Metallothionein-Null Mice Are More Sensitive than Wild-Type Mice to Liver Injury Induced by Repeated Exposure to Cadmium

Sultan S. Habeebu, Jie Liu, Yaping Liu and Curtis D. Klaassen1

Center for Environmental and Occupational Health, Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas 66160–7417

Received September 28, 1999; accepted December 22, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Liver is a major target organ of cadmium (Cd) toxicity following acute and chronic exposure. Metallothionein (MT), a low-molecular-weight, cysteine-rich, metal-binding protein has been shown to play an important role in protection against acute Cd-induced liver injury. This study investigates the role of MT in liver injury induced by repeated exposure to Cd. Wild-type and MT-I/II knockout (MT I/II-null) mice were injected sc with a wide range of CdCl2 doses, 6 times/week, for up to 10 weeks, and their hepatic Cd content, hepatic MT concentration, and liver injury were examined. Repeated administration of CdCl2 produced acute and nonspecific chronic inflammation in the parenchyma and portal tracts and around central veins. Higher doses produced granulomatous inflammation and proliferating nodules in liver parenchyma. Apoptosis and mitosis occurred concomitantly in liver following repeated Cd exposure, whereas necrosis was mild. As a result, significant elevation of serum enzyme levels was not observed. In wild-type mice, hepatic Cd concentration increased in a dose- and time-dependent manner, reaching 400 µg/g liver, along with 150-fold increases in hepatic MT concentrations, the latter reaching 1200 µg/g liver. In contrast, in MT I/II-null mice, hepatic Cd concentrations were about 10 µg/g liver. Despite the lower accumulation of Cd in livers of MT I/II-null mice, the maximum tolerated dose of Cd was one-eighth lower than that for wild-type mice at 10 weeks, and liver injury was more pronounced in the MT I/II-null mice, as evidenced by increases in liver/body weight ratios and histopathological analyses. In conclusion, these data indicate that (1) nonspecific chronic inflammation, granulomatous inflammation, apoptosis, liver cell regeneration, and presumably, preneoplastic proliferating nodules are major features of liver injury induced by repeated Cd exposure, and (2) intracellular MT is an important protein protecting against this Cd-induced liver injury.

Key Words: cadmium (Cd) toxicity; metallothionein (MT); liver toxicity; apoptosis; necrosis.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Liver is a major target organ for Cd toxicity after acute exposure (Hoffmann et al., 1975Go; Dudley et al., 1982Go, 1984Go). Acute Cd exposure produces hepatocyte swelling and fatty change, as well as focal, zonal, and massive necrosis, resulting in marked elevation of serum enzymes (Dudley et al., 1982Go). Electron microscopic examination reveals the early changes to be dilatation of rough endoplasmic reticulum, increased perichromatin granules, loss of membrane-associated ribosomes, and mitochondrial swelling with loss of cristae, pyknotic nuclei, and clumps of coagulated chromatin (Dudley et al., 1984Go). We demonstrated recently that apoptosis is prominent in Cd-induced acute liver injury (Habeebu et al., 1998Go). Both apoptosis and necrosis are modes of cell death in acute Cd poisoning. During acute Cd exposure, apoptosis occurs in a dose-dependent manner, and precedes necrosis of hepatocytes (Habeebu et al., 1998Go). However, the presence and role of apoptosis in chronic Cd-induced liver injury needs to be investigated.

As in acute Cd toxicity, liver is also a target organ following repeated Cd exposure. However, compared to acute Cd-induced liver injury, the pathology of liver injury following repeated Cd exposure has not been fully investigated. In marked contrast to the ultrastructural changes and massive necrosis seen in Cd-induced acute liver injury, the liver necrosis of chronic Cd exposure is mild. The lesions currently described include those of focal necrosis with mild or no increase in serum enzyme activity, parenchymal-cell swelling and cytoplasmic eosinophilia, enlarged nuclei, a slightly dilated rough endoplasmic reticulum, proliferation of connective-tissue fiber bundles, and interstitial fibrosis surrounding central veins and portal triads (Dudley et al., 1984Go, 1985Go; Faeder et al., 1977Go).

Most of the total body burden of Cd is associated with metallothionein (MT). MT is a low-molecular-weight protein ubiquitous in mammals (Kagi, 1993Go). The amino acid composition of MT is unusual in that it has no aromatic amino acids and one-third of its amino acids are cysteine residues. MT is highly conserved in its sequence, cysteine content, and capacity to bind metals (Kagi, 1993Go). One molecule of MT is capable of binding 7 atoms of Cd or Zn. MT is induced by Cd, and binds the metal with high affinity (Waalkes et al., 1984Go).

MT has been proposed as a key player in Cd detoxication. Pretreatment of animals with low doses of Cd induces MT, and produces tolerance to subsequent high, hepatotoxic, and lethal doses of Cd (Goering and Klaassen, 1984Go). It is thought that the increased MT binds Cd in the cytosol, thus reducing Cd content in critical organelles (Goering and Klaassen, 1983Go). This hypothesis is further supported by results from studies with MT-transgenic animal models; the constitutive overexpression of MT protects against Cd-induced lethality and liver injury (Liu et al., 1995Go), while lack of MT renders MT I/II-null mice highly susceptible to Cd toxicity (Liu et al., 1996bGo; Masters et al., 1994Go; Michalska and Choo, 1993Go). Thus, the protective role of MT in acute Cd-induced liver injury is well established.

In contrast to acute Cd exposure, very little is known about the role of MT in protecting against chronic Cd-induced liver injury. In fact, doubt has been expressed over whether MT can provide long-term protection against the toxicity of Cd (Cherian, 1995Go; Petering and Fowler, 1986Go). This study, using wild-type and MT I/II-null mice, was designed to investigate the features of liver injury following repeated Cd exposure, and the role of MT in this Cd-induced liver injury. Our results demonstrate that chronic inflammation, granulomas, and proliferating nodules are the major features of the liver injury induced by repeated exposure to Cd, necrosis is not prominent, and MT protects against this Cd-induced liver injury.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
CdCl2 was obtained from Fisher Scientific Co (Fair Lawn, NJ). CdCl2 solution was prepared in 0.9% NaCl. 109CdCl2 (5.12 mCi/mg) was obtained from New England Nuclear (Boston, MA). All other chemicals were of reagent grade.

Animals.
Homozygous MT-I and -II knockout mice (129/SvPCJ background; Masters et al., 1994) were obtained from Jackson Laboratories (Bar Harbor, ME). Mice were housed in AAALAC-accredited rooms with a 12-h light/dark cycle at 70°F. 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 aged 6 to 8 weeks were used in this study. In 3 preliminary studies, homozygous MT-I and -II knock-out mice (129/Ola x C57BL/6J background, Michalska and Choo, 1993), obtained from the Murdoch Institute, Royal Children's Hospital (Parkville, Australia), were used and gave similar results (data not shown). Genetic background-matched mice were bred as controls, with both sex and age matched to MT I/II-null mice.

Animals were given CdCl2 over a wide dose range: 0.05 to 2.4 mg Cd/kg, 0.01 µCi 109Cd/kg, sc for wild-type mice; and 0.0125 to 0.8 mg Cd/kg, 0.01 µCi 109Cd/kg, sc for MT I/II-null mice. Control mice (wild-type and MT I/II-null) were given saline (10 ml/kg, sc). Animals were dosed 6 times/week for up to 10 weeks.

The animals were observed daily and body weights were recorded weekly to determine the effect of Cd on their general health. Moribund animals were euthanized with CO2, and necropsy was performed. Although tissues were collected every week, the data from the 3 major tissue collections, namely 3-week (70 mice), 6-week (98 mice), and 10-week (84 mice) groups that were administered Cd, are presented.

Tissue and blood analysis.
At the end of the exposure to Cd, animals were anesthetized, decapitated to collect blood, and necropsied. A portion of liver was fixed in 10% neutral buffered formalin, and Cd content was determined by 109Cd analysis. Liver samples were processed by standard histological techniques, and were stained with hematoxylin and eosin (H&E) for light microscopic examination. A portion of liver was homogenized in 10 mM Tris–HCl, pH 7.4, and cytosols were prepared by ultracentrifugation. Metallothionein concentrations in liver cytosols were determined by the Cd/hemoglobin radioassay (Eaton and Toal, 1982Go). Serum was analyzed for alanine aminotransferase activity using a commercially available kit from Sigma (St. Louis, MO).

Cytosolic distribution of Cd in liver.
A portion of liver was placed in 0.25 M sucrose containing 10 mM Tris-acetate, pH 7.4 (1:4, w:v), and homogenized with a Teflon pestle-glass homogenizer. Various fractions were prepared by differential centrifugation at 4°C. The resultant pellets were defined as nuclei (600 g, 10 min), mitochondria (10,000 g, 10 min), microsomes (100,000 g, 65 min) and cytosol (100,000 g supernatant). The distribution of 109Cd in the hepatic cytosolic fraction was analyzed by Sephadex G-75 chromatography (Pharmacia Fine Chemicals, Piscataway, NJ). Elution was performed with 10 mM Tris-acetate, pH 7.4, at a rate of 30 ml/h, at 4°C, and ninety 5-ml fractions were collected for 109Cd analysis as previously described (Goering and Klaassen, 1984Go).

In situ apoptosis detection.
Apoptosis was demonstrated in situ by the TUNEL (TdT-mediated dUTP-digoxigenin Nick End Labeling) assay, using a kit from Oncor (Gaithersburg, MD) according to their instructions. Briefly, sections were dewaxed in xylene, hydrated in graded alcohol series, and endogenous peroxidase blocked with H2O2 in PBS. Terminal deoxynucleotidyl transferase (TdT) enzyme and digoxigenin-labeled dUTP were applied to the sections for 1 h at 37°C. Sections were washed in Wash Buffer and treated with peroxidase-conjugated anti-digoxigenin antibody for 1 h at room temperature. They were next treated with HistoMark® Black chromogen solution according to the manufacturer's instructions. Sections were stained lightly in H&E, dehydrated in alcohol series, cleared in xylene, and mounted in Permount. Hepatocytes (2000–2500/animal) in 20–24 randomly selected fields were counted under 400x magnification, aided by a grid of 100 squares. The apoptotic index was determined by dividing the total number of hepatocytes showing staining for apoptosis by the total number of cells in the fields examined.

Statistics.
Data are expressed as mean ± standard error of mean. Comparisons between wild-type and MT I/II-null mouse groups (given the same dose of Cd) were performed with the Student's t-test. Statistical significance was set at p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Repeated administration of CdCl2 resulted in a dose-dependent lethality in both wild-type and MT I/II-null mice (Liu et al., 1999bGo). The maximum tolerated dose of Cd (above which there is 40% lethality or greater) in wild-type mice was approximately 2.4, 1.6, and 0.8 mg Cd/kg for 3-, 6-, and 10-week exposures, respectively. In comparison, the maximum tolerated dose of Cd in MT I/II-null mice was 6–8 times lower, at 0.4, 0.2, and 0.1 mg Cd/kg for 3, 6, and 10-week exposures, respectively. There was a dose-dependent loss of body weight at the highest tolerated doses (Table 1Go; Liu et al., 1999b). Two of the doses administered to MT I/II-null mice (0.0125 and 0.025 mg Cd/kg) were not administered to wild-type mice because our preliminary studies indicated that these doses were too low to produce any significant effects in wild-type mice.


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TABLE 1 Effect of up to 10 Weeks of Repeated Cadmium Exposure on Mice Body Weight and Liver/Body Weight Ratios
 
Repeated administration of Cd produced much less accumulation of Cd in livers of MT I/II-null than in wild-type mice (Fig. 1Go). For example, in wild-type mice receiving 10 weeks exposure to 0.05, 0.1, 0.2, 0.4, and 0.8 mg Cd/kg, Cd concentration was 32, 73, 157, 302, and 424 µg/g liver, respectively. In comparison, in MT I/II-null mice receiving 10 weeks exposure to 0.0125, 0.025, 0.05, and 0.1 mg Cd/kg, hepatic Cd concentration was only 5, 6, 8 and 12 µg/g liver, respectively. Liver Cd accumulation was also much lower in the MT I/II-null than wild-type mice at various times of exposure. For example, at the dose of 0.1 mg Cd/kg, hepatic Cd concentration in wild-type mice was 17, 38, and 74 µg/g liver after 3, 6, and 10 weeks of Cd injection, respectively. Whereas in MT I/II-null mice receiving the same dose of Cd, hepatic Cd concentration was only about 10 µg/g liver during the 3 to 10 weeks of exposure to Cd.



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FIG. 1. Hepatic Cd concentrations following repeated CdCl2 administration to wild-type (0.05–2.4 mg Cd/kg, sc, 6 times/week) and MT I/II-null mice (0.0125–0.4 mg Cd/kg, sc, 6 times/week) for 3, 6 and 10 weeks. Data are mean ± SE of 6–8 mice. *MT I/II-null mice significantly different from wild-type mice (p < 0.05).

 
Repeated administration of CdCl2 enlarged the liver in a dose-dependent manner, with up to 100% increase in liver/body weight ratios in both wild-type and MT I/II-null mice (Table 1Go). For example, liver/body weight ratios in wild-type mice increased from 34.3 to 37.5, 39.8, 46.6, and 69.3 g/kg after sc injection of Cd at 0.2, 0.4, 0.8, and 1.6 mg/kg respectively, for 6 weeks. At the same duration of Cd exposure, MT I/II-null mice had higher liver/body weight ratios than wild-type mice, at approximately one-tenth the dose given to the wild-type mice.

Histopathological examination showed the lesions of liver injury induced by repeated Cd exposure to be progressive and diverse. The earliest lesions observed in both wild-type and MT I/II-null mice included focal necrosis of parenchymal cells, apoptosis, and foci of acute inflammation (collections of polymorphonuclear leucocytes, PMNs, mainly neutrophils) in the parenchyma, portal tracts, and around central veins (Fig. 2AGo). With increasing dose and time, the lesions progressed to subacute inflammation (collections of mixed acute and chronic inflammatory cells), then to nonspecific chronic inflammation (collections of mainly chronic inflammatory cells, that is, lymphocytes and macrophages, Fig. 2BGo), granulomas in liver parenchyma (Fig. 2CGo), proliferating nodules (Fig. 3AGo), and scattered bizarre multinucleate giant cells (Figs. 3B and 3CGo). Subacute peritonitis (Fig. 3DGo) was observed at higher doses. It is noteworthy that peritonitis was present, although Cd had been administered sc, not ip. Liver cell regeneration was observed at all doses at later time points. The severity of inflammation and the incidence of other morphologic lesions (cell proliferation, bizarre giant cells, and proliferating nodules) were determined semi-quantitatively by analyzing 20 (mitosis, for cell proliferation) or 50 representative high-power fields (hpf) of liver sections from mice administered Cd repeatedly for 10 weeks. The severity of inflammation and liver cell regeneration (mitosis) showed dose-dependent increases, both more marked in MT I/II-null mice than wild-type mice at corresponding doses of Cd (Table 2Go; also compare Figs. 2A and 2C with Fig. 2DGo). In contrast, the incidence of giant cells and proliferating nodules did not show a discernible dose-response relationship (Table 2Go), but were higher in MT I/II-null mice than wild-type mice given identical doses of Cd.



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FIG. 2. Representative photomicrographs of liver sections from mice administered Cd, sc, 6 times/week for 10 weeks. (A) Liver section from MT I/II-null mouse (0.1 mg Cd/kg), showing a collection of polymorphonuclear cells, PMNs, mainly neutrophils; magnification x240. (B) Liver section from wild-type mouse (0.8 mg Cd/kg), showing a collection of mainly chronic inflammatory cells, that is, lymphocytes and macrophages; x240. (C) Liver section from MT I/II-null mouse (0.1 mgCd/kg) showing a granuloma, including a Langhan's-type giant cell; x240. (D) Liver section from wild-type mouse (0.1 mg Cd/kg) showing focal necrosis (arrows); x120; lesions in wild-type mice at this dose, were minimal compared to the advanced lesions in MT I/II-null mice (A) and (C) at the same dose.

 


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FIG. 3. Representative photomicrographs of liver sections from mice administered Cd, sc, 6 times/week for 10 weeks. (A) Liver section from wild-type mouse (0.8 mg Cd/kg) showing a proliferating nodule, with two cells in mitosis; original magnification x580. (B and C) Liver sections from MT I/II-null mice (0.1 mg Cd/kg) showing bizzare multinucleate giant cells; x580. (D) Liver section from MT I/II-null mouse showing subacute peritonitis; solid arrows indicate infiltration of inflammatory cells beneath the liver capsule, and open arrows indicate folded fibrinopurulent membrane (inflammatory exudate) deposited on the liver surface; x230; note that mice were injected sc, not ip.

 

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TABLE 2 Quantitative Analysis of Morphologic Lesions Produced by Repeated Daily Injection of Cadmium for 10 Weeks
 
Apoptosis was quantified immunohistochemically (TUNEL-stained sections, Figs. 4A and 4BGo) by determining the apoptotic index in mice exposed to repeated Cd injections for 10 weeks. Apoptotic index was defined as the percentage of cells undergoing apoptosis in randomly selected, representative fields. In wild-type and MT I/II-null mice, the dose-dependent increase in apoptotic index was gradual, except at the highest tolerated doses where there was a sharp increase (Fig. 5Go). Mitosis increased with dose and duration of exposure, and appeared to depend mainly on the severity of the pathologic lesions. In acute Cd-induced liver injury, necrosis is prominent and inflammation is minimal (Habeebu et al., 1998Go). In contrast, in liver injury following repeated Cd exposure, necrosis occurs mainly as a consequence of the active inflammation rather than as a direct effect of Cd hepatotoxicity. Serum alanine aminotransferase (ALT) activity, an indicator of parenchymal cell necrosis, was significantly increased only in mice receiving the highest dose of Cd (Fig. 6Go).



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FIG. 4. Representative photomicrographs of liver sections showing immunohistochemical demonstration of apoptotic cells and bodies (arrows) following subchronic administration of Cd (0.1 mg Cd/kg, sc, 6 times/week for 10 weeks); magnification x280. (A) Wild-type mouse, (B) MT I/II-null mouse.

 


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FIG. 5. Hepatic apoptotic index following repeated CdCl2 administration to wild-type (0.05–0.8 mg Cd/kg, sc, 6 times/week) and MT I/II-null mice (0.0125–0.1 mg Cd/kg, sc, 6 times/week) for10 weeks. Data are mean ± SE of 6–8 mice. *MT I/II-null mice significantly different from wild-type mice (p < 0.05).

 


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FIG. 6. Serum alanine aminotransferase (ALT) activities following repeated CdCl2 administration to wild-type (0.05–2.4 mg Cd/kg, sc, 6 times/week) and MT I/II-null mice (0.0125–0.4 mg Cd/kg, sc, 6 times/week) for 3, 6, and 10 weeks. Data are mean ± SE of 6–8 mice.

 
Corresponding to increased hepatic Cd concentration, hepatic metallothionein (MT) concentration was markedly increased following repeated Cd exposure in wild-type mice (Fig. 7Go). Hepatic MT induction also showed a dose- and time-dependent pattern, reaching 1200 µg/g. In comparison, there was no MT in the liver of MT I/II-null mice either before or during Cd exposure. The intracellular MT in wild-type mice resulted in preferential distribution of cellular Cd to the cytosol in wild-type mice when compared to MT I/II-null mice. Much of the cytosolic Cd was bound to MT in wild-type mice (Fig. 8Go, fractions 40–50), but in MT I/II-null mice there was no association of Cd with MT (Fig. 8Go) because of the absence of MT.



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FIG. 7. Concentrations of MT in the liver following repeated CdCl2 administration to wild-type (0.05–2.4 mg Cd/kg, sc, 6 times/week) and MT I/II-null mice (0.0125–0.4 mg Cd/kg, sc, 6 times/week) for 3, 6, and 10 weeks. Data are mean ± SE of 6–8 mice. *MT I/II-null mice significantly different from wild-type mice (p < 0.05).

 


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FIG. 8. G-75 chromatography profiles of Cd distribution in liver cytosol following repeated Cd exposure (0.1 mg Cd/kg, sc, 6 times/week) for 6 weeks. The peak over fractions #20–30 indicates Cd bound to high molecular weight proteins, while the peak over fractions #40–50 indicates Cd bound to metallothionein.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The purpose of the present study was 2-fold: (1) to characterize liver injury in mice following repeated Cd exposure, and (2) to determine the role of MT in the Cd-induced liver injury.

The most apparent change following repeated exposure to Cd is hepatomegaly, which is dependent on the dose and duration of Cd exposure (Table 1Go). The increase in liver/body weight ratio is consistent with similar findings by Kamiyama's group, who studied the effects of one-year ip administration of Cd to rats (Kamiyama et al., 1995Go). The increase in liver mass is due to inflammation, granulomas, and proliferation of hepatocytes.

The primary pathologic lesion is inflammation of the liver parenchyma and portal tracts. This starts as acute inflammation (collections of mainly polymorphonuclear leukocytes, PMNs, Fig. 2AGo), progresses to subacute inflammation (collections of mixed PMNs and mononuclear cells), then to nonspecific chronic inflammation (collections mainly of mononuclear cells, that is, lymphocytes and macrophages, Fig. 2BGo), and finally, to granulomas (Fig. 2CGo). The presence of PMNs indicate on-going active destruction of liver cells while the mononuclear cells and granulomas indicate chronicity of the liver's inflammatory response. The liver's inflammatory response and repair (liver cell regeneration) following Cd exposure have been shown to be mediated by cytokines (Dong et al., 1998Go; Kayama et al., 1995Go) .

In addition to inducing inflammation, 10 weeks of repeated exposure to Cd also induced other lesions in the liver, including apoptosis, liver cell regeneration, multinucleate giant cells with bizarre nuclei, proliferating nodules, and subacute peritonitis. The significance of the multinucleate giant cells, scattered singly in the liver parenchyma, is unknown at this time. The mechanism of induction of the giant cells by Cd is also unknown. Multinucleate giant cells were similarly observed in the spleens of these mice (Liu et al., 1999aGo). The presence of granulomas suggests that cell-mediated hypersensitivity is involved in the pathogenesis of the liver injury induced by repeated exposure to Cd. This contrasts with acute Cd-induced liver injury where the observed leukocytoclastic vasculitis (hypersensitivity angiitis, normally caused by deposition of antigen-antibody complexes in small blood vessels) suggests the involvement of humoral mechanisms in acute Cd toxicity. Splenic enlargement (due to lymphoid hyperplasia) is observed in both acute and subchronic Cd poisoning (Liu et al., 1999aGo), lending further support to the involvement of the immune system in the pathogenesis of Cd toxicity.

The proliferating nodules observed in the liver consist of closely packed hepatocytes, sometimes with one or more cells in mitosis (Fig. 3AGo). The nodules were observed at all doses of Cd administered in MT I/II-null mice and all doses except the lowest (0.05 mg/kg) in wild-type mice. However, a dose-response pattern was not evident. The incidence of these nodules may appear to be low, but when it is noted that the repeated exposure to Cd was for 10 weeks only, it becomes remarkable that proliferative lesions could be induced by Cd within this time frame. The incidence of proliferative nodules was significantly higher in MT I/II-null mice than wild-type mice at identical doses of Cd. This suggests that intracellular metallothionein may play a protective role against the induction by Cd of proliferative lesions in mice liver. These nodules represent either nodular hyperplasia or benign adenomas. The difficulty in the differential diagnosis of these nodules is well documented (Moch et al., 1996Go; Popp and Cattley, 1991Go). If these nodules are adenomas, then they are neoplastic. On the other hand, if the nodules represent nodular hyperplasia, then presumably, the nodules are preneoplastic lesions. Cadmium is an established carcinogen in humans and laboratory animals; it is known to cause tumors of the testis and ventral prostate in rodents and lung cancer in humans (IARC, 1993Go). Cd has also been associated with tumors of the hematopoietic system, that is, leukemias and lymphomas in rodents (Waalkes and Rehm, 1994Go) and with injection site tumors (sarcomas). Waalkes et al. (1999) recently showed that chronic Cd exposure also causes tumors of the adrenal gland and kidney, as well as proliferative, presumably preneoplastic, lesions in the dorsolateral prostate in the Noble rat. Furthermore, epidemiological studies have linked Cd with cancer of the urinary bladder (Darewicz et al., 1998Go) and prostate (Brys et al., 1997Go) in humans. One mechanism by which Cd is thought to induce tumors is the inhibition of DNA repair systems, leading to decreased removal of endogenous DNA lesions and of DNA damage caused by environmental toxicants including Cd (Hartwig, 1998Go). Cd is thought to displace Zn from zinc-finger structures of DNA repair enzymes. Also, Cd is known to be genotoxic in vitro, causing frame-shift mutations (Biggart and Murphy, 1988Go), single strand breaks (Coogan et al., 1992Go), and chromosomal aberrations (Hartwig, 1994Go). To our knowledge, there are no reports associating Cd with tumors of the liver in humans, rodents, or any other animal species. This makes our finding of proliferating nodules in liver very interesting, and highlights the need for further research in this area.

The results of the present study indicate that the liver injury induced by repeated exposure to Cd is quite different from the injury of acute Cd exposure. Parenchymal-cell necrosis with marked elevations of serum enzymes is the major feature of acute Cd-induced liver injury (Dudley et al., 1982Go; Habeebu et al., 1998Go; Liu et al., 1996bGo). In contrast, following repeated Cd exposure, liver necrosis is mild and there is no significant increase in serum ALT activity (Fig. 6Go). Apoptosis is a feature of both acute Cd exposure and 10 weeks of repeated Cd injections in mice; in both instances, the incidence of apoptosis is dose-dependent. Apoptosis of hepatocytes provides a means of elimination of critically damaged liver cells without disturbing liver structure and function. The incidence of apoptosis (apoptotic index) was higher in MT I/II-null than in wild-type mice (Fig. 5Go). As in this study, apoptosis has been shown to be enhanced in MT I/II-null cells or animals exposed to oxidative stress (Kondo et al., 1997Go), cisplatin (Kondo et al., 1997Go; Liu et al., 1998aGo), and subchronic CdMT (Liu et al., 1999bGo), suggesting that intracellular MT protects against apoptosis. Apoptosis, by itself, would be expected to protect against the induction of tumors, first by counter-balancing the effect of cell proliferation, and secondly, by eliminating the cells with critical damage to their DNA. However, the incidence of apoptosis (apoptotic index) may not be sufficiently high to cope with the rate of acquisition of new cells with irreversible damage to their DNA and DNA-repair mechanisms. This could explain the higher incidence of proliferative nodules in the liver of MT I/II-null mice despite their having higher apoptotic indices than wild-type mice at identical doses of Cd.

Dramatic differences in hepatic Cd and MT concentrations were observed between wild-type and MT I/II-null mice. In wild-type mice, hepatic Cd concentration increased in both a dose- and time-dependent manner, along with marked induction of hepatic MT. In contrast, there was no MT in MT I/II-null mice, and Cd concentration in liver was much lower in MT I/II-null mice than in wild-type mice. MT has been shown not to affect the absorption and initial distribution of Cd in rodents following exposure to Cd (Liu et al., 1996aGo). It is, therefore, unlikely that MT affects the transport of Cd into cells. On the other hand, MT has been shown to decrease the excretion of Cd (Liu et al., 1996aGo). It is thought that the binding of Cd to intracellular MT, forming CdMT complex, traps the Cd in cells and prevents it from being excreted. The absence of MT in MT I/II-null mice, therefore, facilitates the excretion of Cd from their liver cells, and explains why the MT I/II-null mice have lower Cd accumulation than wild-type mice.

Though there is much lower Cd concentration in the livers of MT I/II-null than wild-type mice, the liver injury was much more severe in the MT I/II-null mice. At the same duration of exposure, MT I/II-null mice demonstrated far more advanced pathologic lesions in their livers than wild-type mice exposed to the same dose of Cd. Lesions were observed in MT I/II-null mice at about one-tenth the dose that produced similar lesions in wild-type mice, and the maximum tolerable dose of 10 weeks of repeated Cd exposure was one-eighth in MT I/II-null mice of that in wild type mice. These data clearly demonstrate that deficiency in MT makes animals more vulnerable to liver injury from repeated exposure to Cd. We have reported similar findings in mouse kidneys (Liu et al., 1998bGo), spleen, thymus and hemopoietic system (Liu et al., 1999aGo), and bone (Habeebu et al., 2000Go).

The mechanism by which MT protects against acute Cd toxicity is presently thought to be due to the ability of MT to bind Cd and sequester the Cd in the cytosol, thus reducing Cd content in critical organelles (Goering and Klaassen, 1983Go; Liu et al., 1995Go). In the present study with repeated administration of Cd to mice, a similar phenomenon was observed. In wild-type animals, the majority of Cd was bound to MT in the hepatic cytosol. In contrast, no Cd was bound to MT in the cytosol of MT I/II-null mice (as MT is absent). It therefore appears that the mechanism by which MT protects cells against injury from repeated Cd exposure is the same as the mechanism by which MT protects cells against injury from acute Cd exposure.

In conclusion, the present study using MT I/II-null mice has demonstrated that (1) the major features of liver injury following 10 weeks of repeated Cd exposure include chronic inflammation, granulomas, proliferating nodules, apoptosis, and liver cell regeneration; (2) intracellular MT is an important protein protecting against this Cd-induced liver injury; and (3) Cd might have a carcinogenic potential in mouse liver following repeated Cd exposure.


    ACKNOWLEDGMENTS
 
This study was supported by NIH Grant ES-01142. S.S.H. was supported by NIH grant ES-07079 and the Kansas Health Foundation Scholarship. The authors thank Ms. Sandra Markham for her technical assistance.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (913) 588-7501. E-mail: cklaasse{at}kumc.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Biggart, N. W., and Murphy, E., Jr. (1988). Analysis of metal-induced mutations altering the expression or structure of a retroviral gene in a mammalian cell line. Mutat. Res. 198, 115–129.[ISI][Medline]

Brys, M., Nawrocka, A. D., Miekos, E., Zydek, C., Foksinski, M., Barecki, A., and Krajewska, W. M. (1997). Zinc and cadmium analysis in human prostate neoplasms. Biol. Trace Elem. Res. 59, 145–152.[ISI][Medline]

Cherian, M. G. (1995). Metallothionein and its interaction with metals. In Toxicology of Metals: Biochemical Aspects (R. A. Goyer and M. G. Cherian, Eds.), pp. 121–138. Springer-Verlag., New York.

Coogan, T. P., Bare, R. M., and Waalkes, M. P. (1992). Cadmium-induced DNA strand damage in cultured liver cells: Reduction in cadmium genotoxicity following zinc pretreatment. Toxicol. Appl. Pharmacol. 113, 227–233.[ISI][Medline]

Darewicz, G., Malczyk, E., and Darewicz, J. (1998). Investigations of urinary cadmium content in patients with urinary bladder carcinoma. Int. Urol. Nephrol. 30, 137–139.[Medline]

Dong, W., Simeonova, P. P., Gallucci, R., Matheson, J., Flood, L., Wang, S., Hubbs, A., and Luster, M. I. (1998). Toxic metals stimulate inflammatory cytokines in hepatocytes through oxidative stress mechanisms. Toxicol. Appl. Pharmacol. 151, 359–366.[ISI][Medline]

Dudley, R. E., Gammal, L. M., and Klaassen, C. D. (1985). Cadmium-induced hepatic and renal injury in chronically exposed rats: Likely role of hepatic cadmium-metallothionein in nephrotoxicity. Toxicol. Appl. Pharmacol. 77, 414–426.[ISI][Medline]

Dudley, R. E., Svoboda, D. J., and Klaassen, C. (1982). Acute exposure to cadmium causes severe liver injury in rats. Toxicol. Appl. Pharmacol. 65, 302–313.[ISI][Medline]

Dudley, R. E., Svoboda, D. J., and Klaassen, C. D. (1984). Time course of cadmium-induced ultrastructural changes in rat liver. Toxicol. Appl. Pharmacol. 76, 150–160.[ISI][Medline]

Eaton, D. L., and Toal, B. F. (1982). Evaluation of the Cd/hemoglobin affinity assay for the rapid determination of metallothionein in biological tissues. Toxicol. Appl. Pharmacol. 66, 134–142.[ISI][Medline]

Faeder, E. J., Chaney, S. Q., King, L. C., Hinners, T. A., Bruce, R., and Fowler, B. A. (1977). Biochemical and ultrastructural changes in livers of cadmium-treated rats. Toxicol. Appl. Pharmacol. 39, 473–487.[ISI][Medline]

Goering, P. L., and Klaassen, C. D. (1983). Altered subcellular distribution of cadmium following cadmium pretreatment: Possible mechanism of tolerance to cadmium-induced lethality. Toxicol. Appl. Pharmacol. 70, 195–203.[ISI][Medline]

Goering, P. L., and Klaassen, C. D. (1984). Tolerance to cadmium-induced hepatotoxicity following cadmium pretreatment. Toxicol. Appl. Pharmacol. 74, 308–313.[ISI][Medline]

Habeebu, S. S., Liu, J., and Klaassen, C. D. (1998). Cadmium-induced apoptosis in mouse liver. Toxicol. Appl. Pharmacol. 149, 203–209.[ISI][Medline]

Habeebu, S. S., Liu, J., Liu, V., and Klaassen, C. D. (2000). Metallothionein-null mice are more susceptible than wild-type mice to chronic CdCl2-induced bone injury. Toxicol. Sci. (in press).

Hartwig, A. (1994). Role of DNA repair inhibition in lead- and cadmium-induced genotoxicity: a review. Environ. Health. Perspect. 3, 45–50.

Hartwig, A. (1998). Carcinogenicity of metal compounds: possible role of DNA repair inhibition. Toxicol. Lett. 102103, 235–239.

Hoffmann, E. O., Cook, J. A., di, Luzio, N. R., and Coover, J. A. (1975). The effects of acute cadmium administration in the liver and kidney of the rat: Light and electron microscopic studies. Lab. Invest. 32, 655–664.[ISI][Medline]

IARC. (1993). International Agency for Research on Cancer Monographs on the Evaluation of the Carcinogenic Risks to Humans. Volume 58, Beryllium, Cadmium, Mercury, and Exposures in the Glass Industry, pp. 119–238. IARC Scientific Publications, Lyon.

Kagi, J. H. R. (1993). Evolution, structure, and chemical activity of class I metallothioneins: an overview. In Metallothionein III: Biological Roles and Medical Implications (K. T. Suzuki, N. Imura, and M. Kimura, Eds.), pp. 29–56. Birkhauser Verlag, Berlin.

Kamiyama, T., Miyakawa, H., Li, J. P., Akiba, T., Liu, J. H., Liu, J., Marumo, F., and Sato, C. (1995). Effects of one-year cadmium exposure on livers and kidneys and their relation to glutathione levels. Res. Commun. Mol. Pathol. Pharmacol. 88, 177–186.[ISI][Medline]

Kayama, F., Yoshida, T., Elwell, M. R., and Luster, M. I. (1995). Role of tumor necrosis factor-alpha in cadmium-induced hepatotoxicity. Toxicol. Appl. Pharmacol. 131, 224–234.[ISI][Medline]

Kondo, Y., Rusnak, J. M., Hoyt, D. G., Settineri, C. E., Pitt, B. R., and Lazo, J. S. (1997). Enhanced apoptosis in metallothionein null cells. Mol. Pharmacol. 52, 195–201.[Abstract/Free Full Text]

Liu, J., Liu, Y., Habeebu, S. S., and Klaassen, C. D. (1998a). Metallothionein (MT)-null mice are sensitive to cisplatin-induced hepatotoxicity. Toxicol. Appl. Pharmacol. 149, 24–31.[ISI][Medline]

Liu, J., Liu, Y., Habeebu, S. S., and Klaassen, C. D. (1998b). Susceptibility of MT-null mice to chronic CdCl2-induced nephrotoxicity indicates that renal injury is not mediated by the CdMT complex. Toxicol. Sci. 46, 197–203.[Abstract]

Liu, J., Liu, Y., Habeebu, S. S., and Klaassen, C. D. (1999a). Metallothionein-null mice are highly susceptible to the hematotoxic and immunotoxic effects of chronic CdCl2 exposure. Toxicol. Appl. Pharmacol. 159, 98–108.[ISI][Medline]

Liu, Y., Liu, J., Habeebu, S. S., and Klaassen, C. D. (1999b). Metallothionein protects against the nephrotoxicity produced by chronic CdMT exposure. Toxicol. Sci. 50, 221–227.[Abstract]

Liu, Y., Liu, J., Iszard, M. B., Andrews, G., Palmiter, R. D., and Klaassen, C. D. (1995). Transgenic mice that overexpress metallothionein-I are protected from cadmium lethality and hepatotoxicity. Toxicol. Appl. Pharmacol. 135, 222–228.[ISI][Medline]

Liu, J., Liu, Y., Michalska, A. E., Choo, K. H., and Klaassen, C. D. (1996a). Distribution and retention of cadmium in metallothionein I and II null mice. Toxicol. Appl. Pharmacol. 136, 260–268.[ISI][Medline]

Liu, J., Liu, Y., Michalska, A. E., Choo, K. H., and Klaassen, C. D. (1996b). Metallothionein plays less of a protective role in cadmium-metallothionein-induced nephrotoxicity than in cadmium chloride-induced hepatotoxicity. J. Pharmacol. Exp. Ther. 276, 1216–1223.[Abstract]

Masters, B. A., Kelly, E. J., Quaife, C. J., Brinster, R. L., and Palmiter, R. D. (1994). Targeted disruption of metallothionein I and II genes increases sensitivity to cadmium. Proc. Natl. Acad. Sci. U S A 91, 584–588.[Abstract]

Michalska, A. E., and Choo, K. H. (1993). Targeting and germ-line transmission of a null mutation at the metallothionein I and II loci in mouse. Proc. Natl. Acad. Sci. U S A 90, 8088–8092.[Abstract/Free Full Text]

Moch, R. W., Dua, P. N., and Hines, F. A. (1996). Problems in consideration of rodent hepatocarcinogenesis for regulatory purposes. Toxicol. Pathol. 24, 138–145.[ISI][Medline]

Petering, D. H., and Fowler, B. A. (1986). Uptake of galactose, ouabain, and taurocholate into centrilobular- and periportal-enriched hepatocyte subpopulations. Environ. Health Perspect. 65, 217–224.[ISI][Medline]

Popp, J. A., and Cattley, R. C. (1991). Hepatobiliary system. In Handbook of Toxicologic Pathology (W. M. Haschek and C. G. Rousseaux, Eds.), pp. 279–314. Academic Press, San Diego.

Waalkes, M. P., Anver, M. R., and Diwan, B. A. (1999). Chronic toxic and carcinogenic effects of oral cadmium in the Noble (NBL/Cr) rat: induction of neoplastic and proliferative lesions of the adrenal, kidney, prostate, and testes. J. Toxicol. Environ. Health 58, 199–214.[ISI]

Waalkes, M. P., and Diwan, B. A. (1999). Cadmium-induced inhibition of the growth and metastasis of human lung carcinoma xenografts: Role of apoptosis. Carcinogenesis 20, 65–70.[Abstract/Free Full Text]

Waalkes, M. P., and Rehm, S. (1994). Chronic toxic and carcinogenic effects of cadmium chloride in male DBA/2NCr and NFS/NCr mice: Strain-dependent association with tumors of the hematopoietic system, injection site, liver, and lung. Fundam. Appl. Toxicol. 23, 21–31.[ISI][Medline]

Waalkes, M. P., Diwan, B. A., Rehm, S., Ward, J. M., Moussa, M., Cherian, M. G., and Goyer, R. A. (1996). Down-regulation of metallothionein expression in human and murine hepatocellular tumors: association with the tumor-necrotizing and antineoplastic effects of cadmium in mice. J. Pharmacol. Exp. Ther. 277, 1026–1033.[Abstract]

Waalkes, M. P., Harvey, M. J., and Klaassen, C. D. (1984). Relative in vitro affinity of hepatic metallothionein for metals. Toxicol. Lett. 20, 33–39.[ISI][Medline]