metallothionein and Hepatitis--Animal

Chronic cholangiohepatitis (CCH) is a common pathological condition in cats with a guarded prognosis and unknown etiology. Recently, in human medicine, there has been increased interest in enhancing liver defense mechanisms as an effective treatment strategy to control liver diseases that have a poor prognosis. Metallothionein (MT) is a ubiquitous protein, which has been widely researched for its role in liver defense through heavy metal detoxification, neutralization of reactive oxygen species, and liver regeneration. In this study, immunohistochemistry was used to evaluate the role of MT in CCH and hepatocellular regeneration in 34 cats histologically diagnosed with this condition by assessing the correlation between hepatocellular MT and Ki-67 (marker for cellular proliferation) expression with histological parameters of CCH, such as inflammation, fibrosis, and bile duct proliferation. Statistical analysis was performed using the Spearman-rank correlation test. A significant positive correlation was observed between inflammation and the number of MT-positive hepatocytes (. La cholangiohépatite chronique (CCH) est une affection pathologique courante chez les chats avec un pronostic réservé et une étiologie inconnue. Récemment, en médecine humaine, il y a eu un intérêt accru pour l’amélioration des mécanismes de défense hépatique en tant que stratégie de traitement efficace pour contrôler les maladies du foie qui ont un mauvais pronostic. La métallothionéine (MT) est une protéine omniprésente, qui a été largement étudiée pour son rôle dans la défense du foie par la détoxification des métaux lourds, la neutralisation des espèces réactives de l’oxygène et la régénération du foie. Dans cette étude, l’immunohistochimie a été utilisée pour évaluer le rôle de la MT dans la CCH et la régénération hépatocellulaire chez 34 chats diagnostiqués histologiquement avec cette condition en évaluant la corrélation entre l’expression hépatocellulaire de la MT et du Ki-67 (marqueur de la prolifération cellulaire) avec les paramètres histologiques de la CCH, comme l’inflammation, la fibrose et la prolifération des voies biliaires. L’analyse statistique a été réalisée à l’aide du test de corrélation de rang de Spearman. Une corrélation positive significative a été observée entre l’inflammation et le nombre d’hépatocytes MT-positifs (

Topics: Animals; Biliary Tract Diseases; Cat Diseases; Cats; Chronic Disease; Female; Hepatitis, Animal; Ki-67 Antigen; Male; Metallothionein

Long-Evans Cinnamon (LEC) rats are characterized by an abnormal hepatic deposition of copper (Cu) due to a lack of the Cu-transporter P-type adenosine triphosphatase: accordingly, the strain is a good animal model of Wilson's disease. The effect of oral zinc (Zn) acetate treatment on the development of acute hepatitis and the biochemical parameters of Cu-induced liver damage was studied in 5-week-old LEC rats (n=52).. Rats receiving 50 or 80 mg/ml/day Zn acetate by gavage and control rats receiving a daily dose of glucose solution 0.02 g/ml by gastric intubation were killed at 1, 2 or 8 weeks after the start of treatment.. Treatment with Zn acetate resulted in the prevention of acute hepatitis: 10 of the 13 untreated rats developed signs and symptoms compatible with acute hepatitis between the 6th and 7th week of treatment. Tissue metallothionein (MT) significantly increased in the treated rats and positively correlated with Zn concentrations within the liver. Control rats had a significantly higher iron concentration in the liver and kidneys compared with supplemented rats, after both short- and long-term experiments. 8-hydroxy-2'-deoxyguanosine amounts were significantly lower in untreated rats.. Zn acetate prevents acute hepatitis, by increasing tissue MT concentrations, reducing Cu absorption and interfering with Fe metabolism.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Administration, Oral; Animals; Chemical and Drug Induced Liver Injury; Deoxyguanosine; Disease Models, Animal; Dose-Response Relationship, Drug; Hepatitis, Animal; Iron; Kidney; Liver; Male; Metallothionein; Protective Agents; Rats; Rats, Inbred LEC; Zinc; Zinc Acetate

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.. 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.. 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.. 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.

Topics: Animals; Chelating Agents; Copper; Cytoplasmic Granules; Cytosol; Hepatitis, Animal; Liver; Lysosomes; Metallothionein; Microscopy, Electron; Penicillamine; Rats; Rats, Long-Evans; Rats, Wistar

To study effects of dietary Cu and Fe levels on the onset of hepatitis in Long-Evans Cinnamon (LEC) rats, female rats (40 days old) were fed a semipurified diet containing 0.1 or 10 mg Cu/kg and 1.5 or 150 mg Fe/kg in a 2 x 2 factorial arrangement for 35 days. At 75 days after birth, LEC rats (+Cu-Fe) fed a Cu-sufficient but Fe-deficient diet (Cu, 10 mg/kg; Fe, 1.5 mg/kg) showed jaundice, with lethargy, anorexia, and malaise. The biochemical variables relating to liver function were significantly increased compared to three other groups, a Cu- and Fe-deficient (-Cu-Fe) group, a Cu-deficient but Fe-sufficient (-Cu+Fe) group, and a Cu and Fe sufficient (+Cu+Fe) group. Furthermore, the +Cu-Fe rat liver showed massive necrosis with huge nuclei. The other three groups presented no biochemical and histological findings of hepatitis. Hepatic Cu and metallothionein concentrations were 289 +/- 87 (mean +/- SD) microg/g liver and 8.7 +/- 1.8 mg/g liver, respectively, in the +Cu-Fe rats. However, in the +Cu+Fe group the values were 196 +/- 28 microg Cu/g liver and 10.8 +/- 1.0 mg/g liver. Hepatic Fe deposition was not influenced significantly by the dietary Cu level. The +Cu-Fe group with jaundice showed the highest free Cu concentration in the liver among the four groups, but the hepatic free Fe concentration was similar to those in the -Cu+Fe and +Cu+Fe groups. Our results indicate that an Fe-deficient diet enhances the deposition of hepatic Cu due to increased absorption of Cu from the gastrointestinal tract. This deposition stimulated the onset of hepatitis.

Topics: Animals; Body Weight; Copper; Female; Ferritins; Hepatitis, Animal; Hepatolenticular Degeneration; Intestinal Absorption; Iron; Iron Deficiencies; Iron, Dietary; Liver; Metallothionein; Rats; Rats, Long-Evans; Specific Pathogen-Free Organisms

The Long-Evans cinnamon (LEC) rat has a mutation homologous to the human Wilson's disease gene, leading to copper-induced hepatotoxicity. The mechanism of how excess copper damages the liver or what chemical form of copper is toxic is still unclear.. In liver cytosol, copper levels were highest just before the onset of hepatitis and declined thereafter. In cytosol, total copper was bound to metallothionein (MT). Considerable amounts of both copper and iron accumulated in lysosomes with increasing age and development of liver damage. Lysosomal levels of presumably reactive non-MT-bound copper were increased. In severely affected livers, large amounts of copper were associated with insoluble material of high density which, upon ultrastructural information, was found to be derived from the lysosomes of Kupffer cells. This copper-rich material is considered to consist of polymeric degradation products of copper-MT.. We suggest that chronic copper toxicity in LEC rats involves the uptake of copper-loaded MT into lysosomes, where it is incompletely degraded and polymerizes to an insoluble material containing reactive copper. This copper, together with iron, initiates lysosomal lipid peroxidation, leading to hepatocyte necrosis. Subsequent to phagocytosis by Kupffer cells, the reactive copper may amplify liver damage either directly or through stimulation of these cells.

Topics: Animals; Copper; Cytosol; Female; Hepatitis, Animal; Hepatolenticular Degeneration; Humans; Kupffer Cells; Liver; Lysosomes; Male; Metallothionein; Mitochondria, Liver; Rats; Rats, Mutant Strains; Rats, Wistar

Long-Evans Cinnamon (LEC) rats are characterized by the sudden onset of hepatitis around 4 months after birth and the gross accumulation of hepatic copper (Cu) accompanied by metallothionein (MT). The biliary excretion of manganese (Mn) and cadmium (Cd) injected intravenously was studied in 3-month-old LEC rats without signs of hepatitis. Injected Mn was excreted into the bile in LEC and Fischer rats used for comparison. However, increased biliary excretion of Cd was found not in the LEC rat but in the Fischer rat. Excretion of horseradish peroxidase (HRP) injected along with the metal mixture was significantly lower in the LEC group than in the Fischer group. Our results suggest that Mn excretion is not related to the existence of a gross amount of Cu-MT. Reduced excretion of Cd may be partly due to binding to Cu-MT in the liver. Decreased excretion of HRP implies the existence of an inherent defect in the bile excretion route for endo- and exogenous substances.

Topics: Animals; Bile; Cadmium; Copper; Hepatitis, Animal; Horseradish Peroxidase; Injections, Intravenous; Manganese; Metallothionein; Rats; Rats, Inbred F344; Species Specificity; Specific Pathogen-Free Organisms

We determined the copper (Cu) and metallothionein (MT) concentrations in the liver and kidney supernatants of Long-Evans rats with a cinnamon-like coat color (LEC rats), and also measured the Cu and MT levels in the serum of these rats. Seven-week-old rats had abnormally high levels of both substances in the liver. The levels in the liver supernatant were over 80- and 16-fold higher, respectively, in LEC rats than in normal 7-week-old Wistar rats. LEC rats suffering from acute hepatitis or hepatoma had a much higher level of hepatic MT, but the Cu level was higher only in the liver of those with hepatoma. The serum levels of Cu and MT in LEC rats with acute hepatitis were more than 10-fold higher than those in normal LEC rats. These levels were decreased in the rats with chronic hepatitis or hepatoma. In the liver of LEC rats with hepatoma, the area of hepatocellular carcinoma and of noncancerous liver showed over twice higher Cu and MT levels than the area of cholangiofibrosis. The Sephadex G-75 elution profile from the liver supernatant of a normal LEC rat showed that the peak of Cu closely corresponded to that of MT recognized with anti-MT antiserum. The levels of Cu and MT in the kidney supernatant of LEC rats with acute hepatitis were more than 25-fold higher than in that of normal LEC rats. However, there were no marked increases in the levels in the kidney supernatant of LEC rats with chronic hepatitis or hepatoma.

Topics: Animals; Carcinoma, Hepatocellular; Chromatography, Ion Exchange; Copper; Female; Hepatitis, Animal; Kidney; Liver; Liver Function Tests; Liver Neoplasms; Male; Metallothionein; Radioimmunoassay; Rats; Rats, Inbred Strains; Rats, Wistar; Spectrophotometry, Atomic; Zinc

Tetrathiomolybdate (TTM) was injected at a dose of 10 mg/kg bw daily for eight consecutive days into Long-Evans Cinnamon (LEC) rats, which inherently abnormally deposit Cu (260 micrograms/g) in the liver. The hepatic Cu (100 micrograms/g) and metallothionein (MT) bound Cu (from 2,600 to 540 micrograms/g protein) concentrations were decreased greatly by the injection. On the other hand, the renal Cu concentration increased significantly, but the brain Cu concentration only very slightly. The reduction of the hepatic Cu concentration was accompanied by reductions of Zn and Fe concentrations in the liver, kidney and brain. The TTM compound slightly stimulated excretion (about 3-fold) of Cu into the bile, but greatly (about 40-fold) into the blood. In rats not treated with TTM, most biliary (100%) and serum (78%) Cu was recovered in the trichloroacetic acid (TCA) soluble fraction. On the other hand, in rats treated with TTM, bile and serum Cu were recovered overwhelmingly in the TCA insoluble fraction, probably in the form of a Cu-TTM-albumin complex. Our results suggest that although there is an inherent failure in the intrinsic secretory process of Cu from the liver in LEC rats, the TTM compound can remove Cu from Cu-MT, resulting in a decrease of hepatic Cu.

Topics: Animals; Bile; Brain; Chelating Agents; Copper; Female; Hepatitis, Animal; Iron; Kidney; Metallothionein; Molybdenum; Rats; Serum Albumin; Zinc

Liver slices from Wistar and Long-Evans Cinnamon (LEC) rats were incubated while open to the atmosphere to assess the liver function in LEC rats. Leakages of glutamic-oxaloacetic transaminase (GOT) and lactic dehydrogenase (LDH) into the medium were significantly lower in the LEC rat than in the Wistar rat. Furthermore, no pronounced enhancement of the concentration of thiobarbituric acid-reactive substances (TBARS) was found in the LEC rat. Hepatic Cu and Cu-metallothionein (Cu-MT) concentrations were 355.0 +/- 18.7 micrograms/g liver and 2559 +/- 181 micrograms/g protein in the LEC rats, whereas Wistar rats showed 4.1 +/- 0.1 Cu microgram/g liver accompanied by 16 +/- 4 micrograms/g protein of MT. The decrease of intrahepatic Cu-MT in LEC rats was stimulated by incubation with Fenitrilotriacetate (Fe-NTA). There was a direct correlation between the enhancement of TBARS and disappearance of Cu-MT. Our results suggest that hepatic Cu-MT in LEC rats protects against liver injury stimulated by oxidative stress.

Topics: Animals; Aspartate Aminotransferases; Carcinogens; Copper; Ferric Compounds; Hepatitis, Animal; L-Lactate Dehydrogenase; Liver; Male; Metallothionein; Mutation; Nitrilotriacetic Acid; Rats; Rats, Wistar; Thiobarbituric Acid Reactive Substances

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-metallothionein (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 H2O2, 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.

Topics: Animals; Copper; Electron Spin Resonance Spectroscopy; Free Radicals; Hepatitis, Animal; Hydrogen Peroxide; Hydroxides; Liver; Male; Metallothionein; Rats; Rats, Mutant Strains; Rats, Wistar

The concentration of biliary Cu was 0.12 +/- 0.01 microgram/ml in male LEC rats aged 14 weeks and 0.43 +/- 0.09 micrograms/ml in Fischer rats of the same age. When copper chloride (170 micrograms/kg b.w. as Cu) was infused intravenously (i.v.), the concentration of biliary Cu increased to only 0.21 +/- 0.06 microgram/ml 30 min after the infusion in LEC rats. In contrast, Fischer rats showed a concentration about 10 times higher (4.02 +/- 2.2 micrograms/ml) than that before the infusion. In Fischer rats pretreated with cadmium chloride, the biliary Cu concentration was 1.04 + 0.43 micrograms/ml 30 min after infusion of copper. Horseradish peroxidase (E.C.1.11.1.7) infused iv along with copper chloride was excreted into bile at a low level in LEC rats compared to Fischer rats. Our results suggest that the gross accumulation of hepatic Cu in the new, mutant LEC rats is due to a low excretion of Cu into bile and that the hepatobiliary dysfunction is related to spontaneous hepatitis.

Topics: Animals; Bile; Cadmium; Cadmium Chloride; Chlorides; Copper; Hepatitis, Animal; Horseradish Peroxidase; Injections, Intravenous; Liver; Male; Metallothionein; Rats; Rats, Inbred Strains

Copper (Cu) accumulating in the liver of LEC (Long-Evans with a cinnamon-like coat color) rats due to a hereditary metabolic disorder is assumed to cause acute hepatitis with severe jaundice or chronic hepatitis leading to cancer. Changes in concentrations and distributions of Cu, zinc and iron in the liver of LEC rats were determined to find the relationship between the chemical forms and the toxicity. Female rats after delivery were used because of high susceptibility to acute hepatitis. They were divided into four stages according to the development of jaundice. Cu concentrations in the whole liver and the supernatant decreased with development of jaundice. Distribution profiles of Cu, zinc, iron and sulfur on a gel filtration column by HPLC-ICP showed that Cu in the liver supernatant was mostly bound to metallothionein (MT) before jaundice (stage 1), high molecular weight proteins and MT at the beginning of jaundice (stages 2 and 3), and then mostly to MT at severe jaundice (stage 4) though the concentration of Cu at this stage was decreased to about 50% of stage 1. The results suggest that Cu accumulating as MT in the liver is liberated drastically after exceeding the capacity of MT synthesis, and the liberated Cu causes acute hepatitis.

Topics: Animals; Copper; Female; Hepatitis, Animal; Iron; Jaundice; Liver; Metallothionein; Rats; Rats, Inbred Strains; Sulfur; Zinc

Copper (Cu) accumulating bound to metallothionein (MT) in the liver of LEC (Long-Evans with cinnamon-like coat color) rats due to a hereditary metabolic disorder is assumed to lead to acute hepatitis with severe jaundice. The metal was shown to be present in the liver in a form not bound to MT at the beginning of hepatitis after first delivery and lactation. Following this change in the distribution of Cu from MT-bound to non-MT bound form in the liver, changes in the concentrations and distributions of Cu, zinc (Zn) and iron in the plasma and kidneys of LEC rats were also observed. Cu plasma distribution on a gel filtration column by HPLC-ICP revealed that the holo-form of ceruloplasmin (Cp) was present before hepatitis and increased with its development, indicating the availability of Cu for Cp by hepatitis. Cu-binding proteins migrating at the same retention times as those of hepatic Cu-MT and Cu,Zn-superoxide dismutase (SOD) were detected in plasma during hepatitis. Albumin was largely present in the form of nonmercaptoalbumin, reflecting that the bloodstream was under oxidative stress. A sudden increase in the concentration of Cu in the kidneys occurred with hepatitis, and the metal came to be distributed more to high molecular weight proteins with its development.

Topics: Acute Disease; Animals; Copper; Female; Hepatitis, Animal; Iron; Kidney; Metallothionein; Rats; Rats, Inbred Strains; Sulfur; Zinc

The Cu concentration was about 40 and 60 times higher in the liver in Long-Evans with a cinnamon-like coat color (LEC) rats aged 80 days (without hepatitis) and 130 days (with hepatitis), respectively than in the liver in Fischer rats. Most hepatic Cu was recovered in the cytosol fraction. Furthermore, about 96% and 84% of the cytosolic Cu was found in the metallothionein region on a Sephadex G-75 column in LEC rats aged 80 and 130 days, respectively. The hepatic metallothionein concentration was about 130 to 140 times higher in LEC rats than in Fischer rats when the concentration was expressed as metallothionein-bound Cu. Three forms of Cu-metallothionein were isolated by DEAE-cartridge. Although the concentration of hepatic Cu-metallothionein and its composition of polymorphic form were not changed greatly in hepatitis phase (in the 130-day-old LEC rats), activities of serum enzymes, aspartate aminotransferase (GOT) and alanine aminotransferase (GPT) were increased significantly. The LEC rat showed a significantly low concentration of biliary Cu and markedly low activity of ceruloplasmin (as ferroxidase). Serum Cu showed a low concentration in the 80-day-old LEC rats, but recovered to the control level in the 130-day-old LEC rats. The abnormal accumulation of Cu may be due to the inherent reduction of excretion of Cu into the bile and blood. Such deposition may be a trigger for the onset of the spontaneous hepatitis occurring at 90-120 days after birth and for the onset of hepatoma later.

Topics: Animals; Ceruloplasmin; Copper; Female; Hepatitis, Animal; Liver; Metallothionein; Rats; Species Specificity; Spectrophotometry, Atomic; Tissue Distribution

Recently, copper (Cu) was found to be unusually accumulated, suggesting the induction of metallothionein (MT) in the liver of LEC rats (Long-Evans rats with a cinnamon-like coat color), which develop spontaneous jaundice with hereditary hepatitis. Thus, the direct relationship between the unusual Cu accumulation and the induction of Cu-MT was investigated by giving LEC rats Cu-overloaded or Cu-deficient diets. Results based on the determinations of Cu and MT levels in several organs, as well as the gel-filtration profiles of the cytosols of liver homogenates, showed that dietary Cu induced Cu-MT and development of hepatic injury associated with jaundice.

Topics: Animals; Copper; Cytosol; Hepatitis, Animal; Jaundice; Liver; Metallothionein; Rats; Rats, Mutant Strains

To study the effects of increased expression of major histocompatibility complex class I molecules on the development of self-tolerance, transgenic mice were produced that expressed the H-2Kb gene under the control of the metallothionein promoter. Administration of zinc enhanced transgene expression in liver, kidney and exocrine pancreas. No evidence suggestive of an autoimmune response was found in transgene-expressing tissues in mice otherwise allogeneic to H-2Kb. Despite this lack of responsiveness in vivo, T cells could be stimulated in vitro to lyse H-2Kb-bearing target cells. No infiltration was detected in transgenic mice after irradiation and reconstitution with bone marrow cells. When spleen cells were used for reconstitution, however, dense lymphocytic infiltration was seen, particularly in the portal tracts of the liver, and this was accompanied by piecemeal necrosis and apoptosis of periportal hepatocytes. This aggressive response progressively diminished with time, and by 12 weeks after reconstitution many of the portal tracts were free of infiltration while the others showed no accompanying necrosis. The picture at this stage was similar to that seen in chronic persistent hepatitis. These results suggest that, in addition to negative selection in the thymus, peripheral mechanisms not involving clonal deletion or permanent clonal anergy can prevent immune responses to self molecules.

Topics: Animals; Bone Marrow Transplantation; Chronic Disease; Gene Expression Regulation; Graft vs Host Disease; H-2 Antigens; Hepatitis, Animal; Immune Tolerance; Metallothionein; Mice; Mice, Transgenic; Promoter Regions, Genetic; Radiation Chimera; Spleen