Excessive Copper Accumulation Research Articles

Limited yield of diagnoses of intrahepatic infectious causes of canine granulomatous hepatitis from archival liver tissue

Canine granulomatous hepatitis is an uncommon morphologic diagnosis that has been associated with a variety of diseases, including a number of systemic infectious etiologies. Formalin-fixed, paraffin-embedded (FFPE) tissues are typically the only source of liver tissue remaining for additional testing for the presence of infectious disease within granulomas. It is unclear if the more common infectious culprits of granulomatous hepatitis can be identified from such specimens. The aim of the current study was to retrospectively investigate archival FFPE liver tissue from dogs with granulomatous hepatitis for the presence of infectious agents. Semiquantitative analysis of copper accumulation in liver specimens was also performed. Medical records were examined for recorded evidence of systemic infectious disease diagnosis. Formalin-fixed, paraffin-embedded liver was prospectively evaluated for infectious agents via differential staining techniques (n = 13), eubacterial fluorescent in situ hybridization (n = 11), and Bartonella polymerase chain reaction assays (n = 15). An infectious cause of granulomatous hepatitis was not identified within liver tissue from any dog using these diagnostic methodologies. Six out of 25 (24%) dogs were diagnosed with concurrent systemic or localized bacterial infections at the time of presentation. Nine out of 17 (53%) dogs had excessive hepatic copper accumulation when evaluated by a semiquantitative histologic grading scheme or quantitative copper analysis. As definitive infectious causes of granulomatous hepatitis were not identified within archival liver biopsy samples, it was concluded that investigation of infectious etiologies within FFPE liver specimens using these diagnostic approaches may be of low yield.

In Vivo Detection of Copper Ions by Magnetic Resonance Imaging Using a Prion-Based Contrast Agent

Abnormal distributions of transition metals inside the body are potential diagnostic markers for several diseases, including Alzheimer's disease, Parkinson's disease, Wilson's disease, and cancer. In this article, we demonstrate that P57/Gd, a novel prion-based contrast agent, can selectively image tissues with excessive copper accumulation using magnetic resonance imaging (MRI). P57/Gd selectivity binds copper(II) over other physiologically relevant cations such as zinc, iron, manganese, and calcium. To simulate a metabolic copper disorder, we treated mice with an intraperitoneal injection of a CuSO(4) solution to induce a renal copper overload. The MRI signal intensities from the renal cortex and medulla of copper spiked animals that were administered P57/Gd were found to correlate with the ex vivo copper concentrations determined by inductively coupled plasma mass spectrometry.

The diagnostic value of multimodal evoked potentials in the determination of subclinical neurological involvement of Wilson’s disease

Wilson's disease (WD) is a progressive degeneration of hepatolenticular tissue caused by excessive tissue-damaging copper accumulation and in which liver involvement most frequently presents in childhood. Neurological signs also accompany liver disease with time. However, subclinical neurological involvement may occur earlier and diagnostic methods that reveal this subclinical involvement are not well established. The aim of the current study is to assess the subclinical neurological involvement by using multimodality evoked potential (EP) measurements and to explore the relationship between neurological disease and the severity of liver damage. The patient group included 28 children (mean age 11.8 ± 2.9 years, range 5.5-17) diagnosed with WD and a control group included 24 age-matched healthy children. Multimodality EP tests (Nihon Kohden Neuropack 8 4200K) of both groups were performed at the Department of Neurology Electrophysiology Laboratory of Ege University. At least one abnormal EP value was observed in 53.5% of the children in the patient group. At least on one side, there were abnormal values for visual evoked potential (VEP) P100, brainstem auditory evoked potential (BAEP) and somatosensory evoked potential (SEP), where the ratios were 25, 28.5, and 11%, respectively. Absolute latency values of patients with right-side VEP P100 and left-side BAEP I, and the interpeak latency values of right-side BAEP I-III were significantly high. The difference in right-side BAEP I-III interpeak latency between cirrhotic and noncirrhotic groups was found to be statistically significant (P<0.05). The EP examinations can be an indicator of subclinical brain damage in non-neurological WD; however, cirrhosis because of WD does not cause an increase in the EP values. Detection of changes in the EP values periodically, especially at the time of diagnosis and during the treatment follow-up, may be valuable for revealing subclinical impairment.

Work Ability Assessment in a Patient with Wilson's Disease

Wilson's disease (WD) is a rare, progressive autosomal recessive disorder characterised by impaired transport and excessive accumulation of copper in the liver, brain, and other tissues. The disease is diagnosed based on clinical manifestations and screening tests results. Work ability assessment of patients with WD is based on the analysis of liver, kidney, neurological, and cognitive impairments, and takes into account patient's level of education.This article presents a case with a 48-year-old male patient, who was admitted for work ability assessment due to polymorphic symptoms. The patient had been working as a salesman for 28 years. A detailed interview and examination by occupational health and other medical specialists revealed that the patient had been suffering from Wilson's disease from the age of 13, and had now developed hepatic manifestations (compensated liver cirrhosis with portal hypertension), neurological manifestations (dystonia, dysarthria, muscle weakness, vertigo), and psychiatric manifestations (depression, insomnia, cognitive impairment) of the disease, including problems partially caused by long-lasting treatment with copper chelating agents (neurological and haematological manifestations). There were no ocular manifestations of Wilson's disease (Kayser-Fleischer rings or sunflower cataract).The patient was assessed as having drastically diminished general work ability, dominantly due to neurological and psychiatric impairments caused by Wilson's disease.

Proteomic analysis of sera of asymptomatic, early‐stage patients with Wilson's disease

Wilson's disease (WD) is characterized by excessive accumulation of intracellular copper in liver and extrahepatic tissues, leading to significant oxidative stress and tissue damage. To date, several diagnostic biomarkers for WD such as serum ceruloplasmin, serum or urine copper levels and copper content in liver have been identified. However, these biomarkers may not be convincing for the diagnosis in some WD patients. To identify additional novel diagnostic biomarkers, we compared the serum protein profiles of asymptomatic childhood WD patients (n=20), without neurologic manifestation or liver cirrhosis, with normal controls (n=13). Fourteen spots, five up-regulated and nine down-regulated (>2-fold), were differentially expressed in WD patients in comparison to normal control on 2-DE. Among them, three spots were down-regulated in both male and female WD. MS/MS analysis revealed that the three spots were complement component C3, complement factor B and alpha-2 macroglobulin. By comparative proteome analysis, complement component C3, complement factor B and alpha-2 macroglobulin, which are related to oxidative stress and inflammation, turned out to be good candidates for novel diagnostic biomarkers for early stages of WD.

Copper Transport Systems are Involved in Multidrug Resistance and Drug Transport

Copper is an essential trace element and several copper containing proteins are indispensable for such processes as oxidative respiration, neural development and collagen remodeling. Copper metabolism is precisely regulated by several transporters and chaperone proteins. Copper Transport Protein 1 (CTR1) selectively uptakes copper into cells. Subsequently three chaperone proteins, HAH1 (human atx1 homologue 1), Cox17p and CCS (copper chaperone for superoxide dismutase) transport copper to the Golgi apparatus, mitochondria and copper/zinc superoxide dismutase respectively. Defects in the copper transporters ATP7A and ATP7B are responsible for Menkes disease and Wilson's disease respectively. These proteins transport copper via HAH1 to the Golgi apparatus to deliver copper to cuproenzymes. They also prevent cellular damage from an excess accumulation of copper by mediating the efflux of copper from the cell. There is increasing evidence that copper transport mechanisms may play a role in drug resistance. We, and others, found that ATP7A and ATP7B are involved in drug resistance against the anti-tumor drug cis-diamminedichloroplatinum (II) (CDDP). A relationship between the expression of ATP7A or ATP7B in tumors and CDDP resistance is supported by clinical studies. In addition, the copper uptake transporter CTR1 has also been reported to play a role in CDDP sensitivity. Furthermore, we have recently found that the effect of ATP7A on drug resistance is not limited to CDDP. Using an ex vivo drug sensitivity assay, the histoculture drug response assay (HDRA), the expression of ATP7A in human surgically resected colon cancer cells correlated with sensitivity to 7-ethyl-10-hydroxy-camptothecin (SN-38). ATP7A-overexpressing cells are resistant to many anticancer drugs including SN-38, 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11), vincristine, paclitaxel, etoposide, doxorubicin (Dox), and mitoxantron. The mechanism by which ATP7A and copper metabolism modulate drug transport appears to involve modulation of drug cellular localization via modulation of the vesicle transport system. In ATP7A overexpressing cells, Dox accumulates in the Golgi apparatus. In contrast, in the parental cells, Dox is localized in the nuclei, where the target molecules of Dox, topoisomerase II and DNA, are found. Disruption of the intracellular vesicle transport system with monensin, a Na+/H+ ionophore, induced the relocalization of Dox from the Golgi apparatus to the nuclei in the ATP7A overexpressing cells. These data suggested that ATP7A-related drug transport is dependent on the vesicle transport system. Thus copper transport systems play important roles in drug transport as well as in copper metabolism. Components of copper metabolism are therefore likely to include target molecules for the modulation of drug potency of not only anti-cancer agents but also of other drugs.

Aceruloplasminemia: a case report

Hereditary aceruloplasminemia is a rare autosomal recessive disease, firstly identified by Miyajima et al. in Japan in 1987 [1]. The disease is caused by the absence of an a2glycoprotein, the ceruloplasmin (Cp), a copper-containing ferroxidase, mainly synthesized in hepatocytes and widely expressed, including the central nervous system, which catalyses the oxidation of ferrous to ferric iron, a change required for release of iron to plasma transferrin [2]. It is hypothesized that in reticuloendothelial (RE) cells and hepatocytes Cp cooperates to export iron with the iron exporter protein ferroportin 1 (FPN1) [3]. As a consequence, Cp deficiency results in iron deposition in the liver, pancreas, basal ganglia, and other organs [2]. The Cp gene maps to chromosome 3q21–24 [4], consists of 19 exons [5] and encodes a protein of 1,046 amino acids [6]. Recently 20th exon has been recognized as the result of a brain-specific alternative splicing. This adds to the protein an aminoacid sequence that makes Cp a glycosylphosphatidylinositol (GPI)-anchored protein in the brain [7]. Under normal circumstances, infact, circulating Cp does not cross the blood–brain barrier. Through the oxidation mediated by GPI-linked Cp expressed in the astrocytes, iron could be taken from the circulation, incorporated into transferring derived from oligodendrocytes and then transferred to the neuronal cells [2]. Ceruloplasmin deficiency is a characteristic feature in copper metabolic disorders, including Wilson’s disease and Menkes’ disease. In Wilson’s disease, an inability to transfer copper into the Cp precursor protein, apoceruloplasmin, and a decrease in biliary copper excretion results in serum ceruloplasmin deficiency and excess copper accumulation. In Menkes’ disease, copper absorption from the intestine is decreased, leading to copper and Cp deficiencies in the body. In contrast, aceruloplasminemia, is an iron metabolic disorder in which ceruloplasmin deficiency is caused by a lack of apoceruloplasmin biosynthesis and copper metabolism is not disturbed. In Wilson’s disease and Menkes’ disease, several gene mutations in the coppertransporting ATPases (ATP-7B and ATP-7A) have been found within the last 10 years. Aceruloplasminemia is characterized by mutations in the ceruloplasmin gene itself [8]. Aceruloplasminemia, after the first description, has been reported mainly in Japanese patients [1, 2, 9–18] and rarely in whites [10, 19]. More than 30 aceruloplasminemiacausing mutations have been identified. Some of the mutations have been found in several families throughout the world, while other have been detected in a single patient. The majority of mutations in the CP gene are the truncated mutations leading to the formation of a premature stop codon. These mutations would be predicted to result in formation of a protein lacking the copper-binding sites resumed to be critical for enzymatic function. First clinical manifestation is usually diabetes mellitus secondary to pancreatic involvement [2]. A progressive neurological disease begins a few years later, typically with focal dystonia, followed by dysarthria, extrapyramidal D. Di Raimondo A. Pinto A. Tuttolomondo P. Fernandez G. Licata Dipartimento Biomedico di Medicina Interna e Specialistica, AOU Policlinico ‘‘P. Giaccone’’, Universita degli Studi di Palermo, Palermo, Italy

Identification of novelATP7Bgene mutations and their functional roles in Korean patients with Wilson disease

Wilson disease (WND), an autosomal recessive disorder of copper transport, is characterized by excessive accumulation of intracellular copper in liver and extrahepatic tissues because of impaired biliary copper excretion and disturbed incorporation of copper into ceruloplasmin. Hepatic cirrhosis and neuronal degeneration are the major symptoms of WND, and mutations in the ATP7B gene are associated with WND. We have identified 28 different mutations in the ATP7B gene, including six novel variations, in 120 unrelated Korean patients with WND. Molecular defects in ATP7B were present in only 75.0% of Korean WND patients, with the most common mutation, p.Arg778Leu, having an allele frequency of 39.2%. To evaluate the functional defects of ATP7B caused by novel mutations, we used a yeast complementation system, and we used confocal microscopy to localize each mutation after transient expression in mammalian cells. Six novel variations were cloned into a yeast expression vector and two into a mammalian expression vector for confocal analysis. We found that c.2785A>G (p.Ile929Val) and c.3316G>A (p.Val1106Ile) were rare polymorphisms, whereas the others were novel variations disturbing ATP7B function.

Ocular motility and Wilson’s disease: a study on 34 patients

Background:Wilson’s disease is an autosomal recessive genetic disorder resulting from an abnormality of copper metabolism. The excessive accumulation of copper in the brain induces an extrapyramidal syndrome. Oculomotor abnormalities occur...

Whipple’s triad as a clinical manifestation of hepatolenticular degeneration

Hepatolenticular degeneration (Wilson's disease) is a rare condition characterised by a defect in biliary excretion of copper resulting in excessive copper accumulation and toxicity. To the most frequent symptoms of this disorder belong liver, neurological or psychiatric disturbances, although other less common clinical features may sometimes be present. Since the clinical presentation of the disease is highly heterogeneous, it may mimic the symptoms of many various disorders. Diagnosis of the condition depends primarily on clinical features, biochemical parameters and the presence of the Kayser-Fleischer ring. Early detection and treatment protect patients from devastating organ damage. We describe an atypical case of Wilson's disease in a 23-year-old woman, whose clinical presentation suggested the presence of an insulin-secreting tumour. After the diagnosis was established and zinc sulphate treatment implemented, her clinical status improved remarkably. The presented case suggests that hepatolenticular degeneration should be taken into consideration in a differential diagnosis of hypoglycaemia of an unknown origin.

Copper homeostasis in Drosophila by complex interplay of import, storage and behavioral avoidance

Copper is an essential but potentially toxic trace element. In Drosophila, the metal-responsive transcription factor (MTF-1) plays a dual role in copper homeostasis: at limiting copper concentrations, it induces the Ctr1B copper importer gene, whereas at high copper concentrations, it mainly induces the metallothionein genes. Here we find that, despite the downregulation of the Ctr1B gene at high copper concentrations, the protein persists on the plasma membrane of intestinal cells for many hours and thereby fills the intracellular copper stores. Drosophila may risk excessive copper accumulation for the potential benefit of overcoming a period of copper scarcity. Indeed, we find that copper-enriched flies donate a vital supply to their offspring, allowing the following generation to thrive on low-copper food. We also describe two additional modes of copper handling: behavioral avoidance of food containing high (>or=0.5 mM) copper levels, as well as the ability of DmATP7, the Drosophila homolog of Wilson/Menkes disease copper exporters, to counteract copper toxicity. Regulated import, storage, export, and avoidance of high-copper food establish an adequate copper homeostasis under variable environmental conditions.

Erythrocyte Metabolism and Antioxidant Status of Patients with Wilson Disease with Hemolytic Anemia

Wilson disease (WD) is an autosomal recessive disorder due to the defect in ATP7B gene characterized by excessive accumulation of copper in the liver with progressive hepatic damage and subsequent redistribution to various extrahepatic tissues including the brain, kidneys, and cornea. Strikingly, the total serum copper concentration is always low in WD, even though the non-ceruloplasmin copper level is still expected to be high. To assess the role of free radical reactions catalyzed by non-ceruloplasmin copper, we investigated erythrocyte metabolism and oxidative stress as a mechanism for hemolysis in eight WD patients during episodes of acute hemolysis and compared them with eight follow-up cases of WD on d-penicillamine therapy and eight healthy, age-matched children. Elevated levels of non-ceruloplasmin copper were found in all the WD patients during an episode of hemolytic anemia. There was marked inhibition in erythrocyte enzymes, namely, hexokinase, total adenosine triphosphatase (ATPase), and glucose-6-phosphate dehydrogenase (G-6-PD) from WD patients compared with patients on penicillamine and healthy children, indicating altered erythrocyte metabolism during a hemolytic crisis. Antioxidant status was also found to be compromised as is evident from decreased glutathione (GSH) levels, decreased antioxidant enzymes (namely, superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase), increased lipid peroxidation, and deranged plasma antioxidants. Uric acid showed maximum decrease followed by ascorbic acid. These findings suggest that the free radical production by elevated non-ceruloplasmin copper through transition metal catalyzed reactions leads to oxidative injury resulting in altered erythrocyte metabolism and severely compromised antioxidant status of WD patients during hemolytic anemia.

Hepatocyte Transplantation in the Long Evans Cinnamon Rat Model of Wilson's Disease

Wilson's disease (WD), caused by a mutation in the P-type copper transporting ATPase (Atp7b) gene, results in excessive accumulation of copper in the liver. Long Evans Cinnamon rats (LEC) bear a mutation in the atp7b gene and share clinical characteristics of human WD. To explore hepatocyte transplantation (HT) as therapy for metabolic liver diseases, 8-week-old LEC rats (n = 12) were transplanted by intrasplenic injection of hepatocytes from donor Long Evans (LE) rats. Immunosuppression was maintained with intraperitoneal tacrolimus. The success of HT was monitored at 24 weeks of life. Serum aminotransferases and bilirubin peaked at 14-21 weeks in both HT rats and nontransplanted controls, but at 24 weeks, survival was 97% in LEC-HT versus 63% in controls. All transplanted rats showed restored biliary copper excretion and reduced liver iron concentration associated with increased ceruloplasmin oxidase activity. Liver tissue expressed atp7b mRNA (11.9 +/- 13.6%) indicative of engraftment of normal cells in 7 of 12 HT rats, associated with a reduced liver copper concentration compared to untreated LEC rats. Periportal islets of normal appearing hepatocytes, recognized by atp7b antibody, were observed in transplanted livers while lobular host cells showed persistent pleomorphic changes and inflammatory infiltrates. In conclusion, transplantation of normal hepatocytes prevented fulminant hepatitis, reduces chronic inflammation, and improved 6-month survival in LEC rats. Engraftment of transplanted cells, which express atp7b mRNA, repopulated the recipient liver with normal functional capacity.

Suppressive effects of dietary D-penicillamine on the gene expression of hepatic metallothionein-1 and protein level of hepatic metallothionein in Long-Evans Cinnamon rats

We investigated the effects of dietary D-penicillamine on copper and zinc contents in the liver, serum, and urine of Long-Evans Cinnamon (LEC) rats, as well as the gene expression of hepatic metallothionein-1 and hepatic metallothionein protein level in these animals. Young male LEC and Fischer rats were fed a basal or D-penicillamine (5 g D-penicillamine/kg diet) diet for 16 days. The urinary output of copper and zinc was markedly increased by the D-penicillamine diet, and the copper and zinc contents in the liver were significantly decreased in LEC rats. Excess copper accumulation in the liver induced hepatic metallothionein in LEC rats, but the D-penicillamine diet depressed the level of hepatic metallothionein-1 mRNA as well as the hepatic metallothionein protein level. When fed the basal diet, serum ornithine carbamoyltransferase activity was higher in LEC rats than in Fischer rats, but a significant decrease in serum ornithine carbamoyltransferase activity rats was observed in LEC rats on feeding the D-penicillamine diet. These results indicated that dietary D-penicillamine significantly reduced excess copper and zinc in the livers of LEC rats through urinary excretion, and the decrease in hepatic copper and zinc contents caused a decrease in the levels of metallothionein-1 mRNA and metallothionein protein in LEC rats.

The Copper Toxicosis Gene Product Murr1 Directly Interacts with the Wilson Disease Protein

Copper toxicosis in Bedlington terriers is an autosomal recessive disorder characterized by excessive hepatic copper accumulation in association with a marked decrease in biliary copper excretion. Recent genetic data have revealed that MURR1, a single copy gene on dog chromosome 10q26, is mutated in this disorder. This gene encodes a 190-amino acid open reading frame of unknown function that is highly conserved in vertebrate species. The Wilson disease protein is a copper transporting ATPase shown to play a critical role in biliary copper excretion. Here we demonstrate that the Wilson disease protein directly interacts with the human homologue of Murr1 in vitro and in vivo and that this interaction is mediated via the copper binding, amino terminus of this ATPase. Importantly, this interaction is specific for this copper transporter, a finding consistent with the observation that impaired copper homeostasis in affected terriers is confined to the liver. Our findings reveal involvement of Murr1 in the defined pathway of hepatic biliary copper excretion, suggest a potential mechanism for Murr1 function in this process, and provide biochemical evidence in support of the proposed role of the MURR1 gene in hepatic copper toxicosis.

Correction of the copper transport defect of Menkes patient fibroblasts by expression of two forms of the sheep Wilson ATPase

The Wilson disease (WD) protein (ATP7B) is a copper-transporting P-type ATPase that is responsible for the efflux of hepatic copper into the bile, a process that is essential for copper homeostasis in mammals. Compared with other mammals, sheep have a variant copper phenotype and do not efficiently excrete copper via the bile, often resulting in excessive copper accumulation in the liver. To investigate the function of sheep ATP7B and its potential role in the copper-accumulation phenotype, cDNAs encoding the two forms of ovine ATP7B were transfected into immortalised fibroblast cell lines derived from a Menkes disease patient and a normal control. Both forms of ATP7B were able to correct the copper-retention phenotype of the Menkes cell line, demonstrating each to be functional copper-transporting molecules and suggesting that the accumulation of copper in the sheep liver is not due to a defect in the copper transport function of either form of sATP7B.

Excessive hepatic copper accumulation in jaundiced rats fed a high-copper diet.

The response of copper metabolism to dietary copper challenge was investigated in jaundiced rats with elevated plasma concentrations of conjugated bilirubin as a result of impaired canicular transport of bilirubin glucuronides. Control and jaundiced rats were fed purified diets with either normal (64 micromol Cu/kg) or high (640 micromol Cu/kg) concentration of added copper. Copper loading produced a greater increase in hepatic copper concentrations in the jaundiced than in control rats. The greater dietary-copper-induced increase in hepatic copper in the jaundiced rats can be explained by the observed smaller rise in biliary copper excretion and a greater efficiency of dietary copper absorption. In individual rats, there was a positive relationship between hepatic copper concentrations and biliary copper concentrations. It is suggested that not the transport of copper from liver cells to bile but that from plasma to bile is diminished in the jaundiced rats. The elevated plasma copper concentrations in the jaundiced rats may support this suggestion.

Copper-induced apical trafficking of ATP7B in polarized hepatoma cells provides a mechanism for biliary copper excretion

Background & Aims: Mutations in the ATP7B gene, encoding a copper-transporting P-type adenosine triphosphatase, lead to excessive hepatic copper accumulation because of impaired biliary copper excretion in Wilson's disease. In human liver, ATP7B is predominantly localized to the trans-Golgi network, which appears incompatible with a role of ATP7B in biliary copper excretion. The aim of this study was to elucidate this discrepancy. Methods: Immunofluorescence and electron-microscopic methods were used to study the effects of excess copper on ATP7B localization in polarized HepG2 hepatoma cells. Results: ATP7B is localized to the trans-Golgi network only when extracellular copper concentration is low (<1 μmol/L). At increased copper levels, ATP7B redistributes to vesicular structures and to apical vacuoles reminiscent of bile canaliculi. After copper depletion, ATP7B returns to the trans-Golgi network. Brefeldin A and nocodazole impair copper-induced apical trafficking of ATP7B and cause accumulation of apically retrieved transporters in a subapical compartment, suggesting continuous recycling of ATP7B between this vesicular compartment and the apical membrane when copper is increased. Conclusions: Copper induces trafficking of its own transporter from the trans-Golgi network to the apical membrane, where it may facilitate biliary copper excretion. This system of ligand-induced apical sorting provides a novel mechanism to control copper homeostasis in hepatic cells.GASTROENTEROLOGY 2000;119:782-793

Cloning, mapping and expression analysis of the sheep Wilson disease gene homologue

Copper homeostasis in mammals is maintained by the balance of dietary intake and copper excretion via the bile. Sheep have a variant copper phenotype and do not efficiently excrete copper by this mechanism, often resulting in excessive copper accumulation in the liver. The Wilson disease protein (ATP7B) is a copper transporting P-type ATPase that is responsible for the efflux of hepatic copper into the bile. To investigate the role of ATP7B in the sheep copper accumulation phenotype, the cDNA encoding the ovine homologue of ATP7B was isolated and sequenced and the gene was localised by fluorescence in situ hybridisation to chromosome 10. The 6.3 kb cDNA encoded a predicted protein of 1444 amino acids which included all of the functional domains characteristic of copper transporting P-type ATPases. ATP7B mRNA was expressed primarily in the liver with lower levels present in the intestine, hypothalamus and ovary. A splice variant of ATP7B mRNA, which was expressed in the liver and comprised approximately 10% of the total ATP7B mRNA pool, also was isolated. The results suggest that ATP7B is produced in the sheep and that the tendency to accumulate copper in the liver is not due to a gross alteration in the structure or expression of ATP7B.

Storage of lipid droplets in and production of extracellular matrix by hepatic stellate cells (vitamin A-storing cells) in Long-Evans cinnamon-like colored (LEC) rats.

LEC rats spontaneously develop hepatocellular carcinoma with cholangiofibrosis after chronic hepatitis, but the mechanism of development of the hepatic injury is not clear. To investigate the role of hepatic stellate cells in induction or suppression of hepatic fibrosis, we morphologically examined the liver of LEC rats. Accumulation of copper was analyzed by the Danscher-Timm's sulfide-silver method. Histopathological changes were evaluated by hematoxylin and eosin staining, and by Masson's trichrome method. Activated stellate cells were identified by immunostaining method for alpha-smooth muscle actin. Cytological alterations of the stellate cells were investigated by transmission electron microscopy. To evaluate the lipid content in the stellate cells, we analyzed the area of lipid droplets of the cells by morphometric analysis. Also for evaluation of the changes in the number of stellate cells, the numbers of nucleated stellate cells and parenchymal cells were counted and statistically analyzed. Hepatic parenchymal cells showed excessive accumulation of copper at 5 weeks of age. Submassive necrosis was observed at 19 weeks of age. The liver of LEC rats 1.5 years of age showed cholangiofibrosis and subcellular injury of hepatic parenchymal cells. However, no diffuse hepatic fibrosis was observed in the liver, and hepatic stellate cells around the regions of cholangiofibrosis were negative for alpha-smooth muscle actin. The area of lipid droplets of a stellate cell in the liver of LEC rats was 1.6 to 1.8 times as large as that of normal Wistar rats. The hepatic stellate cells did not participate in the accumulation of collagen fibers around themselves when the cells contained a large amount of vitamin A-lipid droplets, even though the development of hepatic lesions was in progress. Our present data are consistent with our previous hypothesis that there is an antagonistic relationship between the storage of vitamin A and the production of collagen in stellate cells.