Increased radiation-induced apoptosis in mouse thymus in the absence of metallothionein

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 γ-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.

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.