the samples were estimated by atomic absorption spectrophotometry (AAS) in a Varian Spectra 30 (Georgetown, Ontario, Canada) with an air–acetylene flame. Metal levels were expressed as mg metal/g wet tissue.
2.4. Immunohistochemical localization of MT and apoptotic cell counts A section of the thymus was fixed in 10% buffered formalin and embedded in paraffin. 5mm-thick sections were cut consecutively from the tissue blocks, mounted onto glass slides and were used for MT staining. The immunohistochemical localization of MT was performed as described earlier from our laboratory (Nartey et al., 1987a,b). Briefly, sections were deparaffinized and rehydrated first, and then were immersed in 3% hydrogen peroxide (H2O2) with methanol for 30 min to remove the endogenous peroxidase activity. Sections were further incubated with 10% normal goat serum for 1 h, followed by incubation with polyclonal rabbit antiMT serum (1:500) at 4°C overnight. This antibody was generated against a polymer of rat liver MT-II isoform but it cross reacted with both liver MT-I and MT-II isoforms from most mammalian tissues (Nartey et al., 1987a,b). The sections were then washed in Tris–HCl buffer (0.05 M, pH 7.6), and they were sequentially incubated with: (1) biotinylated goat anti rabbit IgG, and (2) avidin-biotin horseradish peroxidase complex according to the manufacturer’s instruction (Elite ABC™ kit, Vector Laboratories, Burlingame, CA). The sections were
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stained by immersing slides in 0.05% 3,3%-diaminobenzidine tetrahydrochloride (DAB) in the presence of 0.33% H2O2. All slides were counterstained with hematoxylin and 0.3% ammonia water, dehydrated and mounted. The apoptotic cells were identified by light microscopic examination at ×1000 magnification. Positive apoptotic cells were confirmed by their exclusive nuclear location with a distinct morphologic appearance, such as cell shrinkage, chromatin compaction and intact cell membrane in hematoxylin & eosin (H&E) stain (Fig. 2). For each slide, ten randomly selected fields were examined. The incidence of thymocyte apoptosis was expressed as numbers of apoptotic cells per 100 nuclei.
57 V for 2.5 h. The gel was then stained with SYBR Green I nucleic acid gel stain (Molecular Probes, Eugene, Oregon) at 1:10,000 concentration in TE buffer and was visualized by ultraviolet light. A special photographic filter (S-7569, Molecular Probes, Eugene, Oregon) was required to achieve a desirable film image.
2.6. Statistical analysis Statistical evaluation of data for three stains of mice was performed by one way analysis of variance using the Sigma statistical package (Jandel Scientific, Corte Madera, CA) and Bonferroni’s multiple comparison. The level of significance was set at PB 0.05. All results were presented as mean 9 S.E.
2.5. DNA isolation and DNA mobility analysis 3. Results For DNA isolation, whole thymus was rapidly frozen in liquid nitrogen after excision, and stored at −80°C for DNA isolation and DNA mobility assay. The standard protocols as described by Margulies in Current Protocols in Immunology were followed for isolation of DNA and resolution of DNA fragments (Margulies, 1992). Approximately 0.1 g snap-frozen tissue was ground with a pre-chilled mortar and pestle. The powdered tissue was then suspended in 1.2 ml digestion buffer (100 mM NaCl; 10 mM Tris – HCl, pH 8; 25 mM EDTA, pH 8; 0.5% SDS; 0.1 mg/ml proteinase K) and incubated at 50°C overnight. The sample was extracted with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) solution and DNA was precipitated by two volumes of cold 100% ethanol (− 20°C). The pellet was air dried and resuspended in TE buffer (10 mM Tris – HCl, 1 mM EDTA, pH 8.0). DNA concentration was determined at A260 and was considered purified when the ratio of A260/A280 reading was between 1.6 and 2.0. Approximately 10 mg DNA mixed with 1/10 vol of loading buffer (20% Ficoll 400; 0.1 M Na2EDTA, pH 8.0; 1% SDS; 0.25% bromophenol blue; 0.25% xylene cyanol) was applied to 2.5% agarose gel (Electrophoresis grade, GIBCO BRL). Electrophoresis was performed at
Immunohistochemical staining for MT in the thymus, using a polyclonal rabbit–anti-rat MT antibody, was observed in MT-I* mouse (Fig. 1A), but not in MT-null (Fig. 1C) mouse. Thymus from C57BL/6J mice showed a weak MT staining (Fig. 1E). 24 h after g-irradiation at 5 Gy, the immunostaining of MT in the thymus was detected in both MT-I* (Fig. 1B) and C57BL/6J (Fig. 1F) mice but not in MT-null (Fig. 1D) mice. Similar results were observed after exposure to 10 Gy radiation (data not shown), suggesting the induction of MT synthesis in both MT-I* and control mice after radiation. Not all cells in thymus were immunopositive for MT, but we have not identified completely the subtype of the cells which express MT. Both nuclear and cytoplasmic MT staining was observed in the cells. For the subtype of cells with MT staining, the large cells (open arrows in Fig. 1A), probably of epithelial origin, were uniformly positive for MT and were resistant to radiation induced apoptosis (open arrows in Fig. 1B). Some of the small thymocytes were MT positive (closed arrows in Fig. 1A). Following radiation, most of the thymocytes showed apoptosis (Fig. 1B–E). Positive MT staining were demonstrable in the surviving thymocytes (closed arrows in Fig. 1B and E).
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The concentration of MT and Zn from the thymus of transgenic MT-I* and control C57BL/6J mice were also measured. The MT-I* thymus contained 66.197.92 mg MT/g wet tissue, 2.4 times higher than that of control C57BL/6J thymus (PB 0.05). The concentration of MT in C57BL/6J
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was 27.09 4.05 mg/g of wet tissue. The detection of MT in the thymus of MT-null mice is minimal. The Zn levels in MT-I*, MT-null and control C57BL/6J thymus were 12.39 2.92, 13.39 5.92, and 15.39 6.06 mg/g wet tissue, respectively. They were not significantly different from each other (P \ 0.05).
Fig. 1. Immunohistochemical detection of metallothionein (MT) proteins in the thymus. The thymus tissues of MT-I* (A,B), MT-null (C,D), and control (E,F) mice were stained for MT. A, C, and E: Prior to 5 Gy g-radiation. Large cells, probably epithelial cells, are uniformly positive for MT staining (open arrows). Some thymocytes were positive for MT staining (closed arrows). Panels B,D, and F. are from mice 24 h after g-irradiation at 5 Gy. Intense MT staining was found in epithelial cells (opened arrows) and thymocytes (closed arrows). The number of apoptotic cells in the thymus of MT-I* and C57BL/6J mice, were smaller than the number of apoptotic cells in the thymus of MT-null mice (stars). The apoptotic cells were devoid of MT immunostaining. Counterstained with hematoxylin. Magnification × 450.
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Fig. 2. Normal thymocytes (A, 1000 X) from C57BL/6J mice, and apoptotic bodies (indicated by arrows) in thymocytes (B, 1000 X) from MT-I* mice at 12 h after exposure to 5 Gy radiation.
The number of apoptotic cells, characterized by chromatin condensation, intact cell organelle membrane, and apoptotic body formation (Fig. 2B), were counted at ×1000 magnification by light microscopy. Very small numbers of apoptotic cells were found in thymus of all strains of mouse before radiation (Fig. 3). There was no significant difference of the percentage of apoptosis in the thymus from MT-I*, MT-null and C57BL/6J mice (2, 4, and 3.8% for MT-I*, MTnull, C57BL/6J thymus, respectively). High numbers of apoptotic cells were observed when the mice were irradiated at 5 Gy, and the highest number was found in thymus of MT-null mice (P B 0.05). The percentages of apoptotic cells were 30 93, 59 9 6, and 36 93% for MT-I*, MT-null, and C57BL/6J thymus, respectively.
When the dose was increased to 10 Gy, a decrease in the number was observed in both the number of apoptotic cells and the total number of cells per field. This indicated that a large number of thymocytes were necrotic and were eliminated from the organ. Apparently, the number of apoptotic cells in MT-null thymus was consistently the highest among all three mice strains after 10 Gy irradiation, but was not statistically significant (P= 0.09, Fig. 3). The DNA gel electrophoresis confirmed the detection of apoptotic bodies by H & E staining, and showed minimal DNA damage in the thymus of mice with no irradiation (Fig. 4). A time course study of apoptosis in the thymus of MT-I*, MT-null, and C57BL/6J by DNA ladder formation after exposure to 5 Gy showed distinct DNA ladder at 3–24 h in MT-null mice while similar pattern was observed only after 6 h in MT-I* mice. The DNA ladder formation in the thymus of MT-null mice at 24 h after exposure to a radiation dose of 5 Gy is shown in Fig. 4. These results were consistent with incidence of apoptotic bodies in MT-null mice as shown in Fig. 3. When the dose was increased to 10 Gy, the increase of DNA mobility on agarose gel was observed in all cases (Fig. 4), suggesting a general necrotic effect.
Fig. 3. Quantitative evaluation of apoptotic cells in thymus tissues from transgenic MT-I*, MT-null mice, and control C57BL/6J mice. Each bar presents the mean 9 S.E. (n =4). * Significantly different from that of MT-I* and control mice (P B0.05).
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Fig. 4. Effect of g-irradiation on thymic DNA mobility. DNA from thymus of MT-1*, MT-null, and C57BL/6J mice was isolated at 24 h after g-irradiation. 10 mg DNA per sample was loaded onto a 2.5% agarose gel, electrophoresed at 57 V, and visualized by SYBR Green I nucleic acid dye under UV light. From left to right: lane 1,4,7: MT-I*; lane 2,5,8: MT-null; lane 3,6,9: C57BL/6J. Photograph was typical of three separate experiments.
4. Discussion The present study demonstrates the constitutive expression of MT in thymus of MT-I* mouse. The constitutive overexpression of both MT mRNA (Palmiter et al., 1993) and protein (Iszard et al., 1995) has been reported in other organs of the MT-I* mouse, including liver, kidney, pancreas, heart, intestine, testis, and brain. The overexpression of MT, however, is not consistent among the organs. Pancreas and liver (20× and 16 × , respectively) are the two organs that have the highest basal overexpression as compared to that of age matched control litter mates. A small increase in MT was detected in the lung (1.7×), spleen (2.5× ), and brain (1.4 ×) of the MT-I* mouse. Similarly, we report here about 2.4 fold of MT increase in the thymus of MT-I* mouse. Therefore, it appears that the regulation of MT overexpression may be organ specific. Within thymus, subsets of cells contained different amount of MT (Fig. 1A). The epithelial cells were uniformly positive for MT, while some of the thymocytes were also MT positive, except in the MT-null mice (Fig. 1). The high basal levels of MT in thymus of MT-I* mice can be further increased by exposure to ionizing radiation, as shown on Fig. 1B. Simi-
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larly, the MT expression in the thymus of C57BL/ 6J mice was also increased after radiation as determined by immunohistochemistry (Fig. 1F). MT can be induced by a variety of factors such as metals and oxidative stress (Satoh et al., 1993). It has been shown that MT in thymus was induced by Zn injection in Swiss white mice (Olafson, 1985). MT mRNA in thymus is induced in Wistar rats after whole body X-irradiation (Shiraishi et al., 1989). However, major regulatory factors which are responsible for the induction of MT synthesis in the thymus are unknown. Generally metals and glucocorticoids are considered direct inducers of MT synthesis in mammalian tissues (Chan and Cherian, 1993; Palmiter, 1995). Radiation and other agents such as cytokines, lipopolysaccharide, endotoxin, paraquat and carbon tetrachloride, are considered as indirect inducers for MT synthesis. It has been shown that induction of MT occurred after X-irradiation of mice but not in cells derived from mice, which indicates a physiological factor other than free radicals is responsible for the MT induction (Koropatnick et al., 1989). The incidence of apoptosis, as determined both by the number of apoptotic cells per 100 cells and DNA fragmentation, was high at 24 h after exposure to low dose (5 Gy) of radiation but was decreased at high dose (10 Gy) of radiation. This may be due to increased number of necrotic cells after 10 Gy dose and subsequent elimination of these dead cells from the thymus (Figs. 3 and 4). These results are consistent in all three strains of mice studied. We elected light microscopy for detection and quantification of apoptosis on H&E stain, and did not use techniques such as TUNEL in this study. Although supplemental techniques are sometimes helpful, a carefully studied H&E section gives a definitive assessment of apoptosis. In keeping with this notion, Goldsworthy et al. (1995) have demonstrated that by microscopic examination of H&E stained slides, equivalent results could be obtained as compared to apoptosis detected by TUNEL. In the present study, the results obtained by quantification on H&E stain were confirmed by DNA fragmentation analysis, and suggest that a low dose of radiation is more effective in inducing apoptosis than a high dose of
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radiation. This dose-dependent relation between radiation and apoptosis has been reported previously in other cell lines (HL-60, Hopcia et al., 1996) as well as in certain lymphomas (Mirkovic et al. 1994). In V79 cells, radiation at 3 – 4 Gy caused a significant increase in apoptosis, while radiation at higher dose did not further increase apoptosis, but caused cell necrosis (Ghosh et al., 1996; Jamali and Trott, 1996). Thus, radiation at low and high doses can cause either apoptosis or necrosis in the thymus. The results show an increase in apoptosis in the thymus after radiation in all three strains of mice, but the percentage of apoptosis in thymus was decreased significantly with MT overexpression and significantly increased in the absence of MT expression in MT-null mice (Figs. 3 and 4). This result was consistent at different radiation doses, either with whole body irradiation (present study), or with thymocyte irradiated in vitro. Therefore, MT can protect mouse thymus from apoptosis after g-irradiation. An increase in apoptosis in MT-I* after high radiation dose suggest that the presence of MT alone can not fully protect thymocytes from apoptosis. The mechanisms by which MT can protect cell death are still unclear. Although radiation-induced apoptosis can occur through a variety of mechanisms, the oxidative damage to DNA and cell membrane are important factors for induction of apoptosis (Blank et al., 1997). Many antioxidants such as vitamin E can inhibit apoptosis induced by oxidative stress (Verhaegen et al., 1995; Zhang et al., 1997). Bcl-2, an important factor in regulation of apoptosis, was shown to prevent apoptosis through the anti-oxidative pathway by regulating cellular thiol levels (Hockenbery et al., 1993; Mirkovic et al., 1997; Pourzand et al., 1997). Therefore, it is possible that MT, as an anti-oxidant, may reduce the radiation damage, resulting in low incidence of apoptosis. Recently it has been shown that MT induced by Cd or non-metal inducer, b-thujaplicin, had marked protective effect on UVB-induced apoptosis in keratinocytes both in vivo and in vitro (Baba et al., 1998). MT-null mice exhibit high sensitivity to UVB-induced apoptosis in vivo (Hanada et al., 1998). Thymocytes from p53 null
mice were unable to undergo apoptosis in response to ionizing radiation while they responded to glucocorticoids (Lowe et al., 1993). Further experiments suggest that p53 is required for cells which undergo G1 arrest and apoptosis after radiation (Fan et al., 1994). In MT-null mice, it has been shown that p53 was induced in the liver by cadmium (Zheng et al., 1996). A high rate of apoptosis was detected in MT-null cells exposed to anticancer drugs as compared to controls (Kondo et al., 1997). Therefore, cells which do not have MT may be susceptible to p53 upregulation and apoptosis. Cellular thiols may play an important role in preventing spontaneous or radiation-induced apoptosis (Mirkovic et al., 1994; Sato et al., 1995). Glutathione (GSH) was able to protect PC12 cells from apoptosis induced by ascorbic acid and dopamine (Si et al., 1998). However, depletion of GSH with buthionine sulfoximine alone did not increase either spontaneous or radiation-induced apoptosis in cells (Sato et al., 1995; Mirkovic et al., 1997), suggesting the involvement of other thiols in modulating apoptosis. In the present study, we did not measure GSH levels in thymus. However, others have reported that there are no differences in GSH and other antioxidant levels in tissues such as liver and kidney of control, and MT-null or MT-I* mice (Iszard et al., 1995). The cells derived from these mice also showed no difference in GSH levels as compared to control mice cells (Lazo et al., 1995, 1998). As a major intracellular thiol, MT can scavenge free radicals such as hydroxyl radicals at a much higher efficiency than GSH (Thornalley and Vasak 1985; Miura et al., 1997). Thus, the presence of high content of MT in MT-I* mouse thymus may provide protection while its absence in MT-null mouse thymus may cause increased cellular damage after radiation. In two reports, we have shown a high incidence of apoptosis in both hepatocellular carcinoma and metastatic adenocarcinoma of liver with low MT expression, as compared to control normal human livers (Cai et al., 1998; Deng et al., 1998). In the skin, MT-null mice exhibits high sensitivity to UVB-induced apoptosis in vivo (Hanada et al., 1998). Even, in certain cell lines, a negative corre-
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lation of MT with the incidence of apoptosis has been documented (Abdel-Mageed and Agrawal, 1997; Kondo et al., 1997; Tsangaris and Tzortzatou-Stathopoulou, 1998). Thus, a decrease in apoptosis in the thymus of MT-I* mice as compared to other strains supports published results in other tissues. Zn can either induce or block apoptosis depending on the cellular concentration (Fraker and Telford, 1997). High concentrations of extracellular Zn (500–1000 mM) have been used frequently to block apoptosis in a variety of systems. Zn at physiological concentrations (100 mM or less), on the other hand, could actually induce apoptosis (Fraker and Telford, 1997; Houben et al., 1997). Therefore, the regulation of intracellular free Zn pool by MT may also be a possible mechanism for protective effect of MT against apoptosis. In summary, mouse thymocytes can undergo apoptosis or necrosis after exposure to g-radiation at low (5 Gy) or high dose (10 Gy), respectively. MT-null thymus was most susceptible to apoptosis at low dose of radiation, while MT-I* thymus with high MT content was least damaged at high dose of radiation. These results suggest a protective role for MT in radiation-induced apoptosis and necrosis in thymus. We also demonstrated MT expression in thymus of MT-I overexpressing mice, and MT expression in the thymus was induced further after exposure to g-irradiation in both MT-I* and C57BL/6J mice.
Acknowledgements This work was supported by a research grant from the Medical Research Council of Canada.
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concentration also become resistant to oxidative stress. Biochem. J. 256, 475 – 479. Mirkovic, N., Meyn, R.E., Hunter, N.R., Milas, L., 1994. Radiation-induced apoptosis in a murine lymphoma in vivo. Radiother. Oncol. 33, 11 – 16. Mirkovic, N., Voehringer, D.W., Story, M.D., Mcconkey, D.J., Mcdonnell, T.J., Meyn, R.E., 1997. Resistance to radiation-induced apoptosis in Bcl-2 expressing cells is reversed by depleting cellular thiols. Oncogene 15, 1461 – 1470. Miura, T., Muraoka, S., Ogiso, T., 1997. Antioxidant activity of metallothionein compared with reduced glutathione. Life Sci. 60, 301 – 309. Nartey, N., Cherian, M.G., Banerjee, D., 1987a. Immunohistochemical localization of metallothionein in human thyroid tumors. Am. J. Pathol. 129, 177 – 182. Nartey, N.O., Banerjee, D., Cherian, M.G., 1987b. Immunohistochemical localization of metallothionein in cell nucleus and cytoplasm of fetal human liver and kidney and its changes during development. Pathology 19, 233 – 238. Okada, S., 1970. Radiation-induced death. In: Altman, K.I., Gerber, G.B., Okada, S. (Eds.), Radiation Biochemistry, I. Academic Press, New York, pp. 1 – 76. Olafson, R.W., 1985. Thymus metallothionein: regulation of zinc-thionein in the aging mouse. Can. J. Biochem. Cell. Biol. 63, 91 – 95. Palmiter, R.D., Sandgren, E.P., Koeller, D.M., Brinster, R.L., 1993. Distal regulatory elements from the mouse metallothionein locus stimulate gene expression in transgenic mice. Mol. Cell. Biol. 13, 5266 – 5275. Palmiter, R.D., 1995. Constitutive expression of metallothionein-III (MT-III), but not MT-I, inhibits growth when cells become zinc deficient. Toxicol. Appl. Pharmacol. 135, 139 – 146. Pourzand, C., Rossier, G., Reelfs, O., Borner, C., Tyrrell, R.M., 1997. Overxpression of Bcl-2 inhibits UVA-mediated immediate apoptosis in rat 6 fibroblasts: evidence for the involvement of Bcl-2 as an antioxidant. Cancer Res. 57, 1405 – 1411. Sato, N., Iwata, S., Nakamura, K., Hori, T., Mori, K., Yodoi, J., 1995. Thiol-mediated redox regulation of apoptosis, possible roles of cellular thiols other than glutathione in T cell apoptosis. J. Immunol. 154, 3194 – 3203. Satoh, M., Kloth, D.M., Kadhim, S.A., Chin, J.L., Naganuma, A., Imura, N., Cherian, M.G., 1993. Modulation of both cisplatin nephrotoxicity and drug resistance in murine bladder tumor by controlling metallothionein synthesis. Cancer Res. 53, 1829 – 1832. Shiraishi, N., Hayashi, H., Hiraki, Y., Aono, K., Itano, Y., Kosaka, F., Noji, S., Taniguchi, S., 1989. Elevation in metallothionein messenger RNA in rat tissues after exposure to X-irradiation. Toxicol. Appl. Pharmacol. 98, 501 – 506. Si, F., Ross, G.M., Shin, S.H., 1998. Glutathione protects PC12 cells from ascorbate- or dopamine-induced apoptosis. Exp. Brain Res. 123, 263 – 268. Simonian, N.A., Coyle, J.T., 1996. Oxidative stress in neurodegenerative diseases. Annu. Rev. Pharmacol. Toxicol. 36, 83 – 106.
D. Xi Deng et al. / Toxicology 134 (1999) 39–49 Slater, A.F.C.S., Nobel, I., van den Dobbelsteen, D.J., Orrenius, S., 1995. Signalling mechanisms and oxidative stress in apoptosis. Toxicol. Lett. 82–83, 149–153. Suzuki, Y.J., Forman, H.J., Sevanian, A., 1997. Oxidants as simulators of signal transduction. Free Radic. Biol. Med. 22, 269 – 285. Thornalley, P.J., Vasak, M., 1985. Possible role for metallothionein in protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals. Biochim. Biophys. Acta 827, 36– 44. Tsangaris, G.T., Tzortzatou-Stathopoulou, F., 1998. Metallothionein expression prevents apoptosis: a study with antisense phosphorothioate oligodeoxynucleotides in a human T cell line. Anticancer Res. 18, 2423–2433. Verhaegen, S., McGowan, A.J., Brophy, A.R., Fernandes, R.S., Cotter, T.G., 1995. Inhibition of apoptosis by antiox-
idants in the human HL-60 leukemia cell line. Biochem. Pharmacol. 50, 1021 – 1029. Weil, M.M., Amos, C.I., Mason, K.A., Stephens, L.C., 1996. Genetic basis of strain variation in levels of radiationinduced apoptosis of thymocytes. Radiat. Res. 146, 646 – 651. Wyllie, A.H., Kerr, J.F., Currie, A.R., 1980. Cell death: the significance of apoptosis. Int. Rev. Cytol. 68, 251 – 306. Zhang, D., Okada, S., Yu, Y., Zheng, P., Yamaguchi, R., Kasai, H., 1997. Vitamin E inhibits apoptosis, DNA modification, and cancer incidence induced by iron-mediated peroxidation in Wistar rat kidney. Cancer Res. 57, 2410 – 2414. Zheng, H., Liu, J., Choo, K.H., Michalska, A.E., Klaassen, C.D., 1996. Metallothionein-I and -II knock-out mice are sensitive to cadmium-induced liver mRNA expression of c-jun and p53. Toxicol. Appl. Pharmacol. 136, 229 – 235.
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Increased radiation-induced apoptosis in mouse thymus in the absence of metallothionein Diana Xi Deng, Lu Cai, Subrata Chakrabarti, M. George Cherian * Department of Pathology, Uni6ersity of Western Ontario, London, Ontario, Canada, N6A 5C1 Received 28 November 1998; accepted 14 February 1999
Abstract Metallothionein (MT) has been shown to protect cells from free radical induced DNA damage after exposure to copper, hydrogen peroxide and also radiation. In order to study the role of MT in radiation induced apoptosis, age-matched male control mice (C57BL/6J), MT-I overexpressing (MT-I*) and MT-null transgenic mice were exposed to whole body cobalt 60 g-irradiation at 0, 5, or 10 Gy, and their thymus were removed 24 h later. The basal levels of MT and zinc concentrations in the thymus were measured by 109Cadmium-heme assay and atomic absorption spectrophotometry, respectively. The MT expression after radiation was determined by immunohistochemical staining using a polyclonal antibody to MT. The extent of apoptosis in thymocytes was determined by histology (H&E stain). DNA was isolated from the thymus, and DNA fragmentation was determined by agarose gel electrophoresis. The results showed that the basal level of MT protein in MT-I* thymus was 2.4-fold higher than control mice, and that MT was inducible in both MT-I* and control C57BL6 thymus after radiation exposure. Minimal MT protein was detected in MT-null mice thymus before or after radiation, while, a significantly higher number of apoptotic cells and DNA fragmentation were found in MT-null thymus after whole body irradiation. These data demonstrated a protective role for MT in radiation-induced apoptosis in mouse thymus. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: DNA fragmentation; Apoptosis; Metallothionein; Thymus
1. Introduction Metallothioneins (MTs) are a group of low* Corresponding author. Tel.: +1-519-6612030; fax: +1519-6613370. E-mail address: [email protected] (M.G. Cherian)
molecular-weight (6000–7000 Daltons), cysteine rich (30%) intracellular proteins with high affinity for transitional metals. Four major isoforms of MT have been identified in mammalian tissues, and MT-I and MT-II are the two major isoforms (Kagi, 1993). MT is constitutively expressed in various species and tissues, including mouse thy-
0300-483X/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 9 9 ) 0 0 0 2 6 - 8
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mus (Olafson, 1985). Diverse factors including metals, glucocorticoids, cytokines, and ionizing radiation have been shown to induce MT synthesis (Cherian, 1995). The distribution of MT and the induction in MT levels may vary depending on species and organs (Koropatnick et al., 1989; Cherian, 1995). Several physiologic roles have been proposed for MT, including detoxification of potentially toxic metals, regulation of essential trace metals such as zinc (Zn) and copper, and participation in the cellular antioxidant defense system. Apoptosis or programmed cell death is distinct from necrotic cell death both in morphology and in mechanism (Wyllie et al., 1980; Lowe et al., 1993). Apoptosis plays a key role in a number of immune functions, including antiself or nonsense clone deletions. Abnormal programmed cell death may be a key mechanism of cell loss in several diseases such as autoimmune disease, AIDS, cancer, neurodegenerative disorders. Mild oxidative stress is a well-recognized inducer of apoptosis (Slater et al., 1995; Simonian and Coyle, 1996). Reactive oxygen intermediates generated by exposure to hydrogen peroxide, redox-cycling quinones, thiol-alkylating agents, or nitric oxide can act as second messengers for a variety of signal transduction processes, including programmed cell death (Suzuki et al., 1997). There are reports which suggest a protective role for MT in cellular injury induced by ionizing radiation. For example, induction of MT conferred increased resistance against ionizing radiation in several cell lines (Bakka et al. 1982; Mello-Filho et al., 1988). Mice orally given MTinducing metals such as Zn showed protection against g-irradiation compared to controls (Matsubara et al., 1986). MT can also protect against radiation-induced DNA damage. Zn/CdMT was shown to be an effective radioprotector against damages to the DNA sugar – phosphate backbone (Greenstock et al., 1987). High levels of MT induced by pretreatment with metals in both in vitro and in vivo can protect rabbit lymphocytes from ionizing radiation-induced chromosome aberrations (Cai and Cherian, 1996). However, the role of MT in apoptosis
remains unclear. Apoptosis was enhanced more in MT-null cells than controls when exposed to several anti-cancer agents (Kondo et al., 1997). On the other hand, MT bound to cadmium has been shown to promote apoptosis in human kidney 293 cells (Hamada et al., 1996). Since ionizing radiation can induce both MT synthesis (Shiraishi et al., 1989) and apoptosis (Lowe et al. 1993), present study was undertaken to evaluate the role of MT in radiation induced apoptosis in the thymus. Thymus is one of the most radiosensitive organs both in vivo and in vitro (Okada, 1970). In addition, apoptosis can be induced readily in the thymus by a variety of modulators such as glucocorticoids and ionizing radiation. The present study was undertaken to investigate the role of MT in radiation induced apoptosis in the thymus which is a sensitive organ to radiation injury. The transgenic mice with genetically altered MT genes used in this experiment were an excellent model to study the role of MT in radiation induced apoptosis in the thymus. The MT-I* mice have previously been shown to express MT mRNA and MT protein in a manner similar to control mice with regard to cell specificity and inducibility (Palmiter et al., 1993; Iszard et al., 1995). The MT-null mice used in this experiment transcribe the MT genes, but do not translate MT protein (Masters et al., 1994). In this study, therefore, C57BL/6 mice were used as controls because both transgenes have a partial C57BL/6J background, and the effect of radiation-induced apoptosis was investigated in the thymus in these strains. 2. Materials and methods
2.1. Animals and treatments The two strains of transgenic mice: MT-I* and MT-null, were purchased from Jackson Laboratories (Bar Harbor, ME) and were housed at the animal care facility, University of Western Ontario, London, Ontario in accordance with guidelines established by the Canadian Council on Animal Care (1984). Litters from the inbred transgenic mice were used for
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subsequent experiments. All C57BL/6J mice were purchased from Charles River Laboratories (Montreal, Quebec). Normal thymus from adult male MT-I*, MT-null and control C57BL/6J mice were collected for analysis of the basal levels of MT and Zn concentrations in the thymus. Four age-matched (2 months) male mice per experimental group were exposed to whole body cobalt 60 g-irradiation at a dose rate of 0.65 Gy/min. In previous studies (Liu et al., 1996; Weil et al., 1996), 4 or 5 Gy dose of radiation was used, and induction of apoptosis in the thymus was shown at various time intervals until 24 h after exposure. Therefore, a radiation dose of 5 Gy was used in present study to investigate the influence of different MT levels on radiation-induced apoptosis in the thymus. At 24 h after a whole-body radiation, MT synthesis was induced in thymus and this time was selected to study apoptosis. In addition, we wanted to examine these effects at a higher dose of radiation. Therefore, 10 Gy dose of radiation was also used in this study.
2.2. Estimation of MT Whole thymus was homogenized in 1 ml of a 0.25 M sucrose solution. The homogenate was centrifuged at 10,000×g for 10 min at 4°C. The supernatant was stored at − 80°C for analysis of MT protein. Quantification of MT was performed by a 109cadmium-heme saturation method as described previously (Eaton and Cherian, 1991). Briefly, an aliquot of the resulting supernatant fraction, diluted with 30 mM Tris – HCl buffer (pH 8.0), was incubated with 10 ppm 109Cd solution with known specific activity to saturate the metal-binding sites of MT. Excess Cd was removed by addition of rat hemolysate to the assay tubes followed by heat treatment in a boiling water bath, which caused precipitation of cadmium–hemoglobin and other proteins, except MT which is heat stable. The denatured proteins were removed by centrifugation at 10,000 rpm for 2 min. Hemolysate treatment/heat denaturation/ centrifugation steps were repeated three times. The Cd concentrations in the final supernatant were calculated from the radioactivity of the 109Cd which were measured by a g counter (1272 Clin-
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igamma, LKB Wallac; Turku, Finland), and were converted to MT concentration on the basis of 7 g atoms of cadmium/MT. The total MT concentrations in the thymus were expressed as mg/g wet tissue.
2.3. Estimation of Zn For metal analysis, the whole thymus was digested in 1ml nitric acid overnight at room temperature, heated at 95°C for 1 h and the total volumes were adjusted to 2 ml with distilled water. The Zn concentrations in the samples were estimated by atomic absorption spectrophotometry (AAS) in a Varian Spectra 30 (Georgetown, Ontario, Canada) with an air–acetylene flame. Metal levels were expressed as mg metal/g wet tissue.
2.4. Immunohistochemical localization of MT and apoptotic cell counts A section of the thymus was fixed in 10% buffered formalin and embedded in paraffin. 5mm-thick sections were cut consecutively from the tissue blocks, mounted onto glass slides and were used for MT staining. The immunohistochemical localization of MT was performed as described earlier from our laboratory (Nartey et al., 1987a,b). Briefly, sections were deparaffinized and rehydrated first, and then were immersed in 3% hydrogen peroxide (H2O2) with methanol for 30 min to remove the endogenous peroxidase activity. Sections were further incubated with 10% normal goat serum for 1 h, followed by incubation with polyclonal rabbit antiMT serum (1:500) at 4°C overnight. This antibody was generated against a polymer of rat liver MT-II isoform but it cross reacted with both liver MT-I and MT-II isoforms from most mammalian tissues (Nartey et al., 1987a,b). The sections were then washed in Tris–HCl buffer (0.05 M, pH 7.6), and they were sequentially incubated with: (1) biotinylated goat anti rabbit IgG, and (2) avidin-biotin horseradish peroxidase complex according to the manufacturer’s instruction (Elite ABC™ kit, Vector Laboratories, Burlingame, CA). The sections were
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stained by immersing slides in 0.05% 3,3%-diaminobenzidine tetrahydrochloride (DAB) in the presence of 0.33% H2O2. All slides were counterstained with hematoxylin and 0.3% ammonia water, dehydrated and mounted. The apoptotic cells were identified by light microscopic examination at ×1000 magnification. Positive apoptotic cells were confirmed by their exclusive nuclear location with a distinct morphologic appearance, such as cell shrinkage, chromatin compaction and intact cell membrane in hematoxylin & eosin (H&E) stain (Fig. 2). For each slide, ten randomly selected fields were examined. The incidence of thymocyte apoptosis was expressed as numbers of apoptotic cells per 100 nuclei.
57 V for 2.5 h. The gel was then stained with SYBR Green I nucleic acid gel stain (Molecular Probes, Eugene, Oregon) at 1:10,000 concentration in TE buffer and was visualized by ultraviolet light. A special photographic filter (S-7569, Molecular Probes, Eugene, Oregon) was required to achieve a desirable film image.
2.6. Statistical analysis Statistical evaluation of data for three stains of mice was performed by one way analysis of variance using the Sigma statistical package (Jandel Scientific, Corte Madera, CA) and Bonferroni’s multiple comparison. The level of significance was set at PB 0.05. All results were presented as mean 9 S.E.
2.5. DNA isolation and DNA mobility analysis 3. Results For DNA isolation, whole thymus was rapidly frozen in liquid nitrogen after excision, and stored at −80°C for DNA isolation and DNA mobility assay. The standard protocols as described by Margulies in Current Protocols in Immunology were followed for isolation of DNA and resolution of DNA fragments (Margulies, 1992). Approximately 0.1 g snap-frozen tissue was ground with a pre-chilled mortar and pestle. The powdered tissue was then suspended in 1.2 ml digestion buffer (100 mM NaCl; 10 mM Tris – HCl, pH 8; 25 mM EDTA, pH 8; 0.5% SDS; 0.1 mg/ml proteinase K) and incubated at 50°C overnight. The sample was extracted with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) solution and DNA was precipitated by two volumes of cold 100% ethanol (− 20°C). The pellet was air dried and resuspended in TE buffer (10 mM Tris – HCl, 1 mM EDTA, pH 8.0). DNA concentration was determined at A260 and was considered purified when the ratio of A260/A280 reading was between 1.6 and 2.0. Approximately 10 mg DNA mixed with 1/10 vol of loading buffer (20% Ficoll 400; 0.1 M Na2EDTA, pH 8.0; 1% SDS; 0.25% bromophenol blue; 0.25% xylene cyanol) was applied to 2.5% agarose gel (Electrophoresis grade, GIBCO BRL). Electrophoresis was performed at
Immunohistochemical staining for MT in the thymus, using a polyclonal rabbit–anti-rat MT antibody, was observed in MT-I* mouse (Fig. 1A), but not in MT-null (Fig. 1C) mouse. Thymus from C57BL/6J mice showed a weak MT staining (Fig. 1E). 24 h after g-irradiation at 5 Gy, the immunostaining of MT in the thymus was detected in both MT-I* (Fig. 1B) and C57BL/6J (Fig. 1F) mice but not in MT-null (Fig. 1D) mice. Similar results were observed after exposure to 10 Gy radiation (data not shown), suggesting the induction of MT synthesis in both MT-I* and control mice after radiation. Not all cells in thymus were immunopositive for MT, but we have not identified completely the subtype of the cells which express MT. Both nuclear and cytoplasmic MT staining was observed in the cells. For the subtype of cells with MT staining, the large cells (open arrows in Fig. 1A), probably of epithelial origin, were uniformly positive for MT and were resistant to radiation induced apoptosis (open arrows in Fig. 1B). Some of the small thymocytes were MT positive (closed arrows in Fig. 1A). Following radiation, most of the thymocytes showed apoptosis (Fig. 1B–E). Positive MT staining were demonstrable in the surviving thymocytes (closed arrows in Fig. 1B and E).
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The concentration of MT and Zn from the thymus of transgenic MT-I* and control C57BL/6J mice were also measured. The MT-I* thymus contained 66.197.92 mg MT/g wet tissue, 2.4 times higher than that of control C57BL/6J thymus (PB 0.05). The concentration of MT in C57BL/6J
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was 27.09 4.05 mg/g of wet tissue. The detection of MT in the thymus of MT-null mice is minimal. The Zn levels in MT-I*, MT-null and control C57BL/6J thymus were 12.39 2.92, 13.39 5.92, and 15.39 6.06 mg/g wet tissue, respectively. They were not significantly different from each other (P \ 0.05).
Fig. 1. Immunohistochemical detection of metallothionein (MT) proteins in the thymus. The thymus tissues of MT-I* (A,B), MT-null (C,D), and control (E,F) mice were stained for MT. A, C, and E: Prior to 5 Gy g-radiation. Large cells, probably epithelial cells, are uniformly positive for MT staining (open arrows). Some thymocytes were positive for MT staining (closed arrows). Panels B,D, and F. are from mice 24 h after g-irradiation at 5 Gy. Intense MT staining was found in epithelial cells (opened arrows) and thymocytes (closed arrows). The number of apoptotic cells in the thymus of MT-I* and C57BL/6J mice, were smaller than the number of apoptotic cells in the thymus of MT-null mice (stars). The apoptotic cells were devoid of MT immunostaining. Counterstained with hematoxylin. Magnification × 450.
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Fig. 2. Normal thymocytes (A, 1000 X) from C57BL/6J mice, and apoptotic bodies (indicated by arrows) in thymocytes (B, 1000 X) from MT-I* mice at 12 h after exposure to 5 Gy radiation.
The number of apoptotic cells, characterized by chromatin condensation, intact cell organelle membrane, and apoptotic body formation (Fig. 2B), were counted at ×1000 magnification by light microscopy. Very small numbers of apoptotic cells were found in thymus of all strains of mouse before radiation (Fig. 3). There was no significant difference of the percentage of apoptosis in the thymus from MT-I*, MT-null and C57BL/6J mice (2, 4, and 3.8% for MT-I*, MTnull, C57BL/6J thymus, respectively). High numbers of apoptotic cells were observed when the mice were irradiated at 5 Gy, and the highest number was found in thymus of MT-null mice (P B 0.05). The percentages of apoptotic cells were 30 93, 59 9 6, and 36 93% for MT-I*, MT-null, and C57BL/6J thymus, respectively.
When the dose was increased to 10 Gy, a decrease in the number was observed in both the number of apoptotic cells and the total number of cells per field. This indicated that a large number of thymocytes were necrotic and were eliminated from the organ. Apparently, the number of apoptotic cells in MT-null thymus was consistently the highest among all three mice strains after 10 Gy irradiation, but was not statistically significant (P= 0.09, Fig. 3). The DNA gel electrophoresis confirmed the detection of apoptotic bodies by H & E staining, and showed minimal DNA damage in the thymus of mice with no irradiation (Fig. 4). A time course study of apoptosis in the thymus of MT-I*, MT-null, and C57BL/6J by DNA ladder formation after exposure to 5 Gy showed distinct DNA ladder at 3–24 h in MT-null mice while similar pattern was observed only after 6 h in MT-I* mice. The DNA ladder formation in the thymus of MT-null mice at 24 h after exposure to a radiation dose of 5 Gy is shown in Fig. 4. These results were consistent with incidence of apoptotic bodies in MT-null mice as shown in Fig. 3. When the dose was increased to 10 Gy, the increase of DNA mobility on agarose gel was observed in all cases (Fig. 4), suggesting a general necrotic effect.
Fig. 3. Quantitative evaluation of apoptotic cells in thymus tissues from transgenic MT-I*, MT-null mice, and control C57BL/6J mice. Each bar presents the mean 9 S.E. (n =4). * Significantly different from that of MT-I* and control mice (P B0.05).
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Fig. 4. Effect of g-irradiation on thymic DNA mobility. DNA from thymus of MT-1*, MT-null, and C57BL/6J mice was isolated at 24 h after g-irradiation. 10 mg DNA per sample was loaded onto a 2.5% agarose gel, electrophoresed at 57 V, and visualized by SYBR Green I nucleic acid dye under UV light. From left to right: lane 1,4,7: MT-I*; lane 2,5,8: MT-null; lane 3,6,9: C57BL/6J. Photograph was typical of three separate experiments.
4. Discussion The present study demonstrates the constitutive expression of MT in thymus of MT-I* mouse. The constitutive overexpression of both MT mRNA (Palmiter et al., 1993) and protein (Iszard et al., 1995) has been reported in other organs of the MT-I* mouse, including liver, kidney, pancreas, heart, intestine, testis, and brain. The overexpression of MT, however, is not consistent among the organs. Pancreas and liver (20× and 16 × , respectively) are the two organs that have the highest basal overexpression as compared to that of age matched control litter mates. A small increase in MT was detected in the lung (1.7×), spleen (2.5× ), and brain (1.4 ×) of the MT-I* mouse. Similarly, we report here about 2.4 fold of MT increase in the thymus of MT-I* mouse. Therefore, it appears that the regulation of MT overexpression may be organ specific. Within thymus, subsets of cells contained different amount of MT (Fig. 1A). The epithelial cells were uniformly positive for MT, while some of the thymocytes were also MT positive, except in the MT-null mice (Fig. 1). The high basal levels of MT in thymus of MT-I* mice can be further increased by exposure to ionizing radiation, as shown on Fig. 1B. Simi-
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larly, the MT expression in the thymus of C57BL/ 6J mice was also increased after radiation as determined by immunohistochemistry (Fig. 1F). MT can be induced by a variety of factors such as metals and oxidative stress (Satoh et al., 1993). It has been shown that MT in thymus was induced by Zn injection in Swiss white mice (Olafson, 1985). MT mRNA in thymus is induced in Wistar rats after whole body X-irradiation (Shiraishi et al., 1989). However, major regulatory factors which are responsible for the induction of MT synthesis in the thymus are unknown. Generally metals and glucocorticoids are considered direct inducers of MT synthesis in mammalian tissues (Chan and Cherian, 1993; Palmiter, 1995). Radiation and other agents such as cytokines, lipopolysaccharide, endotoxin, paraquat and carbon tetrachloride, are considered as indirect inducers for MT synthesis. It has been shown that induction of MT occurred after X-irradiation of mice but not in cells derived from mice, which indicates a physiological factor other than free radicals is responsible for the MT induction (Koropatnick et al., 1989). The incidence of apoptosis, as determined both by the number of apoptotic cells per 100 cells and DNA fragmentation, was high at 24 h after exposure to low dose (5 Gy) of radiation but was decreased at high dose (10 Gy) of radiation. This may be due to increased number of necrotic cells after 10 Gy dose and subsequent elimination of these dead cells from the thymus (Figs. 3 and 4). These results are consistent in all three strains of mice studied. We elected light microscopy for detection and quantification of apoptosis on H&E stain, and did not use techniques such as TUNEL in this study. Although supplemental techniques are sometimes helpful, a carefully studied H&E section gives a definitive assessment of apoptosis. In keeping with this notion, Goldsworthy et al. (1995) have demonstrated that by microscopic examination of H&E stained slides, equivalent results could be obtained as compared to apoptosis detected by TUNEL. In the present study, the results obtained by quantification on H&E stain were confirmed by DNA fragmentation analysis, and suggest that a low dose of radiation is more effective in inducing apoptosis than a high dose of
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radiation. This dose-dependent relation between radiation and apoptosis has been reported previously in other cell lines (HL-60, Hopcia et al., 1996) as well as in certain lymphomas (Mirkovic et al. 1994). In V79 cells, radiation at 3 – 4 Gy caused a significant increase in apoptosis, while radiation at higher dose did not further increase apoptosis, but caused cell necrosis (Ghosh et al., 1996; Jamali and Trott, 1996). Thus, radiation at low and high doses can cause either apoptosis or necrosis in the thymus. The results show an increase in apoptosis in the thymus after radiation in all three strains of mice, but the percentage of apoptosis in thymus was decreased significantly with MT overexpression and significantly increased in the absence of MT expression in MT-null mice (Figs. 3 and 4). This result was consistent at different radiation doses, either with whole body irradiation (present study), or with thymocyte irradiated in vitro. Therefore, MT can protect mouse thymus from apoptosis after g-irradiation. An increase in apoptosis in MT-I* after high radiation dose suggest that the presence of MT alone can not fully protect thymocytes from apoptosis. The mechanisms by which MT can protect cell death are still unclear. Although radiation-induced apoptosis can occur through a variety of mechanisms, the oxidative damage to DNA and cell membrane are important factors for induction of apoptosis (Blank et al., 1997). Many antioxidants such as vitamin E can inhibit apoptosis induced by oxidative stress (Verhaegen et al., 1995; Zhang et al., 1997). Bcl-2, an important factor in regulation of apoptosis, was shown to prevent apoptosis through the anti-oxidative pathway by regulating cellular thiol levels (Hockenbery et al., 1993; Mirkovic et al., 1997; Pourzand et al., 1997). Therefore, it is possible that MT, as an anti-oxidant, may reduce the radiation damage, resulting in low incidence of apoptosis. Recently it has been shown that MT induced by Cd or non-metal inducer, b-thujaplicin, had marked protective effect on UVB-induced apoptosis in keratinocytes both in vivo and in vitro (Baba et al., 1998). MT-null mice exhibit high sensitivity to UVB-induced apoptosis in vivo (Hanada et al., 1998). Thymocytes from p53 null
mice were unable to undergo apoptosis in response to ionizing radiation while they responded to glucocorticoids (Lowe et al., 1993). Further experiments suggest that p53 is required for cells which undergo G1 arrest and apoptosis after radiation (Fan et al., 1994). In MT-null mice, it has been shown that p53 was induced in the liver by cadmium (Zheng et al., 1996). A high rate of apoptosis was detected in MT-null cells exposed to anticancer drugs as compared to controls (Kondo et al., 1997). Therefore, cells which do not have MT may be susceptible to p53 upregulation and apoptosis. Cellular thiols may play an important role in preventing spontaneous or radiation-induced apoptosis (Mirkovic et al., 1994; Sato et al., 1995). Glutathione (GSH) was able to protect PC12 cells from apoptosis induced by ascorbic acid and dopamine (Si et al., 1998). However, depletion of GSH with buthionine sulfoximine alone did not increase either spontaneous or radiation-induced apoptosis in cells (Sato et al., 1995; Mirkovic et al., 1997), suggesting the involvement of other thiols in modulating apoptosis. In the present study, we did not measure GSH levels in thymus. However, others have reported that there are no differences in GSH and other antioxidant levels in tissues such as liver and kidney of control, and MT-null or MT-I* mice (Iszard et al., 1995). The cells derived from these mice also showed no difference in GSH levels as compared to control mice cells (Lazo et al., 1995, 1998). As a major intracellular thiol, MT can scavenge free radicals such as hydroxyl radicals at a much higher efficiency than GSH (Thornalley and Vasak 1985; Miura et al., 1997). Thus, the presence of high content of MT in MT-I* mouse thymus may provide protection while its absence in MT-null mouse thymus may cause increased cellular damage after radiation. In two reports, we have shown a high incidence of apoptosis in both hepatocellular carcinoma and metastatic adenocarcinoma of liver with low MT expression, as compared to control normal human livers (Cai et al., 1998; Deng et al., 1998). In the skin, MT-null mice exhibits high sensitivity to UVB-induced apoptosis in vivo (Hanada et al., 1998). Even, in certain cell lines, a negative corre-
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lation of MT with the incidence of apoptosis has been documented (Abdel-Mageed and Agrawal, 1997; Kondo et al., 1997; Tsangaris and Tzortzatou-Stathopoulou, 1998). Thus, a decrease in apoptosis in the thymus of MT-I* mice as compared to other strains supports published results in other tissues. Zn can either induce or block apoptosis depending on the cellular concentration (Fraker and Telford, 1997). High concentrations of extracellular Zn (500–1000 mM) have been used frequently to block apoptosis in a variety of systems. Zn at physiological concentrations (100 mM or less), on the other hand, could actually induce apoptosis (Fraker and Telford, 1997; Houben et al., 1997). Therefore, the regulation of intracellular free Zn pool by MT may also be a possible mechanism for protective effect of MT against apoptosis. In summary, mouse thymocytes can undergo apoptosis or necrosis after exposure to g-radiation at low (5 Gy) or high dose (10 Gy), respectively. MT-null thymus was most susceptible to apoptosis at low dose of radiation, while MT-I* thymus with high MT content was least damaged at high dose of radiation. These results suggest a protective role for MT in radiation-induced apoptosis and necrosis in thymus. We also demonstrated MT expression in thymus of MT-I overexpressing mice, and MT expression in the thymus was induced further after exposure to g-irradiation in both MT-I* and C57BL/6J mice.
Acknowledgements This work was supported by a research grant from the Medical Research Council of Canada.
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