Analysis of Strain Difference in Sensitivity to Cadmium-Induced Hepatotoxicity in Fischer 344 and Sprague-Dawley Rats

Eric B. Harstad and Curtis D. Klaassen,1

Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Boulevard, MS 1018, Kansas City, Kansas 66160-7417

Received October 19, 2001; accepted January 8, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Acute administration of cadmium (Cd) in rats results in hepatotoxicity that appears to involve the activation of Kupffer cells and the subsequent production of proinflammatory chemokines and cytokines. However, the importance of these endogenous mediators in Cd-induced hepatotoxicity is unknown. Therefore, this study was conducted to define and utilize a rat strain difference in sensitivity to Cd-induced hepatotoxicity to elucidate the role of cytokines and chemokines in Cd-induced hepatotoxicity. Doses were selected from a dose-response study of the effect of Cd on serum alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) activities. Hepatotoxic doses of 2.0 mg Cd/kg in Fischer 344 (F344) rats and 3.0 mg Cd/kg in Sprague-Dawley (SD) rats, as well as a relatively nontoxic dose of 2.0 mg Cd/kg in SD rats, were chosen for the time-course experiment. Blood and liver from F344 (saline or 2.0 mg Cd/kg iv) and SD rats (saline or 2.0 or 3.0 mg Cd/kg iv) were collected at 0, 1, 3, 6, 10, 18, 24, and 48 h after Cd administration. Cadmium treatment caused an increase in serum ALT and SDH by 3 h and peaked between 18 and 24 h in both strains. Hepatic Cd content, metallothionein (MT) induction, and nonprotein sulfhydryl (NPSH) content were quantified and determined to be consistent with dosing rather than strain differences. Total RNA samples isolated from liver samples were analyzed for chemokine (CINC-1 and MCP-1) and cytokine (TNF-{alpha}, IL-1ß, IL-6, and IL-10) mRNA levels by the Quantigene branched DNA signal amplification assay. Lipopolysaccharide treatment served as a positive control for chemokine and cytokine induction. After Cd administration, F344 rat livers did not contain higher levels or earlier induction of chemokine and cytokine mRNAs than SD rats. Therefore, this study demonstrates a strain difference in sensitivity to Cd-induced hepatotoxicity that appears to be unrelated to Cd, MT, NPSH, or cytokine expression.

Key Words: cadmium; liver; hepatotoxicity; necrosis; cytokine; chemokine.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The heavy metal cadmium (Cd) is an industrial and environmental pollutant. It is toxic to several tissues, most notably causing hepatotoxicity upon acute administration and nephrotoxicity upon chronic exposure. Histological evaluation of liver injury reveals that acute toxicity is comprised of hepatocellular swelling, sinusoidal congestion, pyknosis, and karyorrhexis (Dudley et al., 1982Go). In a time-course study on Cd-induced hepatotoxicity, early cellular changes occur in the rough endoplasmic reticulum and nucleus (Dudley et al., 1984Go). Later alterations include mitochondrial swelling, further changes in the endoplasmic reticulum, and appearance of fibrillar material within the cytoplasm. These cellular changes may result in both apoptosis and necrosis (Habeebu et al., 1998Go).

Several reports have addressed the direct mechanism of Cd-induced hepatotoxicity at the subcellular and molecular levels. Subcellular localization of Cd demonstrates that Cd distributes to the nucleus, mitochondria, and endoplasmic reticulum, which localizes Cd in target organelles (Goering and Klaassen, 1983Go; Kapoor et al., 1961Go; Klaassen, 1978Go). Cadmium has been shown to cause mitochondrial dysfunction (Diamond and Kench, 1974Go) and more specifically inhibition of electron transport (Miccadei and Floridi, 1993Go). Cadmium administration also may result in DNA damage (Bagchi et al., 1996Go) and lipid peroxidation (Stacey et al., 1980Go), although it is disputed whether oxidative stress plays a role in Cd-induced hepatotoxicity (Harvey and Klaassen, 1983Go; Muller, 1986Go).

Metallothioneins are small, cysteine-rich proteins that bind Cd with high affinity (Klaassen et al., 1999Go). Pretreatment with low doses of zinc or Cd results in protection from subsequent doses of Cd (Goering and Klaassen, 1984aGo,bGo). This protection is a result of the induction of MT and subsequent redistribution of Cd to the cytoplasm where it is bound to MT and thus detoxified (Goering and Klaassen, 1983Go). In MT-null mice, this protective effect is not seen, thus confirming the requirement of MT in the mechanism of this protective effect (Liu et al., 1996Go).

Glutathione (GSH), the primary cellular nonprotein thiol, also plays a role in the detoxication of Cd, although the exact role GSH plays in Cd-induced hepatotoxicity is not accurately defined. Depletion of GSH with phorone, diethyl maleate, or buthionine sulfoximine has been shown to increase sensitivity to both Cd-induced lethality and hepatotoxicity (Dudley and Klaassen, 1984Go; Singhal et al., 1987Go). Glutathione has been shown to be critical for the biliary elimination of Cd from the liver (Dijkstra et al., 1996Go). However, the protective effect may also be due to GSH reducing Cd-induced oxidative stress (Shaikh et al., 1999Go). Therefore, GSH plays an important and perhaps multifunctional role in Cd-induced liver injury.

There also appears to be an indirect component to Cd-induced liver injury. This indirect component appears to involve Kupffer cell activation and subsequent release of endogenous inflammatory mediators. Kupffer cell activation by Cd was first noted by the identification of cytoplasmic vacuolization (Hoffmann et al., 1975Go) and an increase in colloidal carbon clearance (Sauer et al., 1997Go). Elimination of Kupffer cells with gadolinium chloride alleviates Cd-induced hepatotoxicity in rats (Sauer et al., 1997Go; Yamano et al., 1998Go), but fails to protect cultured hepatocytes (Badger et al., 1997Go). This data strongly supports the role of Kupffer cells in the mechanism of Cd-induced hepatotoxicity.

Limited cohesive information is available about the role of proinflammatory cytokines in Cd-induced hepatotoxicity. Tumor necrosis factor-{alpha} (TNF-{alpha}) is weakly induced by Cd both in vitro (Marth et al., 2000Go; Szuster-Ciesielska et al., 2000Go) and in vivo (Kayama et al., 1995Go). Pretreatment with antibodies against TNF-{alpha} mildly decreases Cd-induced hepatotoxicity and abrogated Cd-induced expression of acute phase proteins (Kayama et al., 1995Go). In contrast, Yamano and Rikans determined that TNF-{alpha} is not increased within 24 h of Cd administration (Yamano et al., 2000Go). The concentrations of other cytokines, including interleukin-1{alpha} (IL-1{alpha}), interleukin-1ß (IL-1ß), interferon-{gamma} (IFN-{gamma}), and interleukin-6 (IL-6) also increase after Cd treatment, although their relevance is less well defined (Kayama et al., 1995Go; Liu et al., 1999Go; Marth et al., 2000Go; Yamano et al., 2000Go).

Chemokines are a specific subset of cytokines that primarily function to recruit leukocytes to sites of inflammation. Interleukin-8 and CINC-1 (cytokine-inducible chemoattractant factor-1) are the prototypical neutrophil-specific chemokines for humans and rats, respectively. Because neutrophil accumulation has been reported in sites of inflammation after Cd-administration (Dudley et al., 1982Go; Hoffmann et al., 1975Go; Kayama et al., 1995Go), it is plausible to investigate whether Cd increases expression of neutrophil-specific chemokines. Neutrophil chemokines are induced after Cd treatment of cultured mouse and rat hepatocytes as well as cultured human cells (Dong et al., 1998Go; Horiguchi et al., 1993Go). In addition, CINC-1 protein increases in parallel with increased hepatotoxicity (Yamano et al., 2000Go).

It has been known for some time that sensitivity to Cd-induced hepatotoxicity is dependent upon the strain of mice studied (Nolan et al., 1986; Quaife et al., 1984Go). This strain difference does not appear to be due to a difference in Cd uptake by the liver or induction of MT (Kershaw and Klaassen, 1991Go; Liu et al., 1992Go). The sensitivity of rats to various hepatotoxins is also strain dependent. Fischer 344 (F344) rats are more sensitive than Sprague-Dawley (SD) rats to several chemical hepatotoxins, including 1,2 dichlorobenzene (Stine et al., 1991Go), carbon tetrachloride (Steup et al., 1991Go), diquat (Gupta et al., 1994Go), and paraffin wax (Hoglen et al., 1998Go). Recently, a preliminary report noted that F344 rats are also more sensitive than SD rats to Cd-induced hepatotoxicity (Keuster et al., 1999Go). That study suggested that the rate of MT induction might be causative in the rat strain difference, however, the data was not conclusive.

Therefore this study was conducted to achieve two goals: first, to define and examine the dose- and time-related rat strain difference in sensitivity to Cd-induced hepatotoxicity; and second, to utilize this strain difference to help elucidate the direct versus indirect mechanisms of Cd-induced hepatotoxicity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Male SD and F344 rats were used throughout the study. Both strains of rats were purchased from Sasco, Inc. (Wilmington, MA). Rats were housed in an AAALAC certified facility at 70 ± 2°F with a 12-h light/dark cycle and were fed laboratory Purina rat chow (St. Louis, MO) and water ad libitum. For the dose-response experiment, rats (200–250 g) were dosed with 1.0, 1.5, 2.0, 2.5, and 3.0 mg Cd/kg iv for F344 rats (n = 3) and 1.0, 1.5, 2.0 2.5, 3.0, 3.5, and 4.0 mg Cd/kg iv for SD rats (n = 3). Ten h after Cd administration, the rats were anesthetized with pentobarbital (50 mg/kg, ip) and blood was collected from the descending aorta. Whole blood was centrifuged and the plasma was retained for serum enzyme analysis. Livers were excised and a portion of the left lobe of the each liver was dissected and fixed in 10% neutral buffered formalin. The remainder of the liver was stored at –80°C for subsequent assays. For the time-course experiment, F344 rats were dosed with saline or 2.0 mg Cd/kg iv, and SD rats were dosed with saline or 2.0 or 3.0 mg Cd/kg iv. Rats were anesthetized and blood and liver were removed at 1, 3, 6, 10, 18, 24, and 48 h after Cd treatment (n = 4) and 0, 3, 10, and 24 h after saline treatment (n = 2). After rats were anesthetized, blood and liver were collected and processed as previously described.

Chemicals.
Tris-HCl and CdCl2 were purchased from Fisher Scientific (St. Louis, MO) and 109Cd was purchased from New England Nuclear (Boston, MA). All other chemicals were purchased from Sigma Chemical Company (St. Louis, MO).

Serum enzyme activity assays.
Biochemical evaluation of liver injury was performed by quantifying serum activities of alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) spectrophotometrically, using commercially available test kits (UV detection, kinetic endpoint, Sigma Diagnostics, St. Louis, MO) according to the manufacturer's instructions.

Histopathology.
Liver samples were taken consistently from the left lobe of the liver and processed by standard histological techniques. Briefly, samples were fixed in formalin for 24 h, then switched to ethanol for storage. Liver sections were then processed routinely and embedded in paraffin blocks. Slides were prepared (5 µm thick), stained with hematoxylin and eosin, and analyzed by light microscopy for liver injury.

Hepatic Cd content.
Liver samples (~ 1.0 g) were digested 1:3 (w/v) in concentrated HNO3 at 100°C for 60 min. The digested samples were assayed for Cd content using a Perkin-Elmer atomic absorption spectrophotometer (Norwalk, CT) using an analytical wavelength of 228.8 nm. Sample Cd content was determined by comparing absorbance values to a standard curve of Cd solutions.

Metallothionein assay.
Liver samples were prepared by homogenizing 1:3 (w/v) in 10 mM Tris-HCl buffer (pH 7.4), centrifuged at 13,000 x g, and the supernatant fractions were retained. Protein concentration in supernatants was determined using the bicinchoninic acid method using a BCATM kit (Pierce, Rockford, IL). Supernatants were assayed and hepatic MT content was determined using the Cd-hemoglobin method as previously described (Eaton and Toal, 1982Go).

Hepatic nonprotein thiol (NPSH) content.
Hepatic NPSH was quantified using the DTNB method (Ellman, 1959Go). Briefly, 100 mg liver samples were homogenized using a Kinematica Polytron homogenizer (Littau, Switzerland) in 5% TCA/EDTA, separated by centrifugation at 13,000 x g, and the supernatants retained for NPSH analysis. Each reaction contained 176 µl 0.1 M phosphate buffer (pH 8), 16 µl supernatant, and 8 µl 5 mM 5,5`-dithio-bis(2-nitrobenzoic acid). Absorbance was quantified using a Biotek microtiter plate spectrophotometer (Winooski, VT) at 405 nm (analytical wavelength) and 690 nm (reference wavelength), and compared with a standard curve of known GSH concentrations.

Isolation of total RNA and quantification of specific mRNAs using the branched-DNA (bDNA) method.
Total RNA was isolated using RNAzol B reagent (Tel-Test, Inc., Friendswood, TX) as per the manufacturer's protocol. Each RNA pellet was resuspended in RNase-free water and stored at –80°C. The concentration of total RNA in each sample was quantified spectrophotometrically and RNA integrity was determined by illuminating the 28S and 18S rRNA bands with ethidium bromide after separation by agarose gel electrophoresis. Specific mRNAs encoding individual cytokines were quantified using the QuantigeneTM bDNA signal amplification method (Bayer Diagnostics, East Walpole, MA, www.quantigene.com) as per the manufacturer's protocol. Briefly, 10 µg samples of total RNA were loaded per well of a 96-well microtiter plate containing hybridization buffer and 50 fmol/µl of each probe set. Complimentary probes were designed (see next section) and hybridized to mRNAs encoding for individual cytokines overnight at 53°C. Following hybridization, excess probes were removed by washing with hybridization buffer. The captured mRNAs were then hybridized to branched DNA molecules that contain alkaline phosphatase molecules. After incubation with the chemiluminescent substrate, luminescence was quantified with a Quantiplex 320 bDNA luminometer.

Development of specific oligonucleotide probe sets for bDNA analysis of cytokines.
The gene sequences of interest were accessed from GenBank (www.ncbi.nlm.nih.gov). These target sequences were analyzed by ProbeDesignerTM Software Version 1.0. Multiple, specific probes were developed to each mRNA transcript (Table 1Go). Oligonucleotide probes designed in this manner were specific to a single cytokine transcript. All oligonucleotide probes were designed to have a Tm of approximately 63°C. This feature allows hybridization conditions to be held constant (i.e., 53°C) during each hybridization step for each olignucleotide probe set. Each probe developed in ProbeDesignerTM was submitted to the National Center for Biotechnological Information (NCBI) for nucleotide comparison by the basic logarithmic alignment search tool (BLASTn) to ensure minimal cross-reactivity with other rat sequences. Oligonucleotides with a high degree of similarity (> 80%) to other known rat sequences were eliminated from the design.


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TABLE 1 Oligonucleotide Probes Generated for Analysis of Cytokine Expression by bDNA Signal Amplification
 
Statistics.
Data from the Cd dose-response experiment were analyzed using a two-way ANOVA with a Duncan's multiple range post hoc test. Data from the Cd time-course experiment were analyzed using a three-way ANOVA with a Duncan's multiple range post hoc test. Statistical differences between treatment groups at each time point are presented in the figures. Data from saline-treated control rats were independently analyzed using a two-way ANOVA. Because time had no effect on the data, values for each strain of rats were grouped together; these were considered the control group for each strain. Significance was determined at p < 0.05 for all tests.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dose Response of Cd-Induced Liver Injury in F344 and SD Rats
Hepatotoxicity was assessed by quantifying serum ALT and SDH in F344 and SD rats 10 h after iv administration of various dosages of Cd (Fig. 1Go). In F344 rats, Cd caused severe liver toxicity at doses of 2.0 and 2.5 mg Cd/kg. Minimal liver injury was observed in SD rats at doses 2.0 and 2.5 mg Cd/kg. Doses of 3.0 and 3.5 mg Cd/kg caused hepatotoxicity in SD rats. The dose response of Cd in the two rat strains was terminated at 2.5 mg Cd/kg for F344 rats and at 3.5 mg Cd/kg for SD rats due to lethality above these dosages.



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FIG. 1. Dose response of Cd-induced hepatotoxicity 10 h after iv Cd administration to F344 and SD rats. Fischer 344 rats were administered a dose of 1.0, 1.5, 2.0, or 2.5 mg Cd iv. Spraque-Dawley rats were administered 1.0, 2.0, 2.5, 3.0, or 3.5 mg Cd iv. Hepatotoxicity was assessed by measuring serum indices of hepatotoxicity, (A) alanine aminotransferase activity (ALT) and (B) sorbitol dehydrogenase activity (SDH). Statistical designation: a, F344 rats exhibited significantly greater toxicity than SD rats (p < 0.05).

 
Histopathological scoring of the livers (data not shown) from these rats confirms the serum indicators of hepatotoxicity, ALT and SDH. Sprague-Dawley rats were histologically normal after saline administration (Fig. 2AGo). After saline administration, F344 rat livers are histologically indistinguishable from SD rat livers (not shown). Cadmium (2.0 mg Cd/kg) caused severe hepatotoxicity in F344 rats (Figure 2CGo). This liver injury was consistent with previous reports (Dudley et al., 1982Go) including foci of inflammation, congestion, and various degrees of focal necrosis. On a cellular level, toxicity included hepatocyte swelling, pyknosis, and karyorrhexis. Multifocal necrotic lesions were found throughout the liver without specific zonal localization to any part of the hepatic lobule. An equal dose of Cd (2.0 mg Cd/kg) caused minimal liver injury in SD rats (Fig. 2BGo). The only observed histological changes were mild hepatocellular swelling and congestion. No irreversible changes were noted. A higher dose of Cd (3.0 mg Cd/kg) caused extensive hepatotoxicity in SD rats (Fig. 2DGo). Histopathology in SD rats after 3.0 mg Cd/kg was not substantially different from F344 rats after 2.0 mg Cd/kg.



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FIG. 2. Photomicrographs of Cd-induced hepatotoxicity in F344 and SD rats. Livers were collected 10 h after Cd administration for histological evaluation of liver injury. Livers were fixed in formalin solution and processed routinely for hematoxylin and eosin staining. Photomicrographs are representative of (A) SD rats administered saline, (B) SD rats administered 2.0 mg Cd/kg, (C) F344 rats administered 2.0 mg Cd/kg, and (D) SD rats administered 3.0 mg Cd/kg.

 
Time Course of Cd-Induced Hepatotoxicity
Cd caused severe hepatotoxicity in both F344 rats (2.0 mg Cd/kg) and SD rats (3.0 mg Cd/kg; Fig. 3Go). Liver injury was evident 3 h after Cd administration and became maximal between 18 and 24 h. By 48 h, serum indices of hepatotoxicity were decreased from maximal levels. Sprague-Dawley rats that were administered 2.0 mg Cd/kg had minimal hepatotoxicity at all time points. These data demonstrate that an equal dose of Cd (2.0 mg Cd/kg) causes extensive hepatotoxicity in F344 rats, but only minimal hepatotoxicity in SD rats. In addition, the higher dose of Cd (3.0 mg Cd/kg) also caused extensive hepatotoxicity in SD rats.



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FIG. 3. Time course of Cd-induced hepatotoxicity after iv Cd administration. Fischer 344 rats were administered saline or a hepatotoxic dose of Cd (2.0 mg Cd/kg). Sprague-Dawley rats were administered saline, an equal dose of Cd (2.0 mg Cd/kg), or a hepatotoxic dose of Cd (3.0 mg Cd/kg, iv). Hepatotoxicity was assessed by measuring serum indices of hepatotoxicity, (A) alanine aminotransferase activity (ALT) and (B) sorbitol dehydrogenase activity (SDH). Statistical designations: a, statistical difference from the F344 (2.0 mg Cd/kg) group; b, statistical difference from the SD (2.0 mg Cd/kg) group within each time point (p < 0.05).

 
Time Course of Hepatic Cd Accumulation
Liver samples were individually prepared and analyzed for Cd as described in the Methods section. Hepatic Cd accumulation was maximal 1 h after Cd administration and slowly decreased thereafter in all treatment groups (Fig. 4Go). Hepatic Cd concentrations were not significantly different between F344 and SD rats after 2.0 mg Cd/kg at any time point except for 24 h. Sprague-Dawley rats that were administered the higher dose of Cd (3.0 mg Cd/kg) had significantly higher hepatic cadmium concentrations than F344 rats and SD rats in the 2.0-mg Cd/kg treatment groups.



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FIG. 4. Time course of hepatic Cd accumulation after Cd administration in F344 and SD rats. Fischer 344 rats were administered saline or a hepatotoxic dose of Cd (2.0 mg Cd/kg). Sprague-Dawley rats were administered saline, an equal dose of Cd (2.0 mg Cd/kg), or a hepatotoxic dose of Cd (3.0 mg Cd/kg). Liver samples were individually prepared and analyzed for Cd as described in the Methods section. Statistical designations: a, statistical difference from the F344 (2.0 mg Cd/kg) group; b, statistical difference from the SD (2.0 mg Cd/kg) group within each time point (p < 0.05).

 
Time Course of Hepatic MT and NPSH Concentrations
Liver samples were prepared individually and analyzed for MT and NPSH as described in the Methods section. Metallothionein levels were increased in all Cd-treatment groups in a time-dependent manner that began 6 h after Cd administration and increased throughout the study (Fig. 5AGo). The degree of increase in MT protein concentrations was similar after equal Cd dosages (2.0 mg Cd/kg) as well as after hepatotoxic Cd dosages (2.0 mg Cd/kg in F344 rats and 3.0 mg Cd/kg in SD rats). While SD rats treated with 2.0 mg Cd/kg appear to have greater MT induction than F344 rats at the later time points, it was not significantly different. In all 3 Cd-treated groups, hepatic NPSH remained relatively unchanged through 10 h after Cd administration (Fig. 5BGo). With hepatotoxic doses of Cd, NPSH concentrations decreased at 18, 24, and 48 h. NPSH concentrations remained relatively unchanged in SD rats treated with 2.0 mg Cd/kg.



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FIG. 5. Time course of hepatic metallothionein (MT) and nonprotein thiol (NPSH) concentrations after Cd administration in F344 and SD rats. Fischer 344 rats were administered saline or a hepatotoxic dose of Cd (2.0 mg Cd/kg). Sprague-Dawley rats were administered saline, an equal dose of Cd (2.0 mg Cd/kg), or a hepatotoxic dose of Cd (3.0 mg Cd/kg). Liver samples were individually prepared and analyzed for (A) MT and (B) NPSH as described in the Methods section. Statistical designations: a, statistical difference from the F344 (2.0 mg Cd/kg) group; b, statistical difference from the SD (2.0 mg Cd/kg) group within each time point (p < 0.05).

 
Time Course of Chemokine and Cytokine mRNA Expression
Total RNA samples were isolated from each F344 and SD rat liver from the entire time course study of Cd-induced hepatotoxicity. Additional SD rats were administered lipopolysaccharide (1 mg LPS/kg, ip, Sigma, E. coli 0127:B8, TCA extract) to serve as a positive control for chemokine induction. Livers were collected at 0, 1, and 6 h after LPS administration. Specific mRNAs were quantified using the QuantageneTM bDNA signal amplification method. Total RNA samples from LPS-treated rats were pooled prior to bDNA analysis, whereas total RNA samples from Cd-treated rats were analyzed individually.

Lipopolysaccharide treatment caused a 32-fold increase in CINC-1 mRNA levels at 1 h that decreased 70% by 6 h (Fig. 6AGo). Cadmium treatment increased CINC-1 levels only about 10–20% of the LPS response. This increase was not observed until 18 and 24 h after dosing with 2.0 mg Cd/kg in both F344 and SD rats. The hepatotoxic dose of 3.0 mg Cd/kg in SD rats only increased CINC-1 mRNA levels at 24 h (Fig. 6BGo).



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FIG. 6. Time course of chemokine mRNA after Cd administration in F344 and SD rats. Sprague-Dawley rats were administered lipopolysaccharide (1 mg LPS/kg, ip) to serve as a positive control for hepatic chemokine induction (A and C). Fischer 344 and SD rats were administered Cd and chemokine gene expression was examined at time points between 1 and 48 h (B and D). Total RNAs from all samples were isolated and quantified by the QuantageneTM bDNA signal amplification method. Statistical designations: a, statistical difference from the F344 (2.0 mg Cd/kg) group; b, statistical difference from the SD (2.0 mg Cd/kg) group within each time point (p < 0.05).

 
Lipopolysaccharide treatment caused more than a 600-fold increase in MCP-1 mRNA levels at 1 h that also decreased 70% by 6 h (Fig. 6CGo). Hepatotoxic doses of 2.0 mg Cd/kg in F344 rats and 3.0 mg Cd/kg in SD rats increased MCP-1 mRNA levels only 20–30% of the LPS response at 3, 6, 10, and 18 h in F344 rats and 6, 10, 18, and 24 h in SD rats. Cadmium treatment of the SD rats with 2.0 mg Cd/kg increased MCP-1 mRNA levels only at 6 h (Fig. 6DGo).

Lipopolysaccharide treatment resulted in a 150-fold increase of TNF-{alpha} mRNA levels at 1 h that decreased 80% by 6 h (Fig. 7AGo). Cadmium treatment caused an increase in TNF-{alpha} mRNA levels that was only 5–15% of the response produced by LPS at 3, 6, 10, and 18 h in F344 rats after 2.0 mg Cd/kg, at 3 and 6 h in SD rats after 2.0 mg Cd/kg, and at 3, 6, and 10 h in SD rats after 3.0 mg Cd/kg (Fig. 7BGo).



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FIG. 7. Time course of cytokine mRNA after Cd administration in F344 and SD rats. Sprague-Dawley rats were administered lipopolysaccharide (1 mg LPS/kg, ip) to serve as a positive control for hepatic cytokine induction (A, C, E, and G). Fischer 344 and SD rats were administered Cd and cytokine expression was examined at time points between 1 and 48 h (B, D, F, and H). Total RNA from all samples was isolated and analyzed by the QuantigeneTM bDNA signal amplification method. Statistical designations: a, statistical difference from the F344 (2.0 mg Cd/kg) group; b, statistical difference from the SD (2.0 mg Cd/kg) group within each time point (p < 0.05).

 
Lipopolysaccharide treatment caused a 24-fold increase in IL-1ß mRNA levels at 1 h and decreased 65% by 6 h (Fig. 7CGo). Cadmium treatment caused an increase in IL-1ß mRNA levels only at 3 h in F344 rats after 2.0 mg Cd/kg, and 3 and 6 h in SD rats after 2.0 mg Cd/kg. Cadmium treatment caused an increase in IL-1ß mRNA levels from 1 to 24 h after administering 3.0 mg Cd/kg to SD rats. Cadmium-induced changes in IL-1ß mRNA levels were minimal and did not exceed 15% of the increase induced by administration of LPS (Fig. 7DGo).

Lipopolysaccharide treatment caused a 110-fold increase in IL-6 mRNA levels at 1 h that decreased 55% by 6 h (Fig. 7EGo). Cadmium administration resulted in increased IL-6 mRNA levels in SD rats after 3.0 mg Cd/kg at 6, 10, 18, and 24 h that did not exceed 25% of the LPS response. No significant increases in IL-6 mRNA levels were noted after administration of 2.0 mg Cd/kg to either F344 or SD rats (Fig. 7FGo).

Lipopolysaccharide treatment caused a 15-fold increase in IL-10 mRNA levels at 1 h that continued to increase to 19-fold by 6 h (Fig. 7GGo). Cadmium treatment did not increase IL-10 mRNA levels over control in any dose group at any time point (Fig. 7HGo).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A large body of information is available describing the mechanism by which Cd exerts its deleterious effects upon the rodent liver. It is clear that Cd-induced hepatotoxicity is a complicated process that is conventionally thought to comprise both a direct and indirect mechanism (Rikans and Yamano, 2000Go). It has been proposed that the direct component of Cd toxicity initially manifested in the mitochondria by inhibiting electron transport (Diamond and Kench, 1974Go). This mitochondrial dysfunction may then result in oxidative stress or simply a starving of the hepatocytes for ATP (Muller and Ohnesorge, 1984Go). Subsequent liver injury has been suggested to be a result of Kupffer cell activation and the ensuing inflammation in response to the initial direct toxicity of Cd (Badger et al., 1997Go; Sauer et al., 1997Go; Yamano et al., 1998Go). However, the exact nature of this indirect inflammatory toxicity is as yet unknown.

It is well documented that mouse strains vary widely in their sensitivity to cadmium-induced hepatotoxicity (Nolan and Shaikh, 1986Go; Quaife et al., 1984Go). The mouse strain differences did not appear to be due to differential accumulation of Cd in the liver or induction of MT (Kershaw and Klaassen, 1991Go; Liu et al., 1992Go). However, it is unknown whether there are any strain-related differences in the indirect components of Cd-induced hepatotoxicity.

Therefore, this study was conducted to utilize this rat strain difference in susceptibility to Cd to elucidate the role of cytokines in Cd-induced hepatotoxicity. The first aim of this study was to fully characterize the dose response and time course of Cd-induced liver injury in both F344 and SD rats. These data clearly demonstrate that F344 rats are more sensitive to Cd-induced hepatotoxicity with respect to dose. Cadmium has a similar temporal toxicity in both strains of rats, beginning at about 3 h and becoming maximal between 18 and 24 h. Unfortunately, hepatotoxic doses of Cd could not be compared between F344 and SD rats 48 h after Cd administration due to significant lethality in the SD rats. These data demonstrate a dose-dependent strain difference in sensitivity to Cd-induced hepatotoxicity.

The next aim of this study was to determine whether Cd accumulation was causative for the strain difference. The data show that an equal dosage of Cd (2.0 mg Cd/kg) results in similar hepatic Cd content in the 2 strains during the first 18 h after Cd administration. At later time points there is more Cd in the SD rat livers than F344 rat livers after equal dosages of 2.0 mg Cd/kg. It is plausible that this may simply be due to release of Cd as a result of necrosis in F344 rat livers. Higher dosages of Cd (3.0 mg Cd/kg) in SD rats resulted in higher hepatic cadmium content. Therefore, hepatic Cd accumulation in F344 and SD rats appears to be dependent upon the dose of Cd rather than the strain of rat. These data clearly show that differences in Cd accumulation in the liver do not account for the strain difference in Cd-induced hepatotoxicity.

Thiols are important in Cd toxicity, both as targets for toxicity as well as buffers against toxicity. Metallothionein (MT) is a highly inducible, cysteine-rich protein that binds Cd with high affinity, thus sequestering and detoxifying Cd (Klaassen et al., 1999Go). Therefore, MT was quantified over time after Cd administration. MT was significantly induced in a time-dependent manner, beginning at 6 h after Cd and continuing to increase throughout the time course. However, hepatic MT was not significantly different between the 3 treatment groups at any time point during the study. Nonprotein thiols, such as glutathione, are also known to be important in the detoxification and excretion of Cd (Singhal et al., 1987Go). Therefore, total hepatic nonprotein sulfhydryls (NPSH) were quantified in the time course samples. Hepatic NPSH was similar in naïve F344 and SD rats and remained unchanged from controls through the first 10 h of the study. At and after 18 h, hepatic NPSH was decreased only after hepatotoxic doses of Cd in both F344 and SD rats. NPSH remained unchanged with the lower dose in SD rats. These data indicate that the decrease in NPSH is a result of toxicity rather than a factor that results in the sensitivity of the F344 rats. Therefore, the strain difference in Cd-induced hepatotoxicity is not due to MT induction or alteration in NPSH concentration.

It has been proposed that Kupffer cells are involved in the indirect component of the mechanism of Cd toxicity (Hoffmann et al., 1975Go; Sauer et al., 1997Go; Yamano et al., 1998Go, 2000Go). Kupffer cells have been implicated as the source of the inflammatory response, because they are known to produce proinflammatory mediators such as cytokines and chemokines when activated. Lipopolysaccharide (LPS) is the prototypical activator of Kupffer cells and has been well documented to robustly increase gene expression for proinflammatory cytokines and thus produce liver injury. LPS was used in this study as a positive control to quantify Cd-induced cytokine expression with regard to magnitude and time.

CINC-1 and MCP-1 are the prototypical neutrophil and monocyte chemokines that specifically recruit neutrophils and monocytic macrophages, respectively, to sites of inflammation. The results demonstrate that Cd elicits small increases, relative to LPS, in CINC-1 gene expression that are not substantially different in F344 and SD rats after hepatotoxic dosages of Cd. Moreover, the increases in CINC-1 mRNA did not precede toxicity, and were more likely an effect of, or response to the toxicity. MCP-1 mRNA is increased in F344 rats at slightly earlier time points than in SD rats. While this may contribute to Cd-induced hepatotoxicity, the magnitude of the response is unlikely to be substantial enough to account for the strain difference in toxicity.

Particular attention has been paid to the role of TNF-{alpha} in Cd toxicity, with several reports suggesting that TNF-{alpha} plays a role in the mechanism of hepatotoxicity (Kayama et al., 1995Go; Marth et al., 2000Go; Szuster-Ciesielska et al., 2000Go). In the present study, Cd treatment, when compared to LPS treatment, only mildly increased TNF-{alpha} gene expression in either F344 or SD rats. In addition, the pattern of increased TNF-{alpha} mRNA levels after Cd treatment is not similar to the pattern observed after LPS treatment. Moreover, the strain-related differences in TNF-{alpha} gene expression do not parallel the strain-related differences in hepatotoxicity. Therefore, expression of the TNF-{alpha} gene does not appear to account for the strain difference in sensitivity to Cd-induced hepatotoxicity. Furthermore, these increases are not robust when compared to LPS, and therefore may only be a minor factor in Cd toxicity.

Interleukin-1ß and IL-6 have also been implicated in the mechanism of Cd toxicity (Kayama et al., 1995Go; Marth et al., 2000Go; Yamano et al., 2000Go). Examination of these interleukins in the present study shows that the levels of IL-1ß and IL-6 mRNAs are increased selectively in SD rats after hepatotoxic dosages of Cd. Because IL-1ß and IL-6 are increased in SD rats and not in F344 rats, they can be ruled out as candidates for sensitizing F344 rats to Cd-induced hepatotoxicity. Interleukin-10, the prototypical anti-inflammatory cytokine, was not significantly increased at any time point.

From these data, as well as literature citations, it is clear that Cd toxicity is an extremely complex process. This study was designed to specifically address several potential components of this complex mechanism of hepatotoxicity. This study attempted to utilize this strain difference in sensitivity to Cd-induced hepatotoxicity to tease out important factors in the mechanism of toxicity. Hepatic Cd accumulation, MT induction, and NPSH alteration are similar in both strains and thus do not appear to contribute to the strain difference. More interestingly, this study establishes cytokine profiles over time with regard to both time and magnitude of response. Thus, these chemokines and cytokines may participate in the mechanism of Cd-induced hepatotoxicity; however, it is likely to be only a minor component of the toxicity and does not account for the strain difference in Cd-induced hepatotoxicity.


    ACKNOWLEDGMENTS
 
This research was supported by NIH grant ES-01142. E.B.H. was supported by NIH grant ES-07079.


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


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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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