Increased radiation-induced apoptosis in mouse thymus in the absence of metallothionein
Introduction
Metallothioneins (MTs) are a group of low-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 thymus (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 anti-self 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 MT-inducing metals such as Zn showed protection against γ-irradiation compared to controls (Matsubara et al., 1986). MT can also protect against radiation-induced DNA damage. Zn/Cd-MT 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.
Section snippets
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 subsequent experiments. All C57BL/6J mice were purchased from Charles River Laboratories (Montreal, Quebec). Normal thymus from adult male MT-I*,
Results
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 γ-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),
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
Acknowledgements
This work was supported by a research grant from the Medical Research Council of Canada.
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