Center for Environmental and Occupational Health, Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas 66160-7417
Received December 14, 1999; accepted March 9, 2000
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ABSTRACT |
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Key Words: metallothionein; MT-null mice; cadmium; chronic exposure; bone mass loss; bone calcium loss; bone pathology.
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INTRODUCTION |
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Itai-itai disease was endemic among the elderly women of Toyama Prefecture in Japan after World War II (Nomiyama, 1986). The patients suffered severe movement-induced pain, and X-ray photographs of their bones showed deformation, decreased bone density, fractures, and evidence of bone repair. Itai-itai disease involves multiple organ systems, with severe osteoporosis, varying degrees of osteomalacia, and renal dysfunction with proteinuria, glucosuria, and aminoaciduria (Kjellstrom, 1992
; Nomiyama, 1986
). Itai-itai disease probably has multiple causes, but the main etiologic factor is chronic Cd exposure (Bhattacharyya et al., 1995
; Kjellstrom, 1992
; Nogawa, 1981
).
A lot of effort has been directed, for many years, into reproducing the Cd-induced bone lesions (of Itai-itai disease) in various animal species. Some investigators have succeeded in producing osteomalacia by long-term injection of Cd into rats (Bhattacharyya et al., 1988a; Bhattacharyya et al., 1988b
; Katsuta et al., 1994
; Ogoshi et al., 1992
; Takashima et al., 1980
; Wang and Bhattacharyya, 1993
), while others have been unable to reproduce the bone injury, depending on the animal species, dose, route, and duration of exposure to Cd (Hamada et al., 1991
; Kjellstrom, 1992
; Nomiyama, 1986
; Ogoshi et al., 1989
). In addition, many factors, such as nutritional status, ovariectomy, and sex, are known to influence Cd-induced bone injury (Bhattacharyya et al., 1995
; Whelton et al., 1997
).
Metallothionein (MT) is a low-molecular weight, cysteine-rich, metal-binding protein (Kägi, 1993). Intracellular MT has been shown to be an important protein protecting against the toxicity of heavy metals such as Cd (Goering et al., 1995
). MT is thought to sequester Cd into an inert Cd-metallothionein complex (CdMT), thus reducing the amount of Cd available to interact with target molecules (Goering et al., 1995
). Recent studies have shown that deficiency in MT makes MT-null mice more sensitive to acute Cd-induced lethality and hepatotoxicity (Masters et al., 1994
; Michalska and Choo, 1993
), as well as to chronic Cd-induced nephrotoxicity (Liu et al., 1998
).
Bone has MT-like proteins, but the cysteine content of MT in bones of Cd-treated rats has been reported to be lower than in the liver (Kono et al., 1981), and only 20% of Cd is bound to MT in the cytosol of bone (Kimura et al., 1974
). Exposure of clonal osteogenic cells to Cd increases MT-like protein synthesis, but at a low rate (Miyahara et al., 1986
). The protection by Zn against Cd-induced toxicity in cultured embryonic chick bone is suspected to be mediated by induction of MT (Kaji et al., 1988
). However, no information is available on MT's role in Cd-induced bone injury in intact animals. This study was, therefore, designed to investigate the role of MT in Cd-induced bone injury, using MT-null and their corresponding wild-type mice chronically exposed to Cd. The results indicate that deficiency in MT renders animals more vulnerable to Cd-induced bone lesions, as well as lesions in other tissues (Habeebu et al., 2000
; Liu et al., 1998
; Liu et al., 1999
).
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MATERIALS AND METHODS |
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Animals.
Homozygous MT knock-out mice (129/SvPCJ background) deficient in both MT-I and II proteins (Masters et al., 1994), and their corresponding parental background mice (129/Sv+P+C+MGFSLJ) were obtained from Jackson Laboratories (Bar Harbor, ME). Mice were housed in AAALAC-accredited rooms in metabolic cages with a 12-h light/dark cycle at 22 ± 2°C. Mice were allowed free access to rodent chow and tap water. The homozygous mutants were mated inter se to maintain the line. Male and female mice 68 weeks of age were used in the study. There were four male and four female mice per study group.
Animals were given 109CdCl2 over a wide range of doses: 0.050.8 mg Cd/kg, 0.01 µCi 109Cd/kg, sc for wild-type mice; and 0.01250.1 mg Cd/kg, 0.01 µCi 109Cd/kg, sc for MT-null mice. Controls were given saline (10 ml/kg, sc). All mice were dosed 6 days/week for 10 weeks.
Bone Cd content and histological examination.
At the end of 10 weeks, the mice were anesthetized and decapitated to collect blood, and necropsy was performed. The left femurs and tibias were freed of muscle as much as possible and fixed in 10% neutral buffered formalin. Cd content was determined by measuring the 109Cd content. The bones were then subjected to X-ray photography. The femurs were decalcified in 10% formic acid/10% neutral buffered formalin, followed by washing in neutral buffered formalin. The bones were then processed routinely, embedded in paraffin wax, and sectioned at 5-µm thickness for histological examination.
Bone mass analysis.
The vertebrae, right femurs, and tibias were placed in acid-washed, tarred-glass vials, and dried in an oven at 110°C for 18 h. They were then placed in a desiccator for 6 h and weighed to obtain the dry bone weight, and 10 ml of chloroform/methanol (2:1, v/v) were added to each vial for 48 h to extract fat from the bones. The bones were then rinsed for 2 h. The defatted bones were dried at 110°C for 18 h to obtain the fat-free dry weight. The fat-free bones were ashed for 72 h at 525°C using a muffle furnace to obtain the ash weight (Bhattacharyya et al., 1999; Wang and Bhattacharyya, 1993
). The ash was then dissolved in 10 ml of 1 N HCl, and aliquots were diluted in 0.1 N HCl and 0.1% lanthanum chloride solution for calcium determination by flame atomic absorption spectrophotometry.
Statistics.
Data are expressed as mean ± standard error. Groups of wild-type and MT-null mice were compared using Student's t test. Significance was set at p < 0.05.
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RESULTS |
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The lumbar vertebrae, right femurs, and tibias were used as representative bones for determination of bone mass. The dry and defatted bone weights (expressed as raw data and as percentage of body weight) are shown in Figure 1. Chronic Cd administration produced a dose-dependent decrease in bone mass up to 25%. The decrease in bone mass was more marked in MT-null mice. Furthermore, loss of bone mass occurred in MT-null mice at doses of Cd that were too low to produce detectable bone mass loss in wild-type mice.
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Histopathological examination of decalcified femurs showed thinning of metaphyseal cortical bone and dilatation of haversian canals (Fig. 5). Most dilated haversian canals were surrounded by increased amounts of uncalcified bone matrix (osteoid seams; Figs. 5B and 5C
); this was particularly pronounced in MT-null mice (Fig. 5C
, arrows). Many cortical osteocytes showed increased rounding and pyknosis (osteocytic osteolysis), with expanded pericellular space (Fig. 5C
). There was bone marrow hyperplasia, resulting in marked expansion of marrow space into metaphyseal cortical bone (Fig. 6B
, arrowheads). In MT-null mice there was thinning of the epiphyseal growth plate (Fig. 6D
) due to marrow expansion on both sides of the plate. Along with the thinning, there was extensive loss of the zone of endochondral ossification, resulting in increased direct contact of the growth plate cartilage with bone marrow space (Fig. 6D
). Irregular foci of necrosis were observed in the epiphyseal growth plate cartilage (Fig. 6F
, arrowheads) and metaphyseal cortical bone. There was increase in cancellous (woven) bone in the metaphysis, with resultant thickening of the bony trabeculae histologically. The morphological lesions seen in any treated group of mice were observed in all members of the group; and the severity of the lesions were similar. The lesions were generally more severe in MT-null mice than in wild-type mice.
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DISCUSSION |
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Cd concentration in bone increased in a dose-dependent manner. However, bone Cd concentrations were much lower than hepatic and renal Cd concentrations in the same mice. Tissue Cd levels are usually considerably higher in wild-type than in MT-null mice (Liu et al., 1998). However, in this study, no difference was observed in bone Cd concentrations between wild-type and MT-null mice.
The bone injury induced by Cd includes loss of bone mass (osteoporosis) and calcium (osteomalacia). There was also loss of body weight in these mice (Liu et al., 1998). However, the loss of bone mass is not due simply to weight loss by the mice because the loss of bone mass is evident even when bone weight is normalized to body weight (Fig. 1
). The same is true for bone ash weight and calcium content (Fig. 2
).
In this study we found no difference between male and female mice in the biochemical lesions induced by Cd. In humans, Itai-itai disease is a disease predominantly of postmenopausal women with a history of multiple childbirths (Bhattacharyya et al., 1995; Nomiyama, 1986
). Female mice would therefore be expected to have more severe lesions than male mice. In this study, however, we used mice only 68 weeks of age, which in relative terms is their early adulthood. Furthermore, the mice had no offspring.
Morphological lesions include dilatation of haversian canals, increased osteoid seams around the canals, osteocytic osteolysis, and increased cancellous bone formation in the metaphysis. These findings are consistent with previous reports in mice (Bhattacharyya et al., 1988a) and rats (Katsuta et al., 1994
). Cd-induced thinning of cortical bone and expansion of bone marrow space into metaphyseal cortical bone predisposes the mice to pathological fractures (fractures in the absence of trauma). Bone strength is reduced further by the Cd-induced loss of calcium from bone. Quantitative histopathological studies in humans with Itai-itai disease (autopsy cases) have demonstrated increased osteoid formation and poor mineralization (osteomalacia) occurring concurrently with decreased bone volume (osteoporosis) and thinning of cortical bone (Noda and Kitagawa, 1990
; Noda et al., 1991
). The effects of Cd-induced bone injury in laboratory animals are the same as in humans, thus making these laboratory animals excellent models for the study of Itai-itai disease. The Cd-induced effects may explain the increased incidence of bone fractures in Itai-itai disease patients and laboratory animals.
This study demonstrates that deficiency in MT makes animals more vulnerable to chronic Cd-induced bone injury. In MT-null mice, the dose required to produce toxicity was as low as 0.0125 mg Cd/kg (6 times/week for 10 weeks), close to one order of magnitude lower than the corresponding dose in wild-type mice. There was greater loss of bone mass and bone calcium at all doses in MT-null mice than in wild-type mice. The morphological lesions induced in bone by chronic Cd exposure were also more severe in MT-null than in wild-type mice. For instance, thinning of epiphyseal growth plate by the expanding bone marrow was observed only in MT-null mice. Thus, MT-null mice were more vulnerable than wild-type mice to chronic Cd-induced bone injury, similar to the situation seen in chronic Cd-induced nephrotoxicity (Liu et al., 1998), hematotoxicity (Liu et al., 1999
), and hepatotoxicity (Habeebu et al., 2000
).
The mechanisms by which MT protects against Cd-induced bone injury are not known. Bhattacharyya et al. (1999) suggest that MT's protection against Cd-induced bone calcium loss is probably being exerted at the level of the intestine, where MT sequesters Cd and keeps it from entering the circulation. However, in animals receiving repeated Cd injections, multiple factors may be involved. We suggest that MT's protection against Cd-induced bone injury may be mediated via the following mechanisms: (1) protection against Cd-induced nephrotoxicity, and (2) protection against the direct toxic effects of Cd on bone cells.
Cd-induced bone injury is often associated with renal dysfunction in both Itai-itai patients (Bhattacharyya et al., 1995, Nomiyama, 1986
) and laboratory animals (Katsuta et al., 1994
; Katsuta et al., 1996
). Kidney injury produces bone injury through disturbance of calcium homeostasis, reflected in the calciuria seen in Cd-exposed workers (Kazantzis, 1979
) and laboratory animals (Jin et al., 1992
). The calciuria leads to hypocalcemia, which induces secondary hyperparathyroidism. As a result, bone resorption occurs to maintain serum calcium homeostasis (Sacco-Gibson et al., 1992
; Wang and Bhattacharyya, 1993
). Cd-induced bone resorption can be prevented by Zn, an inducer of MT (Suzuki et al., 1990
). Renal dysfunction also produces impairment of vitamin D metabolism (Aoshima et al., 1993
, Nogawa et al., 1987
; Tsuritani et al., 1992
), which in turn interferes with calcium absorption from the gastrointestinal tract, and with bone formation (Kido et al., 1990
; Nomiyama, 1986
). Because MT-null mice are more sensitive to Cd-induced renal injury, renal dysfunction may potentiate the injury to bone by chronic Cd exposure. We investigated Cd-induced kidney damage in kidneys from the mice used in this study. MT-null mice had more advanced and more widespread kidney lesions (for example, proximal tubule degeneration, glomerular swelling, and interstitial inflammation) than wild-type mice at identical doses and duration of Cd exposure (Liu et al., 1998
). Our results suggest that MT may protect against Cd-induced bone injury indirectly by reducing Cd nephropathy.
Evidence has accumulated that Cd also directly damages bone. In cultured fetal rat limb bones, a very low concentration of Cd (10 nM) produced dramatic demineralization of bone (Bhattacharyya et al., 1988b). In cultured osteoblasts and osteoclasts, Cd produces toxicity at a concentration lower than that toxic to liver (Iwami and Moriyama, 1993
; Wilson et al., 1996
). Induction of MT has been shown to offer cellular protection against Cd toxicity in various cell types (Klaassen and Liu, 1991
), including osteosarcoma cells (Angle et al., 1993
; Thomas et al., 1990
), osteoblastlike cells (Suzuki et al., 1989
), and cultured embryonic chick bone (Kaji et al., 1988
). Thus, MT may also directly reduce the osteotoxic effects of Cd.
In conclusion, this study has demonstrated that (1) chronic Cd exposure produces dose-dependent toxicity to mouse bone, with loss of bone mass, demineralization, and defective repair of morphological lesions, as major features of the bone injury; and (2) lack of MT makes animals more vulnerable to Cd-induced bone injury.
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ACKNOWLEDGMENTS |
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NOTES |
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