Differential Hepatotoxicity Induced by Cadmium in Fischer 344 and Sprague-Dawley Rats

Robert K. Kuester*, Michael P. Waalkes{dagger}, Peter L. Goering{ddagger}, Ben L. Fisher{ddagger}, Robert S. McCuskey* and I. Glenn Sipes*,1

* Department of Pharmacology and Toxicology, Center for Toxicology, The University of Arizona, P.O. Box 210207, Tucson, Arizona 85721-0207; {dagger} National Cancer Institute at the National Institute of Environmental Health, Research Triangle Park, North Carolina 27709; {ddagger} Center for Devices and Radiological Health, Food and Drug Administration, Rockville, Maryland

Received July 9, 2001; accepted October 3, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A number of reports document that Fischer 344 (F344) rats are more susceptible to chemically induced liver injury than Sprague-Dawley (SD) rats. Cadmium (CdCl2), a hepatotoxicant that does not require bioactivation, was used to better define the biological events that are responsible for the differences in liver injury between F344 and SD rats. CdCl2 (3 mg/kg) produced hepatotoxicity in both rat strains, but the hepatic injury was 18-fold greater in F344 rats as assessed by plasma alanine aminotransferase (ALT) activity. This difference in toxicity was not observed when isolated hepatocytes were incubated with CdCl2 in vitro, indicating that other cell types contribute to Cd-induced hepatotoxicity in vivo. Indeed, the sieve plates of hepatic endothelial cells (EC) in F344 rats were damaged to a greater degree than EC in SD rats. Additionally, Kupffer cell (KC) inhibition reduced hepatotoxicity in both strains, suggesting that this cell type is involved in the progression of CdCl2-induced hepatotoxicity. Moreover, enhanced synthesis of heat shock protein 72 occurred earlier in the SD rat. Maximal levels of hepatic metallothionein (MT), a protein associated with cadmium tolerance, were greater in SD rats. These protective factors may limit CdCl2-induced hepatocellular injury in SD compared with F344 rats by reducing KC activation and the subsequent inflammatory response that allows for the progression of hepatic injury.

Key Words: cadmium; liver; Sprague-Dawley; Fischer344; heat shock protein 72; metallothionein; Kupffer cell; endothelial cell.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Metals and metalloids affect almost every organ of the body, including the liver. One such metal is cadmium (Cd), which is of concern because of its increasing prevalence as an environmental contaminant (Jarup et al., 1998Go). Prolonged exposure to Cd results in injury to liver, lung, kidney, and testes (Manca et al., 1991Go). A large bolus dose of Cd causes injury to a number of tissues, including the liver (Dudley et al., 1982Go). Liver injury is manifested by extensive necrosis accompanied by large increases in the plasma levels of alanine aminotransferase (ALT) and other enzymes. (Dudley et al., 1982Go; Skilleter et al., 1985Go). The ultrastructural changes to the hepatocyte following an acute dose of Cd include the formation of intracellular vesicles and destruction of the endoplasmic reticulum (Dudley et al., 1984Go).

Kupffer cells (KC) are known to contribute to the hepatotoxicity of a variety of chemicals, including Cd. When KC are selectively destroyed or inhibited, the hepatotoxicity of Cd is dramatically reduced (Sauer et al., 1997Go, Yamano et al., 1998Go). KC are known to release a variety of cytotoxic mediators that can directly damage hepatocytes. These include reactive oxygen species, nitric oxide, and cytotoxic proteins. In addition, KC can release chemokines that promote the infiltration of other inflammatory cells to the liver (Laskin et al., 1986Go). These, in turn, release a variety of cytotoxic agents that promote the progression of liver injury. For example, infiltrating neutrophils can release large amounts of reactive oxygen species, which can result in oxidative stress and lipid peroxidation (Manca et al., 1991Go). The release of tumor necrosis factor {alpha} (TNF-{alpha}) and interleukin-8 has been associated with Cd-induced liver injury in mice (Kayama et al., 1995Go; Nolan and Shaikh, 1986Go). Collectively, these data suggest that the hepatic injury from acute Cd exposure occurs as a result of cytotoxic mediators released by KC and other inflammatory cells (Rikans and Yamano, 2000Go).

Although Cd may injure hepatocytes directly, there is a growing body of evidence that ischemia may also contribute to Cd-induced liver injury (Liu et al., 1992Go). This ischemia may result from direct effects of Cd on hepatic EC. The extrusion of these damaged cells into the sinusoidal space alters hepatic microcirculation. The resulting hypoxia can further exacerbate hepatic injury (Rikans and Yamano, 2000Go).

Cd can upregulate the expression of a number of genes that produce products that can detoxify Cd and/or repair Cd induced lesions. For example, a critical determining factor in Cd-induced liver injury is the hepatic concentration of metallothionein (MT). The synthesis of this low molecular weight metal-binding protein is induced by Cd exposure. MT forms a complex with Cd and reduces its free concentration within the cell, thus reducing the hepatotoxic potential of Cd. As the binding capacity of MT becomes saturated, the increased availability of unbound Cd initiates a series of events that result in cell injury or death (Chan and Cherian, 1992Go; Coyle et al., 2000Go; Goering and Klaassen, 1984Go). Severe hepatic injury results from activation of the cellular immune system with subsequent infiltration of inflammatory cells (Dudley et al., 1982Go). The induction of MT by Cd appears to be mediated in part by TNF-{alpha}. (Grimble and Bremner, 1989Go) Furthermore, Cd has been shown to activate genes that encode for a group of proteins referred to as stress or heat shock proteins (hsps). Stress proteins are synthesized in response to a wide variety of physical and chemical insults, including inorganic chemicals such as Cd, arsenic, zinc, lead, and copper (Nover, 1991Go; Schlesinger et al., 1982Go). Their upregulation following certain external stimuli results in resistance to subsequent cellular injury (Nover, 1991Go). The synthesis of hsp72 has been reported to precede hepatic necrosis induced by Cd (Goering et al., 1993Go). The timing and extent of synthesis of hsps following exposure to Cd may influence the extent of Cd-induced liver injury.

Male Fischer 344 (F344) rats are generally more sensitive to chemically induced liver injury than male Sprague-Dawley (SD) rats. To date, these strain comparisons have been made using organic chemicals that require bioactivation to elicit toxicity (e.g., carbon tetrachloride, 1,2-dichlorobenzene, and acetaminophen). Exposure to these agents results in hepatocellular necrosis that is clearly more severe in the F344 rat compared with the SD rat (Newton et al., 1983Go; Steup et al., 1991Go; Stine et al., 1991Go). To date, comparison of the sensitivity of the male F344 and male SD rats to inorganic xenobiotics has not been reported. Therefore, the object of these experiments was to determine the relative sensitivities of male F344 and male SD rats to Cd-induced hepatotoxicity and the expression of molecules known to reduce Cd toxicity, e.g., MT and hsps.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Adult male F344 and SD rats (9 weeks of age, Harlan Lab Animals, Inc., Indianapolis, IN) were acclimated for 1 week before each experiment in an AAALAC-approved animal facility and maintained on a 12-h light and dark cycle. Animals were housed in hanging metal wire cages and given free access to food (Teklad 4% mouse/rat diet, Harlan Teklad, Madison, WI) and water throughout the experiment. Rats were identified by a uniquely numbered metal ear tag.

Treatments.
Cadmium, as CdCl2, was diluted in saline and administered iv via the tail vein (1 ml/kg). In dose-response studies, CdCl2 (1.5, 3.0, and 4.5 mg/kg) was administered to SD and F344 rats and livers were removed at 24 h post dose. In time-course studies, livers were removed at 1.5–48 h after CdCl2 (3 mg/kg). To determine how KC may affect Cd hepatotoxicity, male F344 and SD rats were pretreated with 10 mg/kg gadolinium chloride (GdCl3, Aldrich Chemical Co., Milwaukee, WI), an inhibitor of KC activity (Husztik et al., 1980Go). GdCl3 was dissolved in acidified saline and administered by tail vein injection. Twenty-four hours after GdCl3 treatment, animals received a single injection of CdCl2 (3 mg/kg, iv) via the tail vein.

Sample preparation.
At the appropriate time after CdCl2 administration, the rats were euthanized by inhalation of CO2. Blood was collected from the inferior vena cava into glass test tubes containing heparin for measurement of alanine aminotransferase activity (Sigma diagnostic kit procedure No. 59-UV). After collection of blood, a portion of the central lobe of the liver was resected, trimmed, and placed in 10% buffered formalin. Tissues were imbedded in paraffin, and sections (10 µm) were cut, transferred to slides, and stained with hematoxylin and eosin (H&E) for light microscopic examination. An adjacent section of liver was removed and placed in 0.1M cocadylate buffer containing 3% glutaraldehyde for electron microscopy. After fixation, sections were dehydrated in a series of ethanol rinses, cleared with propylene oxide, and embedded in epon. These sections were then evaluated using a Phillips CM-12 transmission electron microscope. Scanning electron microscopy was performed on a Phillips XL30 after the specimens were critical point dried, fractured, and gold coated. Remaining hepatic tissues were snap-frozen in liquid nitrogen and stored at –70°C until analyzed for Cd, MT, and hsp72.

Determination of Cd and MT in liver.
At 3, 6, 12, 24, and 36 h after administration of CdCl2 (3 mg/kg) hepatic Cd and MT were determined by atomic absorption spectroscopy. For Cd analyses, liver tissue (200 mg) was digested overnight in concentrated nitric acid. The solutions were then filtered with a 0.45-µm syringe tip filter and transferred to a flask containing 5 ml distilled deionized (DD) H20. The Cd content of the samples was determined on a Perkin Elmer (model AAnalyst 100) atomic spectrophotometer at 228.8 nm with a Cd lamp. Standards of Cd obtained from Fisher Scientific (Rockville, MD) were prepared in the concentration range of 0–25 µg/l. Concentrations of MT were determined simultaneously in the remaining tissue using the atomic absorption spectrophotometric Cd-hemoglobin assay. This assay detects bound Cd and assumes that 7 atoms of Cd are bound to each molecule of MT, as described by Eaton and Toal (1982). The rate of MT accumulation was determined using area under curve calculations.

Determination of hsp72 in the livers.
Immunological detection of hsp72 was performed as described by Goering et al. (1992). Briefly, equivalent amounts of protein (10 µg) were diluted with 10 mM Tris-HCl, then applied to polyacrylamide gels (12.5% homogenous polyacrylamide). Prestained molecular weight standards were run concurrently on each gel. After electrophoretic separation, proteins were transferred to nitrocellulose membranes and blocked in Tris blocking solution containing 4% nonfat dry milk. The blots were washed two times in DD H2O for 5 min and incubated for 12–15 h with a mouse IgG monoclonal antibody (StressGen, Victoria, BC, Canada) specific for hsp72. Following incubation with the primary antibody, the immunoblots were washed and incubated with goat antimouse IgG conjugated with alkaline phosphatase (Bio-Rad Laboratories, Richmond, CA). After a washing period with deionized H2O, bands were visualized by incubation in a solution of nitroblue tetrazolium/5-bromo-4chloro-3-indolyl, a substrate for alkaline phosphates (Bio-Rad Labs, Richmond, CA).

Cytotoxicity of CdCl2 in primary cultured hepatocytes.
Hepatocytes were isolated from F344 or SD rats using the two-step in situ liver perfusion with collagenase B (Boehringer Mannheim, Mannheim, Germany) as described by McQueen (1989), and plated in Falcon six-well plates (Falcon, Franklin Lakes, New Jersey) at 0.5 x 106 cells/well. Cells were allowed to adhere for 2 h in the presence of 10% bovine calf serum (Hyclone, Logan, Utah), after which the media was replaced with Williams media E (WME; Life Technologies, Grand Island, New York) without bovine calf serum. CdCl2 dissolved in sterile deionized H2O, was added to the monolayers for a final concentration of 0–3 µM. After 3, 6, 12, or 18 h, the medium was removed, and cells were lysed with 0.5% triton x 100/phosphate buffer (pH 7.4) (Sigma Chemical Company). Cytotoxicity was assessed by the leakage of lactate dehydrogenase (LDH) activity into the media using Sigma Number (Procedure No. 228-UV). LDH activity was expressed as percentage of total activity released (LDH in media ÷ total LDH (media + cellular) x 100).

Statistical analysis.
The data are expressed as mean ± SEM. To compare F344 and SD rats, the data were analyzed using two-way analysis of variance followed by Bonferroni's post hoc test. Differences were considered significant if p < 0.05. All statistical analyses were performed using SigmaStat Statistical Software for Windows, Version 1.0 (Jandel Scientific, San Rafael, CA). Calculations of hepatic levels of MT assumed 7 atoms of Cd per molecule of MT and are expressed as rate of accumulation over 48 h test period (using area under curve calculations).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of CdCl2 Treatment on Plasma ALT Activity
Following the administration of CdCl2 (3 mg/kg, iv), plasma ALT activity was markedly higher in male F344 rats than in male SD rats (Fig. 1Go). Elevations in ALT activity were not apparent in the plasma of either strain of rat until 3 h after Cd treatment, when they were 876 ± 314 U/l and 287 ± 44 U/l in F344 and SD rats, respectively. Plasma ALT activity progressively increased from 3 to 24 h in the F344 rats and reached maximal activity at 24 h. At 24 h, plasma ALT activities were 3612 ± 750 and 199 ± 26 U/l in the F344 and SD rats, respectively (Fig. 1Go). Administration of 1.5 mg/kg CdCl2 (iv) did not increase plasma ALT activity in either rat strain at 24 h. At the highest dose tested (4.5 mg/kg, iv) plasma ALT activities were elevated (16 x control) in SD rats, indicative of mild liver injury. This high dose was lethal to all F344 rats (Table 1Go). When KC activity was inhibited in the livers by pretreatment with gadolinium chloride, Cd-induced liver injury (as assessed by plasma ALT activity) was reduced dramatically in both rat strains (Fig. 2Go). Plasma ALT activities were only 56 U/l in F344 rats and 86 U/l in SD rats at 24 h following 3 mg/kg CdCl2.



View larger version (15K):
[in this window]
[in a new window]
 
FIG. 1. Time course of plasma ALT activities in male F344 and male SD rats treated with CdCl2. Rats (n = 4) were administered either CdCl2 (3.0 mg/kg iv) or saline (time 0). Data are expressed as mean ± SEM. (Small error bars are obscured by the symbols.) *Significantly different from SD rats (p < 0.05).

 

View this table:
[in this window]
[in a new window]
 
TABLE 1 Effect of Cd on Plasma Alanine Aminotransferase Activities and Mortality in Male F344 Rats and Male SD Rats 24 Hours after Treatment with CdCl2
 


View larger version (20K):
[in this window]
[in a new window]
 
FIG. 2. Effect of gadolinium chloride (GdCl3) pretreatment on Cd-induced elevations in ALT activity. F344 and SD rats were pretreated with GdCl3 (10 mg/kg iv in saline) 24 h prior to Cd (3 mg/kg iv) or saline. Rats were killed 24 h post CdCl2 treatment and plasma was analyzed for ALT activity. Data are expressed as mean ± SEM; n = 4.

 
Morphological Comparisons
Photomicrographs.
Cd treatment (3.0 mg/kg, iv) substantially altered histopathology in F344 and SD rats over the 24-h time course (Fig. 3Go). At the light microscope level, slight vacuolar degeneration was observed in the livers of both rat strains at 6 h (data not shown). There were marked differences in the progression of liver injury after this point. By 12 h, male F344 rats had moderate centrilobular degeneration, whereas the injury to SD rats was limited to mild multifocal necrosis (Fig. 3CGo). At 24 h, severe injury was apparent in the livers of F344 rats (Fig. 3FGo). Hemorrhagic lesions and coagulative necrosis were widespread throughout the hepatic lobule. Necrosis was substantially less severe and hemorrhage was not apparent in the livers of SD rats at 24 h (Fig. 3EGo).



View larger version (158K):
[in this window]
[in a new window]
 
FIG. 3. CdCl2-induced morphological changes in the livers of SD and F344 rats: At 0, 12, and 24 h after administration of CdCl2 (3 mg/kg iv), rats were killed and liver sections were fixed by immersion, as described in Materials and Methods section. SD rats: (A) control; (C) 12 h after treatment; (E) 24 h after treatment. F344 rats: (B) control; (D) 12 h after treatment; (F) 24 h after treatment. PP, periportal region of rat liver treated with CdCl2 or saline (original magnification x 200). Note the severe, diffuse hepatocellular degeneration progressing into necrosis (arrows), as well as the presence of inflammatory cells.

 
Transmission electron micrographs.
To determine if differences in susceptibility could be observed in individual hepatocytes following Cd treatment, liver sections were prepared for electron microscopy. Representative electron photomicrographs from three animals depicting the ultrastructural changes are shown in (Fig. 4Go). At 3 h after Cd (3 mg/kg, iv) dilated endoplasmic reticulum and translucent vesicles were observed in hepatocytes of both strains of rats (Fig. 4Go). Interestingly, these alterations were still present in the livers of the F344 rats 24 h after CdCl2 administration. In the livers of SD rats, these vesicles had resolved by 24 h (Fig. 4Go).



View larger version (135K):
[in this window]
[in a new window]
 
FIG. 4. Representative transmission electron micrographs of livers from rats treated with CdCl2 (3 mg/kg iv) for various times (0, 3, or 24 h). Arrows indicate vesicles.

 
Scanning electron micrographs of endothelial cells.
Structural changes to the hepatic endothelial cells of male F344 and male SD rats after Cd treatment were investigated via scanning electron microscopy. By 3 h after CdCl2 administration (3 mg/kg), damage to EC of the sinusoidal walls of the livers was evident (Fig. 5Go). Most apparent was the loss of the individual fenestration in the EC that comprised the sinusoids. These were replaced by large gaps. This damage was more severe in livers of F344 rats.



View larger version (91K):
[in this window]
[in a new window]
 
FIG. 5. Representative scanning electron micrographs of hepatic endothelial cells 3 h after CdCl2 (3 mg/kg iv). Arrows indicate destruction of the sieve plate fenestrations.

 
Hepatic Cd and MT Concentrations in F344 and SD Rats
Livers were analyzed for Cd and MT content following administration of CdCl2. The concentration of Cd was not significantly different at any time point in the livers of SD or F344 rats (Table 2Go). At time 0 (control rats), the hepatic concentration of MT was similar in both strains of rats. After exposure to CdCl2, MT appeared to accumulate faster in the livers of SD rats as compared to those of F344 rats, although not significantly so during early time points (< 24 h). However, maximal levels of hepatic MT in the SD rat (at 24 h) were clearly higher than in the F344 rats (Fig. 6Go). When the hepatic levels of MT were expressed as rate of accumulation over 48-h test period (using area under curve calculations), the values were 4007 ± 358 ng/g/h in the SD and 2661 ± 293 ng/g/h in the F344, which are significantly different.


View this table:
[in this window]
[in a new window]
 
TABLE 2 Concentration of Cd in the Livers of F344 and SD Rats at Various Times following Intravenous Administration of 3 mg/kg CdCl2
 


View larger version (25K):
[in this window]
[in a new window]
 
FIG. 6. Metallothionein concentration in livers of F344 and SD rats at various times after administration of CdCl2. Three to 24 h after administration of CdCl2 (3 mg/kg), livers were removed, snap-frozen in liquid nitrogen, and stored at –70°C until levels of metallothionein were determined by atomic absorption spectroscopy. Data are expressed as mean ± SEM of four animals. *Significantly different from SD rats (p < 0.05).

 
Induction of Hepatic hsp72
The de novo synthesis of hepatic hsp72 was compared in both strains of rats after administration of CdCl2 (3 mg/kg, iv). By 3 h, detectable amounts of inducible hsp72 were present in the livers of SD rats. At this time, hsp72 could not be detected in the livers of F344 rats. At 6 h after CdCl2 administration, hsp72 could be detected in the livers of both strains of rats (Fig. 7Go).



View larger version (58K):
[in this window]
[in a new window]
 
FIG. 7. Comparison of hsp72 induction in livers of F344 and SD rat treated with CdCl2. Tissue sections were subjected to SDS-PAGE, blotted to nitrocellulose membrane, and probed with a mouse antihuman IgG antibody specific for hsp72. Time reflects hours after CdCl2 (3 mg/kg iv). Blot is representative of 3 experiments.

 
Cytotoxicity of Cd to Monolayers of Hepatocytes
Incubation of CdCl2 with primary cultures of hepatocytes obtained from untreated F344 and SD rats resulted in cytotoxicity, as assessed by lactate dehydrogenase released into the incubation media. Interestingly, no differences in Cd-induced LDH release was observed between hepatocytes obtained from either strain of rats. The LC50 of CdCl2 was 0.94 µM in F344 rat hepatocytes and 0.89 µM in SD rat (Fig. 8Go). Similarly, when the cytotoxicity of CdCl2 was compared in hepatocytes through time (0–18 h), differences were not observed (Fig. 9Go).



View larger version (14K):
[in this window]
[in a new window]
 
FIG. 8. Dose response of Cd-induced cytotoxicity in monolayers of hepatocytes. Isolated hepatocytes from naïve F344 and SD rats were exposed to Cd at the indicated concentrations (0–3 µM) for 18 h. Data are expressed as percentage of lactate dehydrogenase released into the media, mean ± SEM in triplicate; n = 3.

 


View larger version (18K):
[in this window]
[in a new window]
 
FIG. 9. Time course of Cd-induced cytotoxicity in monolayers of hepatocytes. Isolated hepatocytes from naïve F344 and SD rats were incubated with Cd (0, 1, and 3 µM) for the indicated times. Data are expressed as percentage of lactate dehydrogenase released into the media, mean ± SEM in triplicate; n = 3.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The results of the present study demonstrate dramatic differences in the development of Cd-induced liver injury between male F344 and SD rats. These findings are supported by histological findings and increased ALT activity. In the F344 rat, Cd caused diffuse hepatocellular degeneration by 24 h. In the SD rat, only a moderate degree of injury was present at that time. In both time and dose-response studies, plasma ALT activities in F344 rats treated with Cd were greater in the SD rat. Intracellular effects on the hepatocytes of F344 and SD rats treated with Cd included the formation of clear vacuoles in the endoplasmic reticulum. Dudley et al. (1984) also reported these vacuoles.

Interestingly, when hepatocytes obtained from the livers of F344 or SD rats were incubated with various concentrations of CdCl2, no apparent differences were observed in Cd-induced cytotoxicity. After 18 h of incubation, the LC50 for Cd was similar for F344 and SD rat hepatocytes. Cytotoxicity curves were essentially superimposed throughout the 18-h incubations. One explanation for this lack of correlation between Cd-induced liver injury and its in vitro cytotoxicity is that other cell types play a critical role in Cd-induced liver injury. For example, both hepatic sinusoidal EC and KC have been implicated in Cd-induced liver injury (Rikans and Yamano, 2000Go).

Indeed, damage to hepatic EC of F344 and SD rats was observed within 3 h after Cd (3 mg/kg CdCl2) administration. For F344 rats, there was extensive destruction of fenestrations on the lumenal surface of EC. In contrast, damage to the EC of SD rat livers was much less extensive. Rikans and Yamano (2000) suggested that EC might be the first cellular targets for Cd-induced hepatocellular injury. Once damaged, the injured EC obstruct the capillary lumen and produce local ischemia. This local ischemia may then initiate a number of molecular and cellular events, which results in hepatocellular injury. Differences in sensitivity of the livers of F344 and SD rats to Cd may relate to the initial damage to the EC, which in turn could predispose the F344 rat to subsequent liver injury. Liu et al (1992) reported large differences in Cd-induced liver injury between C3H/HeJ (Cd-sensitive) and DBAj2 (Cd-resistant) mice. This in vivo difference correlated well with the sensitivity of EC incubated in vitro with Cd. EC of C3H/HeJ mice were more sensitive to Cd than those of DBA/2J mice. No differences in sensitivity of hepatocytes from these two mouse strains were observed when they were incubated in vitro with Cd.

In addition to EC, KC are known to play a role in the progression of liver injury caused by a variety of chemicals including Cd (Decker, 1990Go; Laskin et al., 1986Go; McKim et al., 1992Go; Sauer et al., 1997Go; Sipes et al., 1989Go). When KC were inhibited by pretreatment of rats with GdCl3, Cd-induced liver injury was dramatically reduced. This inhibition was apparent in both strains of rats, but more dramatic in F344 rats. Therefore, activation of KC plays a critical role in the development of Cd-induced hepatocellular injury. Whether Cd activates KC directly or indirectly is not known. Incubation of a murine macrophage cell line (RAW 264.7) with soluble or insoluble Cd results in activation of these cells (Goering et al., 2000Go), suggesting that Cd can activate KC directly. KC can also be activated indirectly. For example, hepatocytes treated in vitro with ethanol release factors that stimulate production of interleukin-8 (CINC) by KC (Maher, 1995Go). Furthermore, Cd has been reported to stimulate the production of cytokines from isolated hepatocytes (Dong et al., 1998Go). These cytokines can, in turn, upregulate the activity of KC and promote their involvement in Cd-induced liver injury. Understanding direct and/or indirect mechanisms by which KC are activated during Cd intoxication may reveal the critical events that explain the differences in Cd-induced hepatotoxicity between these two rat strains.

Others have reported that differences in the susceptibility of rat strains to CD-induced injury relates to constitutive levels and/or induction of MT (Klaassen et al., 1999Go; Theocharis et al., 1994Go). For example, the male Wistar rat is more sensitive to Cd-mediated hepatocellular necrosis than Quinster or Lewis rats. When levels of hepatic MT were compared following treatment with Cd, it was shown that low MT levels contributed to the greater hepatic injury in the Wistar rat (Theocharis et al., 1994Go). The present study indicates that, generally speaking, SD rats treated with Cd produced a higher maximal level of MT at an apparently faster rate than in the F344 rat. However, at many time points after Cd exposure, MT levels were not significantly different. The enhanced induction of MT may have played some role in reduced sensitivity in SD rats, although this is clearly not the only factor. In this regard, MT is generally thought to be produced in hepatocytes (Din and Frazier, 1985Go, McKim et al. 1992Go) and it is clear from the present study that EC and KC are important for Cd injury. Therefore, although some differences in production of MT in response to Cd in SD and F344 rats could play a role in strain-related sensitivity, this does not appear to be the main factor.

Our data demonstrated that pharmacokinetic differences in the uptake of Cd by the liver do not account for the differences in susceptibility to Cd-induced liver injury between SD and F344 rats. The hepatic accumulation of Cd was similar in both strains. The possibility exists that the cellular distribution of Cd in the liver could be different on a per-cell basis. EC are known to accumulate more Cd than hepatocytes and KC (Caperna and Failla, 1984Go; McKim et al., 1992Go). Therefore, EC of F344 rats may accumulate more Cd than EC of SD rats. Determining total hepatic Cd concentrations may mask these differences, and uptake of Cd by different cell types in subsequent experiments could address this issue.

Although the detoxification of Cd is due primarily to enhanced MT synthesis, other proteins known as heat shock or stress proteins may contribute to cytoprotection (Goering et al., 1993Go). Inducible hsps have been associated with cytoprotection against a variety of toxic insults (Nover, 1991Go; Welch, 1987Go, 1992Go). Hsps confer cytoprotection through protein–protein interactions that preserve the conformational integrity of proteins. Such interactions can stabilize damaged proteins and promote repair and refolding (Welch, 1987Go, 1992Go). Therefore, the earlier synthesis of hsps might contribute to the ability of the liver to repair early stages of Cd-dependent injury. Induction of hsp72 protein was observed in the livers of F344 and SD rats. However, this induction was apparent 3 h earlier in the livers of SD rats. Therefore, the SD rat responds to Cd with an earlier induction of known hepatoprotective factors such as MT and hsps. These factors may minimize the degree of injury and mitigate its progression. Indeed, at the ultrastructural level, hepatocytes of F344 and SD rats treated with Cd had evidence of injury as early as 3 h after administration of CdCl2. Hepatocytes of both F344 and SD rats displayed abnormal vesicles at this time point. These vesicles were not detected 24 h after administration of Cd to the SD rat but were still present in the F344 rats. The SD rat apparently had mechanisms that repair these early lesions and/or prevented further progression of hepatocellular injury.

Collectively, the delayed expression of hsp72, and possibly MT, may contribute to the increased sensitivity of the F344 to Cd-induced liver injury. The livers of F344 rat appear to respond less efficiently to the induction of these protective responses. Because hepatoprotective measures are expressed earlier in livers of SD rats, the initial injury is minimized and the subsequent inflammatory cascade is blunted.

The initiation and progression of Cd-induced hepatocellular injury involves multiple cell types. EC are the first liver cells that come into contact with free Cd and are clearly involved in the early stages of Cd-induced liver injury. In the current study, the endothelial cells of the F344 were damaged more extensively than the EC of the SD rats. Once damaged, these cells obstruct the hepatic microcirculation, which leads to localized ischemia. This occlusion of hepatic inflow could lead to hypoxic conditions that promote hepatocellular necrosis. The liver responds to these conditions by synthesizing repair proteins. In fact, hsp72 has been shown to be produced early after total hepatic inflow occlusion (Dai et al., 1998Go). The earlier synthesis of MT and hsp72 may contribute to the decreased sensitivity of the SD rat to Cd-induced hepatotoxicity, but at least with regard to MT, this does not appear to be a primary factor. The hepatocytes and EC respond to hepatocellular injury by releasing chemokines and cytokines that result in inflammation orchestrated in part by KC.


    ACKNOWLEDGMENTS
 
The Southwest Environmental Health Science Center (ES-06694) and the Coca-Cola Foundation supported this research.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (520) 626-2466. E-mail: sipes{at}pharmacy.arizona.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Caperna, T. J., and Failla, M. L. (1984). Cadmium metabolism by rat liver endothelial and Kupffer cells. Biochem. J. 221, 631–636.[ISI][Medline]

Chan, M. H., and Cherian, M. G. (1992). Protective roles of metallothionein and glutathione in hepatotoxicity of cadmium. Toxicology 72, 281–290.[ISI][Medline]

Coyle, P., Niezing, G., Shelton, T. L., Philcox, J. C., and Rofe, A. M. (2000). Tolerance to cadmium hepatotoxicity by metallothionein and zinc: In vivo and in vitro studies with MT-null mice. Toxicology 150, 53–67.[ISI][Medline]

Dai, C. L., Kume, M., Yamamoto, Y., Yamagami, K., Yamamoto, H., Nakayama, H., Ozaki N., Shapiro, A. M., Yamamoto, M., and Yamaoka, Y. (1998). Heat shock protein 72 production in liver tissue after experimental total hepatic inflow occlusion. Br. J. Surg. 85, 1061–1065.[ISI][Medline]

Decker, K. (1990). Biologically active products of stimulated liver macrophages (Kupffer cells). Eur. J. Biochem. 192, 245–261.[ISI][Medline]

Din, W. S., and Frazier, J. M. (1985). Protective effects of metallothionein on cadmium toxicity in isolated rat hepatocytes. Biochem. J. 230, 395–401.[ISI][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, 259–266.

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 Cd/hemoglobin affinity assay for the rapid determination of metallothionein in biological tissues. Toxicol. Appl. Pharmacol. 66, 134–142.[ISI][Medline]

Goering, P. L., Fisher, B. R., Chaudhary, P. P., and Dick, C. A. (1992). Relationship between stress protein induction in rat kidney by mercuric chloride and nephrotoxicity. Toxicol. Appl. Pharmacol. 113, 184–191.[ISI][Medline]

Goering, P. L., Fisher, B. R., and Kish, C. L. (1993). Stress protein synthesis induced in rat liver by cadmium precedes hepatotoxicity. Toxicol. Appl. Pharmacol. 122, 139–148.[ISI][Medline]

Goering, P. L., and Klaassen, C. D. (1984). Zinc-induced tolerance to cadmium hepatotoxicity. Toxicol. Appl. Pharmacol. 74, 299–307.[ISI][Medline]

Goering, P. L., Kuester, R. K., Neale, A. R., Chapekar, M. S., Zaremba, T. G., and Hitchins, V. M. (2000). Effect of particulate and soluble cadmium species on biochemical and functional parameters in cultured murine macrophages. In Vitro Mol. Toxicol. 13, 125–136.[ISI][Medline]

Grimble, R. F., and Bremner, I. (1989). Tumor necrosis factor enhances hepatic metallothionein-1 content but reduces that of the kidney. Proc. Nutr. Soc. 48, 64A.

Husztik, E., Lazar G., and Parducz, A. (1980). Electron microscopic study of Kupffer cell phagocytosis blockade induced by gadolinium chloride. Br. J. Exp. Pathol. 61, 624–630.[ISI][Medline]

Jarup, L., Berglund, M., Elinder, C. G., Nordberg, G., and Vahter, M. (1998). Health effects of cadmium exposure: a review of the literature and a risk estimate. Scand. J. Work Environ. Health 24(Suppl.), 1–51.[ISI]

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]

Klaassen, C. D., Liu, J., and Choudhuri, S. (1999). Metallothionein: An intracellular protein to protect against cadmium toxicity. Annu. Rev. Pharmacol. Toxicol. 39, 267–294.[ISI][Medline]

Laskin, D. L., Pilaro, A. M., and Ji, S. (1986). Potential role of activated macrophages in acetaminophen hepatotoxicity. II. Mechanism of macrophage accumulation and activation. Toxicol. Appl. Pharmacol. 86, 216–226.[ISI][Medline]

Liu, J., Kershaw, W. C., Liu Y. P., and Klaassen, C. D. (1992). Cadmium-induced hepatic endothelial cell injury in inbred strains of mice. Toxicology 75, 51–62.[ISI][Medline]

Manca, D., Ricard, A. C., Trottier, B., and Chevalier, G. (1991). Studies on lipid peroxidation in rat tissues following administration of low and moderate doses of cadmium chloride. Toxicology 67, 303–323.[ISI][Medline]

Maher, J. J. (1995). Rat hepatocytes and Kupffer cells interact to produce interleukin-8 (CINC) in the setting of ethanol. Am. J. Physiol. 269, G518–G523.[Abstract/Free Full Text]

McKim, J. M., Jr., Liu, J., Liu, Y. P., and Klaassen, C. D. (1992). Distribution of cadmium chloride and cadmium-metallothionein to liver parenchymal, Kupffer, and endothelial cells; their relative ability to express metallothionein. Toxicol. Appl. Pharmacol. 112, 324–330.[ISI][Medline]

McQueen, C. A. (1989). Hepatocytes in monolayer culture: An in vitro model for toxicity studies. In In Vitro Model Systems and Methods, pp. 131–155. Telford Press, Caldwell, N.J.

Newton, J. F., Yoshimoto, M., Bernstein, J., Rush, G. F., and Hook, J. B. (1983). Acetaminophen nephrotoxicity in the rat. I. Strain differences in nephrotoxicity and metabolism. Toxicol. Appl. Pharmacol. 69, 291–306.[ISI][Medline]

Nolan, C. V., and Shaikh, Z. A. (1986). The vascular endothelium as a target tissue in acute cadmium toxicity. Life Sci. 39, 1403–1409.[ISI][Medline]

Nover, L. (1991). Heat Shock Response. CRC Press, Boca Raton, FL.

Rikans, L. E., and Yamano, T. (2000). Mechanisms of cadmium-mediated acute hepatotoxicity. J. Biochem. Mol. Toxicol. 14, 110–117.[ISI][Medline]

Sauer, J. M., Waalkes, M. P., Hooser, M. P., Kuester, R. K., McQueen, C. A., and Sipes, I. G. (1997). Suppression of Kupffer cell function prevents cadmium-induced hepatocellular necrosis in the male Sprague-Dawley rat. Toxicology 121, 155–164.[ISI][Medline]

Schlesinger, M. J., Ashburner, M., and Tissieres, A. (Eds.). (1982). Heat Shock Proteins: From Bacteria to Man. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Sipes, I. G., Elsisi, A. E., Sim, W., Mobley, S., and Earnest, D. (1989). Role of reactive oxygen species secreted by activated Kupffer cells in the potentiation of carbon tetrachloride hepatotoxicity by hypervitaminosis A. In Cells of the Hepatic Sinusoid (E. Wisse, D. Knook, and K. Decker, Eds.). pp. 376–379. Kupffer Cell Foundation, Rijswijk, The Netherlands.

Skilleter, D., Cain, K., Dinsdale, D., and Paine, A. (1985). Biochemical mechanisms and morphology selectivity in hepatotoxicity: Studies in cultures of hepatic-parenchymal and non-parenchymal cells. Xenobiotica 15, 687–693.[ISI][Medline]

Steup, D., Wiersma, D., McMillan, D., and Sipes, I. G. (1991). Pretreatment with drinking water solutions containing trichloroethylene of chloroform enhances the hepatotoxicity of carbon tetrachloride in Fischer 344 rats. Fundam. Appl. Toxicol. 16, 798–809.[ISI][Medline]

Stine, E. R., Gunawardhana, L., and Sipes, I. G., (1991). The acute hepatotoxicity of isomers of dichlorobenzene in Fischer 344 and Sprague-Dawley rats: Isomer-specific and strain-specific differential toxicity. Toxicol. Appl. Pharmacol. 109, 472–481.[ISI][Medline]

Theocharis, S. E., Margeli, A. P., Giannakou, N., Drakopoulos, D. S., and Mykoniatis, M. G. (1994). Cadmium-induced hepatotoxicity in three different rat strains. Toxicol. Lett. 70, 39–48.[ISI][Medline]

Welch, W. J. (1987). The mammalian heat shock (or stress) response: A cellular defense mechanism. Adv. Exp. Med. Biol. 225, 287–304.[Medline]

Welch, W. J. (1992). Mammalian stress response: Cell physiology, structure/function of stress proteins, and implications for medicine and disease. Physiol Rev. 72, 1063–1081.[Free Full Text]

Yamano, T., Shimizu, M., and Noda, T. (1998). Age-related change in cadmium-induced hepatotoxicity in Wistar rats: Role of Kupffer cells and neutrophils. Toxicol. Appl. Pharmacol. 151, 9–15.[ISI][Medline]