Hepatitis In Long-Evans Cinnamon Rats Research Articles

Iron chelator deferasirox rescued mice from Fas‐induced fulminant hepatitis

Fulminant hepatitis is a disease characterized by development of hepatic failure due to severe liver cell injury. Orthotopic liver transplantation is the therapy proven to improve patient survival; however, less burdensome and safer strategies are required. In a previous study, we showed that iron was intimately involved in hepatocyte apoptosis by demonstrating that spontaneous development of fulminant hepatitis in Long-Evans cinnamon rats was prevented by feeding an iron-deficient diet. Recently, a new iron chelator, deferasirox, has become widely available for the treatment of transfusional hemosiderosis. Deferasirox demonstrated good efficacy and improved compliance due to convenient, once-daily p.o. administration. Our aim was to investigate the efficacy of deferasirox as a therapeutic drug against fulminant hepatitis. Human primary hepatocytes undergoing Fas-stimulated apoptosis were challenged with deferoxamine (DFO) in vitro. In further in vivo experiments, we tested DFO in a mice model of fulminant hepatitis induced by Fas-stimulation. The apoptosis-inducing activity of anti-Fas antibody on human primary hepatocytes was inhibited by the chelation of iron with DFO. DFO suppressed the Fas-induced production of reactive oxygen species (ROS) and the activation of caspase-3, both of which were also suppressed by antioxidant, N-acetyl-L-cystein. In the in vivo experiments, deferasirox effectively reduced hepatic iron concentrations and rescued mice from Fas-induced fulminant hepatitis. These findings indicated that the iron chelation exerted a hepatoprotective effect by scavenging ROS upstream of caspase-3 and that iron chelation with deferasirox is a potential treatment for patients with fulminant hepatitis.

Mild zinc deficiency and dietary phytic acid accelerates the development of fulminant hepatitis in LEC rats

Restriction of copper intake delays hepatic copper accumulation in Long-Evans Cinnamon (LEC) rats, which are animal models of Wilson's disease. Involvement of zinc is suggested to develop hepatitis in the disease; however, this has not been clarified. The aims of this study were to investigate the effects of mild zinc deficiency on the development of hepatitis and to determine the relationship between the absorption and hepatic levels of copper, zinc and iron. Male LEC and F344 (wild type atp7b) rats were fed a low zinc, phytate-containing or control diet. The onset of hepatitis (Experiment 1), and absorptive rates of copper, zinc and iron and hepatitis indices in 4 weeks (Experiment 2) were observed. The onset of fulminant hepatitis in LEC rats was much earlier in the low zinc and phytate groups (mean 94.6 +/- 2.74 days and 82.8 +/- 3.56 days old, respectively) than in the control group (136 +/- 2.11 days old) with worse hepatitis indices. Hepatic copper levels were much higher in LEC rats than F344 rats, but were not largely different among the diet groups without prominent changes in copper absorption. Hepatic levels and intestinal absorption of zinc and iron were lower in the phytate group than in the control group. Mild zinc deficiencies caused by a low zinc or phytate-containing diet accelerate the onset of hepatitis in LEC rats without increasing copper absorption, and zinc and iron metabolism may be involved in the earlier onset of jaundice of LEC rats.

Inhibitory effects of fermented brown rice and rice bran on the development of acute hepatitis in Long-Evans Cinnamon rats

To investigate the effects of fermented brown rice (FBRA) on the development of hereditary hepatitis in Long-Evans Cinnamon (LEC) rats, we compared the incidence and grades of acute hepatitis among rats fed 5% and 10% FBRA in the conventional diet and the conventional diet alone. Both the 5% and 10% FBRA-supplemented diets indicated a tendency to prevent the development of hepatitis, and the significant effect of 10% FBRA was observed until 16-17 weeks of age in the accumulated incidence and survival ratio compared with the unsupplemented conventional diet, although no significant difference was observed between 5% and 10% FBRA-supplemented diets. At the age of 12 weeks, which is just before the rats develop hepatitis, serum copper levels in rats fed either of the test diets were similar to those in rats fed the conventional diet. Furthermore, the copper concentration in liver tissue at 12 weeks of age was not changed by the test diet. These results suggest that FBRA has preventive effects on the development of hepatitis in LEC rats and may play an important role in protecting the liver against the free radicals induced by copper accumulation in the liver.

Prevention of lethal hepatic injury in Long-Evans Cinnamon (LEC) rats by D-galactosamine hydrochloride

Repeated injections of D-galactosamine hydrochloride (GalN) increase the survival rate of Long-Evans Cinnamon (LEC) rats, an animal model of Wilson's disease. The aim of the present study was to investigate the mechanism of GalN for prevention of spontaneous lethal hepatic injury in LEC rats. Male LEC rats were given a single subcutaneous injection of 300 mg/kg of GalN or vehicle (0.9% NaCl) at 14 weeks, and killed at 28 weeks of age. Next, 6-week-old male LEC rats were given weekly subcutaneous injections of 300 mg/kg of GalN or vehicle for 3 or 12 weeks, and their hepatic 8-hydroxydeoxy-2'-guanosine (8-OHdG), glutathione peroxidase (GPX), and catalase activities were measured. None of GalN-treated rats died of hepatic injury (0/12), whereas the mortality rate of control rats given 0.9% NaCl was 17% (2/12). GalN administration for 12 weeks decreased the hepatic 8-OHdG, and GalN administration for either 3 or 12 weeks increased the glutathione peroxidase activity. GalN administration increased the serum level of alanine aminotransferase, and accelerated megalocytic degeneration of the hepatocytes. GalN treatment is effective in preventing lethal hepatitis in LEC rats and decrease of oxidative DNA damage by GalN plays an important role in increase of the survival rate.

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

The objective of this study was to evaluate the potential role of TNF-α 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-α and TNFR1 immunostainings were observed in hepatocytes and Kupffer cells in LEC rats. Hepatic levels of caspase-3 activity, TNF-α 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-α by passive immunization or the inhibition of caspase activity can block the apoptotic process initiated by TNF-α. In this study, we evaluated the effects of passive immunization of LEC rats with weekly administration of anti-rat TNF-α 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-α gene expression. Thus, these results suggest a major role for TNF-α on the pathogenesis of Cu-induced acute hepatitis in LEC rats.

Tetrathiomolybdate in the treatment of acute hepatitis in an animal model for Wilson disease

Background/Aims Tetrathiomolybdate (TTM) is a potent copper-chelating agent that has been shown to be effective in Wilson disease patients with neurological symptoms. Here, we investigate the potential use of TTM in treating the acute hepatic copper toxicosis in Long-Evans Cinnamon (LEC) rats, an authentic model for Wilson disease. Methods After the onset of acute hepatitis, LEC rats were treated once with 10 mg TTM/kg. After 1 and 4 days, parameters of liver toxicity and the subcellular distribution and binding of copper and iron were studied. Results In 11 out of 12 rats TTM rapidly improved acute hepatitis. Hepatic copper decreased through removal from cytosolic metallothionein and lysosomal metallothionein polymers. The remaining lysosomal copper forms a metallothionein–copper–TTM complex. In an almost moribund rat, however, TTM caused severe hepatotoxicity with fatal outcome. Conclusions TTM is effective in treating acute hepatitis in LEC rats when applied before the animals become moribund. TTM appears to act by removing the presumable reactive copper associated to lysosomal metallothionein polymers. The remaining lysosomal copper seems to be inactivated by forming a complex with TTM. Moreover, TTM removes copper from cytosolic copper-containing metallothionein. As a consequence, metallothionein is degraded and the uptake of copper–metallothionein into the lysosomes and the formation of the metallothionein polymer associated copper is reduced.

DNA damage triggers imbalance of proliferation and apoptosis during development of preneoplastic foci in the liver of Long-Evans Cinnamon rats

The mutant strain Long-Evans Cinnamon (LEC) rat accumulates copper, resulting in spontaneous hepatitis and subsequent development of hepatocellular carcinomas (HCCs) in the liver, providing a promising model for investigation of the relationship between hepatitis induced by oxidative stress and hepatocarcinogenesis. We examined DNA strand breaks in peripheral blood cells and p53 expression in livers during acute and chronic hepatitis in LEC rats, along with preneoplastic lesions, and cell proliferation and apoptosis in non-cancerous portions of livers from LEC rats aged 7-115 weeks. Immunohistochemistry using antibodies against glutathione S-transferase placental-form (GST-P), proliferating cell nuclear antigen (PCNA), and in situ DNA nick labeling (TUNEL) were used. Long-Evans Agouti (LEA) rats, a sibling line of the LEC strain, were used as controls. In the LEC rats, DNA strand breaks and expression of p53 were significantly higher than that of LEA rats at 24 weeks of age. The number of GST-P-positive (GST-P+) foci/cm2 increased and peaked at 48 weeks old, and the areas rapidly expanded thereafter. The level of cell proliferation increased with the development of hepatitis and was highest at about 48 weeks old. The induction of apoptosis in LEC rats was transiently higher than that in LEA rats during the period from 24 to 34 weeks of age. However, the ratio of PCNA-positive cells to the apoptotic index showed a growth imbalance in favor of cell proliferation, supporting sustained net growth in LEC rats. These findings suggest that DNA damage, reflected in DNA strand breaks, plays a critical role in the development of hepatocellular preneoplastic foci, with an imbalance between high proliferation and relatively low apoptosis.

In vivo evidence for accelerated generation of hydroxyl radicals in liver of Long-Evans Cinnamon (LEC) rats with acute hepatitis

The Long-Evans Cinnamon (LEC) rats accumulate excess copper (Cu) in the liver in a manner similar to patients with Wilson’s disease (WD) and spontaneously develop acute hepatitis with severe jaundice. Although hydroxyl radicals ( OH) have been proposed to be a cause of hepatitis by the accumulation of Cu, it is not clear whether or not OH can be produced in the liver of hepatitic LEC rats in vivo and also can be involved in the onset of hepatitis. In the present study, OH production in plasma and liver of hepatitic LEC rats was quantified by trapping OH with salicylic acid (SA) as 2, 3-dihydroxybenzoic acid (2, 3-DHBA). The ratios of 2, 3-DHBA/SA were significantly higher in plasma and liver of hepatitic LEC rats than those of Wistar rats and LEC rats showing no signs of hepatitis. Furthermore, the ratios of 2, 3-DHBA/SA in plasma and liver of hepatitic LEC rats were almost the same as those of Wistar rats treated orally with CuSO 4 (0.5 mmol/kg) 2 h before acetylsalicylic acid (ASA) injection. We also evaluated the protective effects of D-mannitol (a OH scavenger) treatment against acute hepatitis in LEC rats. D-mannitol (500 mg/kg) was administered intraperitoneally to 10-week-old LEC rats for 3 weeks. D-mannitol treatment suppressed the increases in serum aspartate aminotransferase activity and total bilirubin concentration. In addition, D-mannitol treatment significantly reduced hepatic mitochondrial lipid peroxidation, which is thought to be important in the pathogenesis of Cu-induced hepatotoxicity. These observations suggest that accelerated generation of OH catalyzed by free Cu in the liver may, at least in part, play a role in the pathogenesis of acute hepatitis in LEC rats.

The antioxidant effect of DL-α-lipoic acid on copper-induced acute hepatitis in Long-Evans Cinnamon (LEC) rats

The Long-Evans Cinnamon (LEC) rats, due to a genetic defect, accumulate excess copper (Cu) in the liver in a manner similar to patients with Wilson's disease and spontaneously develop acute hepatitis with severe jaundice. In this study we examined the protective effect of DL-α-Lipoic acid (LA) against acute hepatitis in LEC rats. LA was administered to LEC rats by gavage in doses of 10, 30 and 100 mg/kg five times per week, starting at 8-weeks-old and continuing till 12-weeks-old. Although LA had little effect against the increases in serum transaminase activities, it suppressed the loss of body weight and prevented severe jaundice in a dose-dependent manner. Antioxidant system analyses in liver showed that LA treatment significantly suppressed the inactivations of catalase and glutathione peroxidase, and the induction of heme oxygenase-1, an enzyme which is inducible under oxidative stress. Furthermore, LA showed dose-dependent suppressive effect against increase in nonheme iron contents of both cytosolic and crude mitochondrial fractions in a dose-dependent manner. Although at the highest dose, LA slightly suppressed the accumulation of Cu in crude mitochondrial fraction, it had no effect on the accumulation of Cu in cytosolic fraction. While LA completely suppressed the increase in lipid peroxidation (LPO) in the microsomal fraction at the highest dose, the suppressive effect against LPO in crude mitochondrial fractions was slight. From these results, it is concluded that LA has antioxidant effects at the molecular level against the development of Cu-induced hepatitis in LEC rats. Moreover, mitochondrial oxidative damage might be involved in the development of acute hepatitis in LEC rats.

Dissolution of copper-rich granules in hepatic lysosomes by D-penicillamine prevents the development of fulminant hepatitis in Long-Evans cinnamon rats

Background/Aim: The Long-Evans cinnamon rat has a mutation homologous to the human Wilson disease gene, leading to gross copper accumulation and the development of hepatitis. D-penicillamine, a copper-chelating drug widely and efficiently used in treating Wilson disease, has also been shown to prevent hepatitis in Long-Evans cinnamon rats. The objectives of this study were: i) to investigate the effectiveness of D-penicillamine when administered to the already affected animals, and ii) to elucidate the mechanism of action of the drug. Methods: Long-Evans cinnamon rats were divided into groups according to age and treatment with D-penicillamine. The drug was administered orally before and after the onset of hepatitis. Livers were examined by light and electron microscopy. The effect of D-penicillamine on the subcellular distribution and binding of copper was investigated in more detail. Finally, the interaction between D-penicillamine and specific hepatic copper-binding proteins was studied in vitro. Results: D-penicillamine when given to either healthy or diseased animals prevented or reversed hepatitis, respectively. The drug particularly inhibited the disease-specific accumulation of copper in lysosomes of hepatocytes, tissue macrophages and Kupffer cells. When administered to diseased animals, the drug sequestered copper particularly from insoluble lysosomal particles. According to results obtained in vitro, the mobilization of this copper is likely to proceed through the solubilization of these particles. In contrast and as supported by the in vitro data, D-penicillamine had only a minor effect on copper bound to metallothionein in the cytosol. Conclusion: Our findings on the Long-Evans cinnamon rat provide some conclusions on the mechanism of action of D-penicillamine in Wilson disease therapy. The drug prevents the formation or promotes the solubilization of copper-rich particles which occur in lysosomes of hepatocytes and Kupffer cells in the livers of patients with Wilson disease. Once chelated with D-penicillamine copper might then be excreted into urine. However, the mobilization of copper by D-penicillamine seems to be limited due to the binding of the metal to metallothionein in liver cytosol. This copper, even at relatively high concentrations, apparently may be well tolerated.

Occurrence of autoimmune antibodies to liver microsomal proteins associated with lethal hepatitis in LEC rats: effects of TJN-101 ((+)-(6S,7S, R-biar)-5,6,7,8-tetrahydro-1,2,3,12-tetramethoxy-6,7-dimethyl-10,11-methylenedioxy-6-dibenzo[ a,c]cyclooctenol) on the development of hepatitis and the autoantibodies

Long Evans Cinnamon (LEC) rats, that spontaneously develop hepatitis, were found to possess autoantibodies to liver microsomal proteins (anti-LM) before the development of hepatitis. Anti-LM antibody was assumed to appear in association with the lethal hepatitis in the LEC rats. Thus, the purpose of this study was to investigate the effects of an anti-hepatitis drug on the development of hepatitis and the occurrence of the antibody in LEC rats. Mortality, blood biochemical parameters and the titer of serum anti-LM antibody were measured. In control LEC rats, 4 of 8 rats died before 20 weeks of age. In rats treated with TJN-101 ((+)-(6S,7S, R-biar)-5,6,7,8-tetrahydro-1,2,3,12-tetramethoxy-6,7-dimethyl-10,11-methylenedioxy-6-dibenzo[ a,c]cyclooctenol), 4 of 7 rats died of hepatitis, but the time of death was delayed by 7–10 weeks compared for the control rats. The titer of the anti-LM antibody increased 3–7 weeks before death in the non-survivors in control and TJN-101-treated rats, supporting the idea that anti-LM antibody occurs in association with acute lethal hepatitis.

A copper deficient diet prevents hepatic copper accumulation and dysfunction in Long-Evans Cinnamon (LEC) rats with an abnormal copper metabolism and hereditary hepatitis.

Long-Evans Cinnamon (LEC) rats that develop spontaneous hepatitis due to an inherently abnormal Cu metabolism have recently been established. This investigation concerns the effects of a Cu-deficient diet on the Cu metabolism linked to hepatic injury in LEC rats. The hepatic Cu concentration at 30 days after birth was 94 +/- 4 Cu micrograms/g liver in LEC rats, whereas that of Fischer rats at the same age was 7 +/- 1 Cu micrograms/g. From 30 days after birth, all rats were fed a semisynthetic diet with two different levels of Cu, 0.5 or 30 micrograms/g food, for 35 days. In LEC rats fed a Cu-deficient diet (0.5 microgram/g), the hepatic Cu concentration was 39 +/- 7 micrograms/g. The Cu-normal diet (30 micrograms/g) LEC group had a concentration of 357 +/- 15 micrograms/g in the hepatic Cu. The group had significantly higher aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT) and gamma-glutamyl transferase (GGT) levels than did the LEC rats given the Cu-deficient diet. These results suggest that the occurrence of acute hepatitis in LEC rats can be prevented by feeding the animals a Cu-deficient diet.

Unusual Generation of Hydroxyl Radicals in Hepatic Copper-Metallothionein of LEC (Long-Evans Cinnamon) Rats in the Presence of Hydrogen Peroxide

A mutant strain of LEC rats (Long-Evans rats with a cinnamon-like coat color) develop spontaneous hepatic injury associated with severe jaundice about 4 months after birth. Recently, we obtained evidence which shows an unusual accumulation of copper (Cu) in the liver of LEC rats, followed by the finding of copper-methallothionein (Cu-MT) induction. To know the mechanism for the development of hepatitis in LEC rats, in relation to induced Cu-MT, we examined whether the generation of active oxygen species is observed. When the Cu-MT was treated with H 2O 2, which is formed by dismutation of superoxide anion radicals or NADPH oxidases in living systems, strong ESR signals due to Cu(II) state appeared when measured at 77K. On the same system, ESR signals due to the spin trapped hydroxyl radicals were observed at room temperature when DMPO (5,5-dimethyl-pyrroline-1-oxide) was used as a spin-trapping agent. The present results suggested that Cu-MT of LEC rat has an important pathogenic role by generating hydroxyl radicals, when hydrogen peroxide is produced in cells or tissues.

Prevention of spontaneous hepatocellular carcinoma in long-evans cinnamon rats with hereditary hepatitis by th administration of d-penicillamine

Acute hepatitis spontaneously develops in the Long-Evans Cinnamon rat at the age of 4 mo, and eventually hepatocellular carcinoma develops after the chronic hepatitis that persists for over a year. Previously, abnormal copper accumulation was found in the livers of Long-Evans Cinnamon rats from birth, and it was reported that short-term administration of D-penicillamine, a copper-chelating agent, prevented acute hepatitis in Long-Evans Cinnamon rats. In this study we investigated whether long-term administration of D-penicillamine could also prevent chronic hepatitis and subsequent hepatocellular carcinoma in Long-Evans Cinnamon rats. During long-term observation, which was continued from 11 to 70 wk after birth, no elevation of serum transaminase levels was observed in the Long-Evans Cinnamon rats treated with D-penicillamine. Moreover, no histological changes characteristic of the chronic hepatitis were observed in D-penicillamine-treated Long-Evans Cinnamon rats, which were killed at 70 wk of age. Furthermore, placental glutathione S-transferase-positive foci, described as a marker for preneoplastic lesions in the liver, were not detected, and thus hepatocarcinogenesis was completely prevented in D-penicillamine-treated Long-Evans Cinnamon rats. We also found that the amount of 8-hydroxy-deoxyguanosine, one of oxidative DNA damage products in the liver, was decreased in the Long-Evans Cinnamon rats treated with D-penicillamine. These findings suggest that a process of the prolonged liver-cell injury and regeneration was essential for spontaneous development of hepatocellular carcinoma in Long-Evans Cinnamon rats with abnormal copper metabolism.

Copper-Metallothionein Distribution in the Liver of Long-Evans Cinnamon Rats: Studies on Immunohistochemical Staining, Metal Determination, Gel Filtration and Electron Spin Resonance Spectroscopy

Distribution of methallothionein (MT) and copper ion (Cu) in the liver of LEC (Long-Evans Cinnamon) rats was investigated to examine the relationship between Cu-MT induction and the development of hepatitis followed by hepatocellular carcinomas. Immunohistochemical studies on MT in the liver of LEC rats indicated that MT is accumulated in nuclei and cytosols. Both MT and Cu, estimated by radioimmunoassy and flameless atomic absorption spectrometry, respectively, in subcellular fractions of the liver were found to be concentrated highest in cytosols, followed by nuclei, mitochondria and microsomal fractions. Gel-filtration (Sephadex G-75) studies demonstrated that MT is induced as the Cu-MT form. Furthermore, the Cu-MT fragment purified by the gel-filtration contains the Cu(I)-MT form, as demonstrated by ESR (electron spin resonance) measurements at 77K. These results will be important for understanding the development of hepatitis in LEC rats.

D-penicillamine prevents the development of hepatitis in Long-Evans Cinnamon rats with abnormal copper metabolism.

The Long-Evans Cinnamon rat is a mutant strain that contracts hereditary hepatitis and, eventually, spontaneous hepatocellular carcinoma. Because we found a corresponding gross copper accumulation in the liver of the rats, we examined whether the development of hepatitis in our rat system could be prevented by administration of D-penicillamine. D-Penicillamine is a copper-chelating agent and one of the drugs effective for human Wilson's disease, in which abnormal copper metabolism is also observed. The results show that D-penicillamine treatment inhibited the elevation of serum transaminases, suppressed abnormal histological changes in the liver and completely prevented the onset of hepatitis in the Long-Evans Cinnamon rats. We further found that the copper concentration in the liver and serum copper and ceruloplasmin levels were decreased, whereas the urinary copper level was increased in the D-penicillamine-treated Long-Evans Cinnamon rats. These findings demonstrate that the pathogenesis of hereditary hepatitis in Long-Evans Cinnamon rats is due to abnormal copper accumulation in the liver.

Spontaneous hepatic copper accumulation in Long-Evans Cinnamon rats with hereditary hepatitis. A model of Wilson's disease.

Long-Evans Cinnamon (LEC) rats, an inbred strain of a mutant rat isolated from Long-Evans rats, develop hereditary hepatitis. To elucidate the role of copper metabolism in the development of the hepatitis in LEC rats, we examined the copper concentration in the tissues and serum levels of copper and ceruloplasmin. Copper concentration in the liver of LEC rats was over 40 times that of normal Long-Evans Agouti (LEA) rats, while the serum ceruloplasmin and copper concentrations in LEC rats decreased significantly. The hepatocytes of LEC rats show steatosis in cytoplasm and pleomorphism of mitochondria, resembling the histologic features of the liver in Wilson's disease. These findings suggest that the hereditary hepatitis in LEC rats is closely associated with copper toxicity, and may be dealing with a rat form of Wilson's disease. Thus the LEC rats will provide a unique and useful animal model for clarifying the mechanism and for developing treatment strategies for Wilson's disease and other abnormal copper metabolism in humans.