Role of tumor necrosis factor-α in the development of spontaneous hepatic toxicity in Long-Evans Cinnamon rats

Toxicology and Applied Pharmacology 200 (2004) 121 – 130 www.elsevier.com/locate/ytaap

Role of tumor necrosis factor-a in the development of spontaneous hepatic $ toxicity in Long-Evans Cinnamon rats Rodolfo Nin˜o Fong, Blanca Patricia Esparza Gonzalez, I. Carmen Fuentealba, and M. George Cherian * Department of Pathology, Faculty of Medicine and Dentistry, University of Western Ontario, London, ON, Canada N6A 5C1 Received 2 March 2004; accepted 31 March 2004 Available online 9 June 2004

Abstract The objective of this study was to evaluate the potential role of TNF-a in the onset of acute hepatitis in the Long-Evans Cinnamon (LEC) rat, an animal model for inherited copper (Cu) toxicosis. In LEC rats, Cu is accumulated in the liver with age, and clinical signs of acute hepatitis were observed as, icterus, reduced body weight, nasal bleeding, dehydration, and reduced food intake at 12 weeks of age. Cellular changes such as apoptosis in the liver were evident in these rats with increasing age. Positive TNF-a and TNFR1 immunostainings were observed in hepatocytes and Kupffer cells in LEC rats. Hepatic levels of caspase-3 activity, TNF-a mRNA, and protein were also increased in LEC rats from 6 to 12 weeks of age as compared with control Long-Evans (LE) rats. The neutralization of TNF-a by passive immunization or the inhibition of caspase activity can block the apoptotic process initiated by TNF-a. In this study, we evaluated the effects of passive immunization of LEC rats with weekly administration of anti-rat TNF-a on Cu-induced acute hepatitis. This treatment resulted in a reduction of the percentage of apoptotic cells in the liver, decreased activity of caspase-3, and also in down-regulation of the TNF-a gene expression. Thus, these results suggest a major role for TNF-a on the pathogenesis of Cu-induced acute hepatitis in LEC rats. D 2004 Elsevier Inc. All rights reserved. Keywords: TNF-a; LEC; Apoptosis; Copper toxicity

Introduction Tumor necrosis factor (TNF) family includes two proteins: TNF-a and TNF-h, which are similar in structure and function. TNF-a is mainly produced by monocytes and/or macrophages, whereas TNF-h is a product of lymphoid cells (Baker and Reddy, 1998; Cuturi et al., 1987; Lindemann et al., 1989). Originally they were characterized by an antitumor activity (Carswell et al., 1975), but later showed their major role on inflammatory, immune, and tissue repair reactions (Tracey and Cerami, 1993). It is now well accepted that trimerization of the respective receptor by TNF (Baker and Reddy, 1998; Leist et al., 1995) leads to the assembly of the death initiating signaling complex, and $ MGC wants to dedicate this paper to late Dr. Francine Denizeau (a toxicology professor at University of Quebec at Montreal) who passed away on March 15, 2004. * Corresponding author. Department of Pathology, Faculty of Medicine and Dentistry, University of Western Ontario, Dental Sciences Building #4044, London, ON, Canada N6A 5C1. Fax: +519-661-3370. E-mail address: [email protected] (M.G. Cherian).

0041-008X/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2004.03.023

subsequently the caspase cascade activation (Griffith et al., 1999; Higuchi and Aggarwal, 1994; Jaeschke et al., 1998; Osawa et al., 2001; Tartaglia et al., 1993), which may result in DNA fragmentation by the endonucleases. In the liver, hepatocytes, Kupffer cells, and infiltrating macrophages can produce TNF-a (Gonzalez-Amaro et al., 1994). It has been suggested that TNF-a plays an important role in the pathogenesis of liver diseases (Spengler et al., 1996). For example, TNF-a-induced hepatocyte apoptosis has been reported in hepatitis C infection (Gonzalez-Amaro et al., 1994). Moreover, the induction of TNF-a synthesis has been reported in liver failure caused by endotoxic shock by lipopolysaccharide (LPS) (Hewett et al., 1993). In alcoholic liver disease, TNF-a has an important role in the inflammatory process and the subsequent development of hepatic cirrhosis (Ferraz et al., 1997). In both in vitro and in vivo experiments, the involvement of TNF-a in liver cell injury with concanavilin A, actinomycin-D, or combination of LPS and galactosamine-D (Bohlinger et al., 1996; Gantner et al., 1995; Kimura et al., 1999) has been shown. However, it is unclear whether TNF-a plays a significant

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role in metal-induced toxic hepatitis. Different animal models have been used to study the pathogenesis of metalinduced hepatoxicity. Long-Evans Cinnamon (LEC) mutant rats spontaneously develop chronic hepatic injury, severe hepatitis, and jaundice at about 4 months of age (Sasaki et al., 1985) with the accumulation of copper (Cu) in the liver and other organs. Impaired Cu metabolism and its increased tissue Cu accumulation are associated with the occurrence of hepatitis, and this acute hepatitis resembles the clinical signs of human fulminant hepatic failure (Sasaki et al., 1985). A single autosomal recessive gene is responsible for the increased accumulation of Cu with age and liver injury (Masuda et al., 1988). The pathogenic mechanism has been attributed to loss of function by mutations in the Cutransporting ATPase gene called atp7b (Cox, 1995; Wu et al., 1994). The objective of this study was to investigate the potential role of TNF-a during the onset of hepatitis with high hepatic Cu accumulation in LEC rats.

Materials and methods Animals, sample collection, and treatment. Female LEC and control Long-Evans (LE) rats were obtained from Charles River Laboratories in Japan and Canada, respectively. Female rats were selected for all experiments because hepatic changes are more prominent in LEC female rats as compared to male (Kasai et al., 1990), and cirrhosis is a common feature reported in female rats (Masuda et al., 1988). Rats were housed two per cage, fed Purina Rat Chow and water ad libitum, and maintained in an environment of 12-h light and dark cycles. All procedures were performed in accordance with the guidelines of the Canadian Council on Animal Care. In the first experiment, 16 LEC rats and 8 control LE rats were used to determine the changes in hepatic Cu levels, TNF-a levels, apoptosis, and liver damage with age. Blood samples from the heart were collected from anesthetized rats after intraperitoneal injection of pentobarbital (100 mg/kg). The samples were allowed to coagulate for at least 30 min and centrifuged at 2500  g to obtain serum fraction. Four LEC rats were killed at 6, 8, 10, and 12 weeks of age. Four LE rats were killed at 6 and 12 weeks of age. Liver samples from both LEC and LE rats were collected for routine histopathological examination. A section of the lower right median lobe of the liver was placed in formalin, and the remainder of the liver was frozen in liquid nitrogen for Cu analysis, caspase-3 activity, TNF-a concentration, and RNA extraction. In the second experiment, the effect of treatment of antiTNF-a on liver damage and apoptosis was studied in LEC and LE rats. Four LEC rats (6 weeks of age) in each group were injected intraperitoneally once a week for 6 weeks with either albumin (group 1), or 50 Ag/kg of anti-rat TNF-a (R&D Systems, Minneapolis, MN) (group 2) and 100 Ag/kg of anti-rat TNF-a (group 3). Six weeks old LE rats in each

group (N = 4) were also treated similarly: control (group 4), 50 Ag/kg of anti-rat TNF-a (group 5), and 100 Ag/kg of antirat TNF-a (group 6). All rats were euthanized at 12 weeks of age. Blood and liver samples were collected as described in the first experiment. Determination of hepatic Cu. Approximately 200 mg of liver samples from LEC and LE rats was weighed and digested in 2 ml of concentrated nitric acid (Syversen and Syversen, 1975) at room temperature for 24 h. These samples were heated to 60jC for 1 h to dissolve semi-solid lipids, and total volumes were adjusted to 10 ml with distilled/deionized water. The metal (Cu) concentration was measured using a Varian Spectra 800 atomic absorption spectrophotometer (AAS) (Varian, Australia), equipped with an air – acetylene flame. Cu concentrations were recorded from a standard curve and calculated as micrograms per gram wet tissue. Liver enzymes. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured in a Hitachi 911 autoanalyser (Bergmeyer et al., 1986). Histological examination. Five-micrometer-thick liver sections from LEC and LE rats were deparaffinized in xylene and rehydrated in consecutive changes of 100 – 70% ethanol before immersion in hematoxylin solution for 5 min. Slides were rinsed in distilled/deionized water for 2 min and destained in 1% HCl in 70% ethanol before being treated with 0.5% ammonia in water and rinsed. These slides were stained with eosin solution for 2 min, and rinsed. Sections were dehydrated through alcohol and three changes of xylene. Slides were mounted with Permount mounting medium (Fisher Scientific, Fair Lawn, NJ) and coverslipped. In situ detection of apoptosis. The nuclear DNA fragmentation of apoptotic cells was labeled in situ using the TUNEL method (Gavrieli et al., 1992). TUNEL staining was done, according to the manufacturer’s protocol (In Situ Cell Death Detection, POD, Boehringer Mannheim). Formalin-fixed liver samples were sectioned at 5 Am, dewaxed, and rehydrated by sequential changes in xylene and ethanol (100%, 95%, 90%, 80%, 70%). Tissue sections were incubated with 20 Ag/ml proteinase K (Sigma Chemical Co., St. Louis, MO) in 10 mM Tris –HCl buffer, pH 7.4– 8.0 for 15– 30 min at room temperature. Endogenous peroxidase was inactivated with 3% hydrogen peroxide (H2O2) (Sigma) in methanol for 10 min at room temperature and cells were permeabilized by treating them with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice. Diluted TUNEL reaction mixture containing terminal deoxynucleotidyl transferase enzyme from calf thymus and fluoresceinlabeled uridine triphosphate (1:3 in 30 mM Tris – HCl buffer, 1 mM cobalt chloride, 140 mM sodium cacodylate) was added to each slide and incubated for 60 min at 37jC in humidified chamber. These slides were incubated with antifluorescein antibody Fab fragments from sheep, conjugated with horseradish peroxidase for 30 min at 37jC in humid-

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ified chamber. The sections were then incubated with DAB solution (1 mg diaminobenzidine, 10 ml PBS, and 6 Al 30% H2O2) for 10 min at room temperature. The sections were counterstained with hematoxylin for 1 min and they were dehydrated, cleared, and mounted (Permount mounting medium, Fisher Scientific). Formalin-fixed, paraffin-embedded sections of pig testis were used as positive controls for apoptosis. Image analysis. TUNEL-stained liver sections were analyzed in 10 random fields using a Bioquant Nova computerized image analyzer (Advanced Image Analysis, R&E Biometrics, Inc., Nashville, TN). Apoptotic cells were counted with SigmaScan/Image measurement software (Jandel Scientific, San Rafael, CA). Immunohistochemical localization of TNF-a and TNFR1. Formalin-fixed liver samples were sectioned at 5 Am, dewaxed, and rehydrated by sequential passes in xylene and ethanol (100%, 95%, 90%, 80%, 70% diluted in distilled water for 5 min each step). After rehydration of the liver samples, endogenous peroxidase was inactivated with 3% H2O2 in 100% methanol for 30 min at room temperature. After washing three times with PBS, liver sections were blocked with universal blocking solution (DAKO Diagnostic Canada Inc., Mississauga, ON, Canada), and incubated at room temperature for 20 min. After removing the blocking solution, the sections were incubated with either anti-rat TNF-a (10 Ag/ml polyclonal rabbit anti-rat TNF-a, Endogen, Woburn, MA) or anti-rat TNFR1 (1:200 of monoclonal antibody from Santa Cruz Biotechnology, Inc.) at room temperature for 90 min. Following washing, the tissue sections were then incubated with the secondary antibodies (either peroxidase-labeled goat anti-rabbit IgG or goat antimouse from Vector Laboratories, Inc., CA) for 30 min at room temperature. These slides were washed in PBS and incubated with DAB solution (1 mg diaminobenzidine, 10 ml PBS, and 6 Al 30% H2O2) for 4 min at room temperature. Finally, slides were counterstained with hematoxylin and mounted (Permount Mounting Medium, Fisher Scientific). The sections without the primary antibody during staining were used as negative controls. The immunohistochemical localization of TNF-a and TNFR1 was graded by three people using a 1– 3 grading system, in which 1 = mild, 2 = moderate, and 3 = strong staining. Determination of hepatic TNF-a. Rat liver TNF-a concentration was determined by ELISA (Quantikine M Murine, R&D Systems), according to the manufacturer’s protocol. Fifty milligrams of frozen liver tissue was homogenized in 100 Al lysis buffer. Protein concentration was determined in liver homogenates by Bradford method (BioRad Protein Assay, Hercules, CA). Fifty micrograms of protein was adjusted in 50 Al calibrator diluent buffer (RD5-17). Fifty microliters of assay diluent buffer followed by 50 Al of standard control or sample per well was added. Each sample, standard, and control was processed in dupli-

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cate. Strips were incubated during 2 h at room temperature. After this, strips were washed 4 times with wash buffer (400 Al buffered surfactant with preservatives), subsequently 100 Al of anti-rat TNF-a conjugated (anti-rat TNF-a conjugated to horseradish peroxidase) was added to each well and incubated for 2 h at room temperature. These strips were washed as mentioned above, and 100 Al of substrate solution (stabilized H2O2 and tetramethylbenzidine) was added to all wells and incubated for 30 min at room temperature (avoid placing the plate in direct light). Last step was the addition of 100 Al of stop solution (hydrochloric acid solution) to each well. Optical density was measured within 30 min using a microplate spectrophotometer reader (Spectro Max 340 M01326) and soft Max Program 1.2.0 software (Molecular Devices Corp.) set to 450 mm with wavelength correction at 540. Increased concentrations of rat TNF-a were used to determine the calibration slope and a known standard concentration of rat TNF-a was used as a positive control. In negative controls, addition of the standard or the anti-rat TNF-a conjugate was omitted. Determination of caspase-3 activity. Caspase-3 activity was determined in liver homogenates by a fluorometric assay (Caspase-3 Fluorometric Assay, R&D Systems) in a 96-well black plate. Briefly, 200 Ag of protein from liver homogenates was adjusted with 50 Al of distilled water. Then, 50 Al of 10 mM DTT diluted in the reaction buffer and 5 Al of caspase-3 fluorogenic substrate (DEVD-AFC) were added to each well. Each sample, and positive and negative controls were processed in duplicate. Plates were incubated at 37jC for 1 –2 h. After incubation, plate was read on fluorescent microplate reader with settings of light excitation at 400 nm wavelength and emitted light at 505 nm wavelength. Recombinant human caspase-3 (R&D Systems) was used as a positive control. Negative controls were included with no addition of sample or caspase-3 substrate. TNF-a gene expression by real time RT-PCR. The total RNA was extracted from frozen liver samples. Fifty to 100 mg of liver tissue was homogenized in 1 ml of TRIzol Reagent (GIBCO BRL, Gaithersburg, MD). After 5 min of incubation at 15– 30jC, 0.2 ml of chloroform was added. After 2– 3 min of incubation at 15 – 30jC, samples were centrifuged at 12,000  g for 15 min at 2 – 8jC. The RNA in the aqueous phase was precipitated with isopropyl alcohol. After centrifugation, RNA samples were washed in 1 ml of 75% ethanol and precipitated by centrifugation. RNA pellets were dried by air and dissolved in RNase-free water. The concentration and purity of the RNA thus isolated were determined by measuring the optical density at 260 and 280 nm in a spectrophotometer. Five micrograms of total RNA was reverse transcribed according to the manufacturer’s protocol (Superscript II Preamplification System; GIBCO BRL), using oligo (dT) for rat TNF-a as a primer. The cDNA product was subjected to PCR for 40 cycles at 60jC using rat specific primers. The forward 5V-GAGATGTGGAACTGGCAGAGG and the reverse 5V-GGTACAGCC-

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CATCTGCTGGTA primers spanned several introns in the TNF gene, and were located at nucleotides 23 – 34 and 386– 406, respectively, of the rat TNF cDNA (Gene Bank accession number X66539). A RT reaction cocktail was prepared by using SYBR Green Taq ReadyMix for Quantitative PCR (Sigma). All the components in the right proportions were added together in the master mix for the number of reactions needed (per reaction: 10 Al SYBR Green Taq ReadyMix, 1.6 Al 25 mM MgCl2, 1 Al of each forward and reverse primers, and 4.4 Al H2O). Statistical analysis. The experimental data were processed using Minitab for Windows software for analysis of variance, and correlation between age and the number of apoptotic cells. Values were considered statistically significant when P < 0.05.

Results An incidental finding of high TNF-a immunoreactivity (data not shown) in the liver tissues from Fischer rats fed with high Cu-containing diet led us to investigate the possible role of TNF-a in the pathogenesis of Cu-induced hepatotoxicity in LEC rats. In the first experiment, we studied the formation of TNF-a and morphological changes in the liver of LE and LEC rats from 6 to 12 weeks of age, to evaluate the potential role of TNF-a on liver damage, especially in hepatocyte apoptosis. There were no clinical signs of acute hepatitis or any other liver damage in control LE rats (6 and 12 weeks of age), or LEC rats at 6 –10 weeks of age. LEC rats at 12 weeks of age, however, showed jaundice, nasal bleeding, dehydration, oliguria, loss of body weight, and low food consumption (data not shown). These signs are compatible with acute hepatitis. This was confirmed by the serum activity of liver enzymes. A significant increase ( P < 0.05) in serum ALT and AST activity was observed in LEC rats at 12 weeks of age as compared with LE and LEC rats from 6 to 10 weeks of age. Cu content in the liver of control LE rats (6 and 12 weeks of age) was 4 Ag/ g. However, the hepatic Cu content in LEC rats was

approximately 41 – 100 times more than age-matched control LE rats. There was a gradual increase in hepatic Cu concentration in LEC rats with age, and it was highest at 12 weeks of age (Table 1). The macro and microscopic observations in livers of control LE and LEC rats at 6 – 10 weeks of age were normal. Macroscopic findings in LEC rats at 12 weeks of age included dehydration, subcutaneous jaundice, presence of clotted blood in nasal ducts, and the liver was smaller and darker as compared with the liver from control LE rats. Microscopic observation of livers showed enlarged hepatocytes with big nuclei, fatty changes, apoptosis, and dysplastic appearance (Fig. 1). No tumors were observed but small areas of necrosis were seen in the parenchyma. Increased Cu accumulation might have caused substantial parenchymal apoptosis in the liver of LEC rats at 12 weeks of age as compared with control LE rats. LEC rats showed a positive correlation between the percentage of apoptotic cells and age. Significant increase in percentage of apoptotic ( P < 0.005) cells was observed in LEC rats at 12 weeks of age as compared with those at 6 and 8 weeks of age (Fig. 2A). The ability of liver to produce TNF-a was confirmed by RT-PCR analysis using h-actin gene expression as control. Data presented in the Fig. 2B demonstrate that small basal levels of TNF-a gene are expressed in the liver from LE and young LEC rats. Results are expressed as ratio of TNF-a:hactin gene expression. Increased TNF-a gene expression was observed in LEC rats with age. No significant difference in the TNF-a gene expression was observed in LEC rats at 6, 8, and 10 weeks of age and LE rats at 6 and 12 week of age. However, significant increase in TNF-a gene expression was detected in LEC rats at 12 weeks of age (Fig. 2B) ( P < 0.005). The concentrations of TNF-a protein in the liver tissues from LEC and LE rats were determined by ELISA, to determine whether TNF-a protein concentrations in the liver tissues can be correlated with the mRNA concentrations. The levels of hepatic TNF-a in control LE rats at 6 and 12 week of age were 40 F 3 and 35 F 4 pg/Ag, respectively. Hepatic TNF-a levels were significantly higher ( P < 0.005) in LEC rats at 10 and 12 week of age (110.68 F 7 and 158 F 4.2 pg/Ag), as compared with LEC

Table 1 Hepatic enzymes activity, liver copper concentration, and liver pathology in LEC and LE rats Age (week)

6

6

8

10

12

12

Rat AST (units/l) ALT (units/l) Cu (mg/g) Histopathology

LEC 385 F 25 132 F 17 166 F 4 Normal

LE 103 F 38 62 F 10 4 F 0.3 Normal

LEC 123 F 15 56 F 10 181 F 5 Normal

LEC 142 F 13 79 F 14 239 F 19 Normal

LEC 957 F 78* 693 F 55* 439 F 8* Apoptosis, enlarged nuclei, dysplasia, and necrosis

LE 95 F 5 51 F 8 4 F 0.7 Normal

Serum ALT and AST activities were measured in a Hitachi 911 autoanalyser (Bergmeyer et al., 1986). In LE rats, serum activity of AST, ALT, hepatic copper levels, and histology were normal. Significant increase in AST and ALT and hepatic copper levels was observed in LEC rats at 12 weeks of age as compared with LEC rats at 6, 8, and 10 weeks of age. Evident histopathology changes were observed in LEC rats at 12 weeks of age only. AST and ALT are expressed in units per litre. Liver copper content is expressed in micrograms per gram of wet weight. Data are presented as the mean of four animals in each group. Data are expressed as the mean value F SD of four animals in each group. * P < 0.05.

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hepatitis in LEC rats. Both LEC and LE rats were treated with two different doses (50 and 100 Ag/kg) of anti-rat TNFa as described in Materials and methods. No clinical signs of acute hepatitis were observed in LEC rats treated with either 50 or 100 Ag/kg of anti-rat TNF-a. Macroscopic appearance of the liver in anti-TNF-a-treated LEC and LE rats was normal at 12 weeks of age. Hepatocytes in LEC rats treated with 50 Ag/kg of anti-rat TNF-a (group 2) showed enlarged cell with big nuclei, fatty changes, small areas of necrosis in the parenchyma, apoptosis, and dysplastic appearance, similar to LEC rats at 12 weeks of age as shown in Fig. 1. However, signs of liver damage were reduced in the

Fig. 1. Light photomicrograph of the liver from LEC rats. Cellular morphological changes were observed in the liver of LEC rats at 12 weeks of age. Hepatocytes were enlarged as well as the nuclei (arrow). Apoptosis (arrowhead), necrosis (asterisk), and vacuolation (open arrow) of the hepatocytes were the most common changes observed in LEC rats with more than 100 times of hepatic Cu level as compared with LE rats. H and E staining, 200 magnification.

rats at 6 and 8 week of age (44.3 F 2.5 and 56.3 F 5 pg/Ag) (Fig. 2C). The formation of TNF-a protein in liver was demonstrated by immunostaining using a polyclonal antibody against rat TNF-a. In the liver of control LE rats at 6 and 12 weeks of age, a weak immunostaining for TNF-a was observed. However, significantly higher TNF-a immunostaining was observed in LEC rats at 6, 8, and 12 weeks of age than age-matched control LE rats. The TNF-a was localized in both hepatocytes and Kupffer cells. The highest immunostaining was observed in LEC rats at 12 weeks of age (Fig. 3A and Table 2), and they had more severe liver damage than younger rats. Because TNFR1 (Type 1 TNF-a receptor) is more important than TNFR2 in triggering cellular apoptosis, a monoclonal antibody against TNFR1 was used to demonstrate the presence of TNFR1 on cell membrane of hepatocytes. Positive immunoreactivity for TNFR1 was observed in hepatic cells in LEC rats at 12 weeks of age (Fig. 3B and Table 2). The activation of the TNFR1 triggers diverse intracellular signals, and caspase cascade activations are the most well-studied parameters in the TNFR1-induced apoptotic process. Livers from LEC rats showed an increased caspase-3 activity by a fluorometric assay as compared with those from control LE rats. There was a significant increase in caspase-3 activity between LEC rats and LE rats at 6 and 12 weeks of age. At 6 weeks of age, caspase-3 activity was 2-fold increased in LEC rats as compared with control LE rats from the same age group. Similar caspase-3 activity was observed at 8 weeks in LEC rats, but an increased activity was observed at 10 (2.53 F 0.24-fold increase) and 12 ( P < 0.05) (3.48 F 0.1-fold increase) weeks of age (Fig. 4). In a second experiment, we tested whether neutralization of TNF-a can reduce the pathological effects of acute

Fig. 2. Comparison of the apoptotic rate, TNF-a mRNA, and TNF-a levels in hepatocytes of LEC and LE rats at different ages. Significant increase in hepatocyte apoptosis (18% F 2%) and TNF-a mRNA levels was observed in LEC rats at 12 weeks of age, as compared with LE rats and LEC rats from 6 to 10 weeks of age (A and B). Significant increase in hepatic TNF-a concentrations (110.68 F 7 and 158 F 4.2 pg/Ag) was observed in LEC rats at 10 and 12 weeks of age as compared to those at 6 and 8 weeks of age (C). Data are represented as mean F SD. *P < 0.005, N = 4.

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Fig. 4. Hepatic caspase-3 activity in liver of LEC and LE rats. Significant increase (*P < 0.05) in hepatic caspase-3 activity was observed in LEC rats at 12 weeks of age as compared with LEC rats at 6, 8, and 10 weeks of age and basal levels of capsase-3 activity in LE rats (39.5 F 1.1 and 37 F 1.7 FU, 6 and 12 weeks of age, respectively) (N = 4). Data are represented as mean F SD. FU = fluorescent units.

Fig. 3. Immunohistochemical localization of TNF-a and TNFR1 in LEC rat liver. Light photomicrograph of positive immunostaining of TNF-a (A) and TNFR1 (B) was observed in hepatocytes and Kupffer cells (arrows) in livers from LEC rats at 12 weeks of age. DAB substrate was used as chromogen. 400 magnification.

group 4, 5, and 6 were 1 F 0.5, 1 F 0.3, and 2 F 0.2, respectively. LEC rats from group 1 and 2 showed similar percentage of apoptotic cells at 12 week of age (18 F 2 and 17 F 1, respectively). The treatment with 100 Ag/kg of antirat TNF-a produced a significant reduction ( P < 0.005) in percentage of apoptotic cells (5 F 1) in LEC rats (group 3) as compared with LEC rats from group 1 and 2 (Fig. 6A). The immunostaining for TNF-a and its receptor in liver sections of LEC rats (group 3) was weak (data not shown) and similar to LEC rats at 6 weeks of age as in the first experiment (Table 2). A significant reduction in the TNF-a gene expression in the liver tissues was observed in LEC

liver tissues of LEC rats treated with 100 Ag/kg of anti-rat TNF-a (group 3). In these rats, hepatocyte vacuolation, enlarged nuclei, and small focal inflammation in the parenchyma were observed in the liver. Furthermore, extra medullary hematopoietic areas were seen in the liver (Fig. 5). The percentage of hepatocyte apoptosis in LE rats from Table 2 Immunohistochemical grading for TNF-a and TNFR1 in liver tissue of LEC and LE rats at different ages Age (week)

6

Rat TNF-a TNFR1

LEC 1 1

LE 1 1

8

10

12

LEC 1 1

LEC 2 2

LEC 3 3

LE 1 1

Light microphotographs of liver tissue were taken at 200 magnification. Immunoreactivity with specific antibodies was developed by enzymatic reaction between peroxidase and diaminobenzidine. TNF-a and TNFR1 immunohistochemical grading: 1 = mild, 2 = moderate, and 3 = strong.

Fig. 5. Light photomicrograph of the liver from LEC rats treated with 100 Ag/kg of anti-rat TNF-a. Histopathological changes such as enlarged nuclei (arrowhead), vacuolation (open arrow), and disseminated foci of extramedullar haematopoiesis were observed in liver of LEC rats at 12 weeks of age treated with 100 Ag/kg of anti-rat TNF-a (arrow). H and E staining, 200 magnification.

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rats treated with 100 Ag/kg of anti-rat TNF-a (group 3) as compared with untreated LEC rats from group 1 and 2 at 12 weeks of age ( P < 0.05) (Fig. 6B). Furthermore, TNF-a

Fig. 7. Changes in hepatic caspase-3 activity in the livers of 12-week-old LEC rats treated with anti-rat TNF-a. Significant reduction in hepatic caspase-3 activity was observed in LEC rats from group 3 (100 Ag/kg antirat TNF-a) (*P < 0.05; N = 4) as compared with LEC rats from groups 1 and 2 (placebo and 50 Ag/kg anti-rat TNF-a) (N = 4). Fold increase in caspase-3 activity of LEC rats (groups 1, 2, and 3) was calculated form caspase-3 activity of LE rats groups 4, 5, and 6 (40.7 F 1.2, 40.6 F 1.9, and 46.11 F 0.9 FU, respectively). Data are presented as mean F SD. FU = fluorescent units.

concentrations in liver homogenates were also significantly reduced ( P < 0.005) in LEC rats treated with 100 Ag/kg of anti-rat TNF-a (group 3) (40 F 19 pg/Ag) (Fig. 6C). Hepatic caspase-3 activity was also reduced significantly ( P < 0.05) in this group of rats (Fig. 7).

Discussion

Fig. 6. Effect of neutralizing antibody to TNF-a on hepatocyte apoptosis, hepatic TNF-a level, and TNF-a protein concentration in LEC rats. Significant reduction on hepatocyte apoptosis, down-regulation of hepatic TNF-a mRNA level, and hepatic TNF-a protein was observed in LEC rats treated with 100 Ag/kg anti-rat TNF-a (group 3) (A – C) as compared with LEC rats group 1 (placebo) and group 2 (50 Ag/kg anti-rat TNF-a). The anti-rat TNF-a treatment in LE rats (group 4: placebo, group 5: 50 Ag/kg anti-rat TNF-a, and group 6: 100 Ag/kg anti-rat TNF-a) did not show any changes in hepatocyte apoptosis, hepatic TNF-a mRNA level, and hepatic TNF-a protein (A – C). Data are presented as mean F SD. *P < 0.05, N = 4.

The LEC rat develops spontaneous hepatitis and liver cancer because of abnormal Cu accumulation with age. These rats exhibit certain clinical features that are similar to those of Wilson’s disease, such as Cu accumulation in the liver, reduced excretion of Cu into bile, a reduced level of serum Cu, and a remarkable decrease in serum ceruloplasmin activity. The atp7b gene of the LEC rat that is homologous to human ATP7B has been found defective (Ono et al., 1994, 1995). The ATP7B protein is localized in the biliary canalicular membrane of the hepatocyte and is involved in both the intracellular transport and excretion of hepatic Cu. The absence or decrease of ATP7B function results in abnormal Cu metabolism in the LEC rat, and in patients with Wilson’s disease. These observations have confirmed that the LEC rat is a good rodent model for Wilson’s disease (Suzuki et al., 1995; Terada and Sugiyama, 1999; Yamaguchi et al., 1994). LEC rats between 11 weeks and 18 weeks of age accumulate 60 times more Cu than age-matched control LE rats (Suzuki et al., 1995). It has been reported that hepatic Cu levels increase after weanling in a linear fashion until 16 weeks of age, followed by hepatitis and a sudden decrease in Cu levels (Nomiyama et al., 1999; Suzuki et al., 1995). Present study showed that the highest hepatic Cu levels in LEC was at 12 weeks of age.

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Increased DNA damage, p53 accumulation, Fas upregulation, and up-regulation of the components of mitochondrial pathways of apoptosis have been suggested to be responsible for the apoptotic death of the hepatic cells (Klein et al., 2003). There are few reports on TNF-a and metal toxicity. Lead (Sieg and Billings, 1997) and uranium (Gazin et al., 2004) have been associated to induce TNF-a synthesis. However, there has been no report so far, on TNF-a-mediated apoptosis in Cu-induced toxicity in LEC rats, for which present study provides the first evidence. It is unclear whether the increased endogenous TNF-a-mediated signalling in the liver of LEC rat results from a direct effect of Cu accumulation or through induction of secondary mediator(s) such as free radicals and oxidative stress. However, the following observations suggest that TNF-a may be central to the pathogenesis of liver toxicity and probably of Wilson’s disease: (a) strong TNF-a expression in the liver of Fischer rats (data not shown) after feeding diet containing high Cu, (b) increased TNF-a expression in LEC rats with age (Table 2, Fig. 2B), (c) increased rate of hepatic apoptosis in LEC rats correlated with the TNF-a expression (Table 2, Figs. 2 and 3), and (d) significant reduction of apoptosis in livers of LEC rats as a result of anti-rat TNF-a therapy (Fig. 6). It is well known that TNF-a induces apoptosis in target cells that express TNF-a receptors on cell membrane and is followed by caspase cascade activation (Cohen, 1997). In this study, using RT-PCR and immunohistochemistry, we found that TNF-a mRNA is expressed in the liver, and that TNF-a protein is localized in hepatocytes and Kupffer cells. Our data demonstrate an increase in hepatic TNF-a concentration and caspase-3 activity in LEC rats with age as compared with control LE rats. Intracellular signalling is triggered by TNF-a with caspase cascade activation and apoptosis. Although different cellular pathways in Cu toxicosis can increase apoptosis, we propose that TNF-a can also mediate apoptosis at the onset of acute hepatitis in LEC rats. It has been suggested that autocrine mechanisms in hepatocytes may result in apoptosis, because hepatocytes themselves may acquire the capacity to synthesize cytokines such as IL-1 and TNF-a (Gonzalez-Amaro et al., 1994). The generation of oxyradicals in liver tissues of LEC rats is characterized by the accumulation of H2O2 that undergoes the Fenton-type reaction in presence of accumulated free Cu+ ions, and thus generating hydroxyl radical in the liver tissue of LEC rats (Yamamoto et al., 1999). The possible interaction of the generated reactive oxygen species (ROS) and TNF-a in liver tissue of LEC rats may explain, at least partially, the pathogenesis of Cu-induced hepatitis. In this study, we found high levels of hepatic TNF-a in LEC rats at 10 and 12 weeks of age. It has been shown that oxyradicals can activate TNF-a converting enzyme (TACE) which is responsible for cleavage of several important surface proteins, including TNF-a (Zhang et al., 2001). In this way, soluble TNF-a can act as a paracrine mediator on surrounding cells. Furthermore, the activation of TNFR1 by TNF-a

can initiate a group of internal signals, which include the caspase cascade and nuclear factor-nB (NF-nB) activation (May and Ghosh, 1998). NF-nB is a transcription factor that plays a pivotal role in the expression of a wide range of genes involved in chronic inflammatory diseases (Barnes and Karin, 1999; May and Ghosh, 1998). In addition, it has been shown that ROS can activate NF-nB and subsequently express of TNF-a (Ye et al., 1999). Finally, it has been shown that TNF-a can generate ROS (Woo et al., 2000). The interaction of ROS with NF-nB and TNF-a may explain the increase of hepatic levels of TNF-a in Cuinduced liver damage in LEC rats. In the present study, because changes in hepatic TNF-a gene expression can be detected as early as 6 weeks of age in LEC rats even before the appearance of any clinical signs of hepatitis, increased expression of TNF-a may be an early cellular response in Cu toxicity. After accumulation of Cu in the LEC rat liver at 6 weeks of age, oxygen free radicals can be generated, and this may induce the synthesis of TNF-a (Neihorster et al., 1992; Tiegs et al., 1989). Neutralization of TNF-a by passive immunization or the inhibition of caspases activity can block the apoptotic process in a murine endotoxin shock model (Jaeschke et al., 2000). In this study, we tested the passive immunization in LEC rats by weekly administration of anti-rat TNF-a antibody on Cu-induced acute hepatitis in LEC rats. To evaluate a possible role of TNF-a in the onset of hepatitis caused by high Cu levels in the liver, we measured the effects of TNF-a neutralization on apoptosis, caspase-3 activity, and TNF-a gene expression. The result showed a reduction of the percentage of apoptotic cells in the liver, decreased activity of caspase-3, down-regulation of the TNF-a gene expression, and lower hepatic TNF-a concentration. These results suggest that TNF-a can contribute to the development of acute hepatitis in Cu toxicosis, or TNFa-induced apoptosis could be an independent mechanism that has synergistic or additive effect with Cu toxicosis to produce hepatic damage in LEC rats. Apoptosis was detected in the present study, using both routine histology and in situ cell death detection TUNEL staining. High percentage of hepatic apoptotic cells coincided with the appearance of biochemical and clinical evidence of liver damage in LEC rats. There was a positive correlation of percentage of apoptotic cells with Cu accumulation and age in LEC rats. Morphological evidence of apoptosis has been described in experimentally induced Cuassociated disease in Wistar rats (Fuentealba and Haywood, 1988), and spontaneous Cu toxicosis in dogs (Haywood et al., 1996), toxic milk mice (Deng et al., 1998), and LEC rats (Bedard et al., 2000). In LEC rats, apoptotic bodies were detected mainly within macrophages (Yamate et al., 1999) similar to those in livers of rats with experimentally induced Cu toxicity (Fuentealba and Haywood, 1988). Yamate et al. (1999) reported that although macrophages might play an important role in liver failure in LEC rats, apoptosis occurred before the increase in numbers of macrophages.

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Results of the present study provide further evidence for localization of TNF-a in both hepatocytes and Kupffer cells before appearance of apoptotic cells in the liver of LEC rats. Studies on LEC rats have reported the expression of a particular group of genes on different stages of liver injury. For instance, the up-regulation of genes related to DNA damage, apoptosis, and inflammation has been shown during the development of hepatitis in LEC rats (Kasai et al., 1990; Yamate et al., 1999). Furthermore, it has been suggested that DNA damage is initiated much earlier than morphological damage due to up-regulation of genes encoding for DNA repair proteins, p53, and interleukin-1h enzyme (Klein et al., 2003). In summary, a potential role for TNF-a in Cu toxicity in LEC rats has been demonstrated in this study. TNF-a has been implicated as an important mediator of cytotoxicity in diverse types of liver diseases and experimental liver injury models (Morio et al., 2001). The expression of TNF-a in livers of LEC rats as early as 6 weeks of age suggest that TNF-a could play an important role in liver injury where apoptosis appears to be an early pathologic finding of Cu toxicity. Although the molecular mechanisms by which TNF-a induces hepatic damage in metal toxicity remains unknown, the formation of ROS may play an important role in TNF-a-induced hepatic Cu toxicity. Rapid rise of ROS levels following stimulation of cells with TNF-a and inhibition of TNF-a-induced cytotoxicity by antioxidants/ ROS scavengers, are in agreement with the above proposition (Manna and Aggarwal, 1998; Moreno-Manzano et al., 2000). Regardless of the mechanisms involved, our data illustrate the pivotal role of TNF-a in the development of hepatic toxicity in LEC rats, and also suggest that neutralizing TNF-a activity can be one of the treatment modalities for Cu-induced hepatotoxicity. Acknowledgments This study was supported by research grant from Canadian Institute of Health Research (CIHR). The authors thank Dr. Chandan Chakraborty (Department of Pathology, UWO) for his helpful suggestions in the preparation of the manuscript. References Baker, S.J., Reddy, E.P., 1998. Modulation of life and death by the TNF receptor superfamily. Oncogene 17, 3261 – 3270. Barnes, P.J., Karin, M., 1999. Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases. N. Engl. J. Med. 336, 1066 – 1071. Bedard, K., Fuentealba, I.C., Cribb, A., 2000. The Long Evans Cinnamon (LEC) rat develops hepatocellular damage in the absence of antimicrosomal antibodies. Toxicology 146, 101 – 109. Bergmeyer, H.U., Horder, M., Rej, R., 1986. International Federation of Clinical Chemistry (IFCC) Scientific Committee, Analytical Section: approved recommendation (1985) on IFCC methods for the measurement

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