Introduction

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MT is a metal-binding protein of low molecular weight, and one-third of its amino acids are cysteine residues (Webb, 1979). The synthesis of MT can be induced by various metals, by xenobiotics and by different types of stress (Kägi, 1991; Sato and Bremner, 1993; Webb, 1979). MT appears to be a multifunctional protein that plays a role in the detoxification of metal compounds and several xenobiotics, in the homeostasis of essential metals, such as zinc and copper, and in the scavenging of reactive oxygen species (Bremner, 1987; Kägi, 1991; Sato and Bremner, 1993; Webb, 1979).

Ionizing radiation generates free radicals in the living cells (Chapman and Gillespie, 1981) and induces injury to the bone marrow (Henshke and Morton, 1957). We reported previously that decrease in the total number of leukocytes in the peripheral blood observed in mice exposed to -irradiation was significantly protected by prior treatment of mice with bismuth compounds (Satoh et al., 1989) which is one of inducers of MT synthesis (Naganuma et al., 1987). In this study, we investigated the possible involvement of MT in the protective effects of BN on the -irradiation-induced decrease in the total number of leukocytes in mice.

	Materials and Methods

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Animals and chemicals. Male ICR mice (6 wk old) were purchased from Charles River Japan, Inc. (Atsugi, Japan). Male B6D2F₁ mice (6 wk old) were purchased from Japan SLC (Hamamatsu, Japan). BN was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan) and dissolved in distilled water. [²⁰³Hg]-HgCl₂ (2.4 mCi/mg) was purchased from New England Nuclear (Boston, MA). Other chemicals were purchased from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan).

Treatment of animals. ICR mice received injections s.c. with 50 or 100 µmol/kg BN once a day for 2 days. Then, 24 hr after the last injection (in most experiments), mice were exposed to whole-body -irradiation (¹³⁷Cs; 3 or 8 grays) from a Gamma Cell 40 (Atomic Energy of Canada Ltd., Ontario, Canada) at a dose rate of 1.117 grays/min.

Assay of CFU-S. To determine the number of multipotential hematopoietic stem cells, we determined numbers of CFU-S as described by Till and McCulloch (1961). The donor mice (B6D2F₁) received injections s.c. with 100 µmol/kg BN once a day for 2 days and then exposed to -irradiation (3 grays) 24 hr after the second injection of BN. The marrow cells of these mice were isolated 1 day after the -irradiation and suspended in saline. The number of nucleated cells among the marrow cells was determined after addition of Turk's reagent. Suspensions of cells were kept on ice before injection into recipient mice. Recipient B6D2F₁ mice were exposed -irradiation (9 grays) and then the marrow cells obtained from the donor mice were transplanted i.v. The recipient mice were killed 10 days after transplantation. Their spleens were removed and fixed in Bouin's solution, and then the number of colonies in each spleen was determined.

Analysis of cellular factors. Bone marrow cells were collected from mice, suspended in saline and then sonicated. The activities of antioxidant enzymes in the samples were determined spectrophotometrically. Activities of catalase and SOD were determined by the methods of Cohen et al. (1970) and Imanari et al. (1977), respectively. The activity of GSH-Px was determined by the method of Lawrence and Burk (1976), with cumene hydroperoxide and hydrogen peroxide as substrates. The activity of GST was determined by the method of Habig et al. (1974) with 1-chloro-2,4-dinitrobenzene as substrate. The concentration of MT in the bone marrow was determined by a ²⁰³Hg-binding assay (Naganuma et al., 1987). The concentration of GSH was determined by method of Tanaka-Kagawa et al. (1993) by high-performance liquid chromatography. The concentration of protein was determined by the method of Lowry et al. (1951) with bovine serum albumin as the standard. The total number of leukocytes in the blood of mice was counted with a Coulter counter.

Northern blot analysis. Mice received injections s.c. with BN (100 µmol/kg) once a day for 2 days. Twelve hours after the second injection of BN, the bone marrow cells and kidney were removed. Total RNA were extracted and the RNA samples (20 µg/lane) were separated by electrophoresis on 1% agarose/formaldehyde gels, transferred to nylon membrane (NYTRAN, Schleicher & Schuell, Inc., Keene, NH), and UV cross-linked. DIG-labeled mouse MT-I and GAPDH (as an internal control) cDNA probes were generated using the PCR (polymerase chain reaction) DIG probe synthesis kit (Boehringer Mannheim, Mannheim, Germany) according to the manufacture's instructions. Hybridization, washing and detection were performed using DIG luminescent detection kit (Boehringer Mannheim, Mannheim, Germany) and membrane was exposed to x-ray film for about 1 hr at room temperature.

	Results

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Table 1 shows the effects of treatment of mice with BN on the lethality of subsequent -irradiation (¹³⁷Cs; 8 grays). Mice were given an injection of BN once a day for 2 days and received whole-body -irradiation 24 hr after the second injection of BN. All mice exposed to -irradiation without prior treatment with BN died within 30 days. The lethal effect of -irradiation was markedly diminished by prior treatment with BN and the effect of BN was dose dependent. This protective effect of BN against the lethality of -irradiation was observed only when BN was administered prior to the -irradiation (table 2). Figure 1 shows the effects of prior treatment with BN on irradiation-induced injury to bone marrow, as indicated by decreases in total numbers of leukocytes. In this experiment, mice received whole-body irradiation from ¹³⁷Cs at a dose of 3 grays, which was toxic but not lethal. A marked reduction in the total of number of leukocytes was observed 1 day after irradiation and subsequently the number of leukocytes was increased gradually in irradiated mice with and without prior treatment with BN. However, the rate of the recovery of the total number of leukocytes, monitored from 5 days after irradiation, in BN-treated irradiated mice was significantly higher than that in the nontreated irradiated mice.

View this table: [in this window] [in a new window]   TABLE 1 Effects of bismuth nitrate (BN) on the lethality of subsequent -irradiation in mice

View this table: [in this window] [in a new window]   TABLE 2 Effects of the schedule for administration of bismuth nitrate (BN) on the lethality of -irradiation in mice

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Effects of treatment with bismuth nitrate (BN) on the total number of leukocytes in mice after subsequent -irradiation. Mice received injections s.c. with 100 µmol/kg of BN or saline once a day for 2 days. They received whole-body irradiation (137Cs; 3 grays) 24 hr after the second injection of BN. Each value represents the mean ± S.D. of four mice. * Significantly different from untreated irradiated mice (P < .025).

To examine the effect of prior treatment with BN on bone marrow cells, we investigated the viability of hematopoietic stem cells by determining number of CFU-S (Till and McCulloch, 1961). The number of colonies formed in spleens of recipient mice 10 days after transplantation of marrow cells obtained from the donor mice after exposure to -rays (3 grays) was about 1% of that in the case of unirradiated control mice (fig. 2). Treatment with BN before -irradiation increased the number of CFU-S to about six times that in the case of BN-untreated irradiated mice (fig. 2). The sizes of the colonies that formed in spleens of recipient mice that had received injections with marrow cells from donor mice after -irradiation without BN-treatment were also significantly smaller than when donor cells came from control mice. However, the sizes of colonies formed in spleens of recipient mice that received injections of marrow cells from mice exposed to -rays after prior treatment with BN was almost the same as that in the case of the control mice (data not shown). These results suggested the possibility that treatment of mice with BN before irradiation protected the bone marrow cells against the toxic effects of -irradiation by scavenging reactive oxygen species because -irradiation is known to increase the rate of generation of reactive oxygen species in living cells (Chapman and Gillespie, 1981) and to induce injury to the bone marrow (Henshke and Morton, 1957).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Effects of bismuth nitrate (BN) on the viability of hematopoietic stem cells, as determined by an assay of colony-forming units in the spleen (CFU-S). Mice received injections s.c. with BN (100 µmol/kg) once a day for 2 days and received whole-body irradiation (137Cs; 3 grays) 24 hr after the second injection of BN. One day after -irradiation, surviving stem cells were examined by the CFU-S assay (see text for details). Each value represents the mean ± S.D. of four mice. * Significantly different from untreated irradiated mice (P < .001).

Table 3 shows the effects of treatment with BN on the activity or concentration of various cellular factors that act as antioxidants in bone marrow cells. Mice were given BN once a day for 2 days and then levels of antioxidant factors were determined 24 hr after the second dose of BN at 100 µmol/kg. BN significantly increased the concentration of MT in the bone marrow cells. However, no significant changes in the activities of catalase, SOD, GST and GSH-Px and the concentration of GSH were observed (table 3). Induction of MT synthesis in the bone marrow cells by BN was also confirmed by an increase in MT-I mRNA levels (fig. 3).

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Northern blot analysis of MT-I mRNA levels in the bone marrow cells and kidney of mice treated with bismuth nitrate (BN). Mice received injections s.c. with BN (100 µmol/kg) once a day for 2 days. Twelve hours after the second injection of BN, the bone marrow cells and kidney were removed. Northern hybridization was performed using digoxigeninlabeled mouse MT-I and GAPDH cDNA probe.

	Discussion

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We previously observed that the decrease in the total number of leukocytes in the peripheral blood, monitored in mice 10 days after -irradiation, could be limited by prior treatment of mice with a bismuth compound (Satoh et al., 1989). In our study, prior treatment with BN did not affect the decrease in the total number of leukocytes determined 1 day after -irradiation (fig. 1). However, the return to normal values of the reduced total number of leukocytes, monitored from 5 days after the -irradiation, was more rapid in BN-treated mice than in untreated mice (fig. 1). This result indicates that the protective effects of bismuth compounds that we had observed previously (Satoh et al., 1989) were not due to protection of leukocytes in the peripheral blood by such compounds. However, significant protection of bone marrow cells against -irradiation was apparent in mice that had been treated with BN before irradiation (fig. 2). These results suggest that prior treatment with BN accelerates the recovery of number of leukocytes in the peripheral blood by protecting the bone marrow against -irradiation.

Treatment of mice with BN had a significant protective effect against lethality of -irradiation, but such protection was observed only when BN was administered prior to -irradiation (tables 1 and 2). Thus, the possibility exists that prior treatment with BN induced synthesis of certain cellular factors that can protect the bone marrow against the reactive oxygen species generated by -irradiation.

Bismuth compounds are known to induce the synthesis of MT via the activation of a metal-response element that is located in the 5'-upstream region of the gene for MT (Palmiter, 1994; Searle et al., 1985). Thornalley and Vasak (1985) noted that MT efficiently eliminates reactive oxygen species, and the rate of reaction of MT with hydroxy radicals is several hundred-fold higher than that of GSH. Moreover, protective effect of MT against DNA damage induced by hydrogen peroxide (Chubatsu and Meneghini, 1993), nitric oxide (Schwarz et al., 1995) or cadmium (Cai et al., 1995; Chubatsu and Meneghini, 1993) has been reported. BN significantly increased the mRNA (fig. 3) and protein levels (table 3) of MT in the bone marrow cells of mice without any effects on activities or levels of other cellular antioxidants such as catalase, SOD, GST, GSH-Px and GSH (table 3). In this experiment, the elevated concentration of MT in the bone marrow caused by treatment with BN was only about twice the control level. In an earlier study of the heart, at only about twice the control level of MT, the peroxidation of cardiac lipid induced by adriamycin was found to be strongly suppressed (Naganuma et al., 1988). These results suggest that BN might protect bone marrow cells against the toxic effects of -irradiation by inducing the synthesis of MT in the bone marrow. MT is a strongly inducible protein and its concentration in tissues is increased by different types of stress (Sato and Bremner, 1993). Thus, MT might play an important role in determining the sensitivity of animals to -irradiation.