Professor of Internal Medicine, Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, University Utrecht
An abnormal copper metabolism can cause hepatic copper accumulation and subsequently an increase in oxidative stress. We therefore analyzed the copper metabolism pathways and (non)-enzymatic defenses against ROS in three different forms of chronic liver failure in the dog. Chronic hepatitis caused by inherited copper toxicosis (Copper toxicosis, CT) was compared to chronic hepatitis of unknown etiology (CH). These two were compared to liver failure due to chronic extrahepatic bile duct obstruction (extra hepatic cholestasis, EC). Copper metabolism was analyzed using histochemical staining (copper levels) and quantitative PCR (Q-PCR) on copper excretory and storage gene products (ATOX1, COX17, ATP7A, ATP7B, CP, MT1A, MURR1, XIAP). Oxidative stress and cellular homeostasis was measured with Q-PCR (SOD1, CAT, GSS, GPX1, CCS, p27KIP, Bcl-2) as well as GSH and GSSG levels.
Results showed massive accumulation of hepatic copper (5+) in CT. In EC and CH no or only slight copper accumulation (1-2+) was observed. Most gene products for copper metabolism remained at control levels. Three clear exceptions were observed in CT; 3-fold mRNA increase of ATP7A and XIAP and complete absence of MURR1. Only quantitative differences between CH, CT, and EC were observed regarding oxidative stress and cellular homeostasis. This was confirmed with GSH/GSSG ratio measurements, were the strongest reduced ratio was seen in CT (8-fold), the least in CH (5-fold). In conclusion, cholestasis and inflammation do not or not significantly increase copper accumulation. All three diseases have reduced protection against oxidative stress, opening a rationale to use anti-oxidants as possible therapy.
Copper is an integral part of many important enzymes involved in several vital biological processes.1 In humans the only copper storage disease of which the molecular background is resolved is Wilson's disease. A major pathogenetic pathway is that accumulated copper catalyzes the formation of highly reactive oxygen species (ROS), like hydroxyl radicals. In dogs like in man, hepatic copper accumulation may cause hepatitis which ultimately causes cirrhosis. Copper associated hepatitis has been described in dog breeds such as Bedlington terriers, Doberman pinschers, Sky terriers, Dalmatians, Anatolian shepherds, and Labrador retrievers. Copper Toxicosis (CT) in Bedlington terriers is an autosomal recessive disorder causing impaired biliary copper excretion. The resulting progressive lysosomal accumulation of copper becomes histologically evident at one year of age. The genetic defect in Bedlington terriers is caused by a deletion of exon 2 of the MURR1 (COMMD1) gene. In all other dog breeds the molecular background of the disease is unknown. Cholestasis is a sequel of most parenchymal liver diseases, and may cause a reduced biliary copper excretion and secondary copper accumulation. For understanding the primary or secondary role of copper it is important to evaluate copper trafficking pathways, oxidative stress, and cholestasis.
Copper is intracellularly bound to specific proteins. Small copper-binding proteins, denoted copper chaperones, distribute copper to specific intracellular destinations. ATOX1 for instance, delivers copper to the ATPases, CCS distributes copper to Cu/Zn superoxide dismutase (SOD1), COX17 delivers the copper to the cytochrome c oxidase in the mitochondria, and MURR1 is implicated in the lysosomal storage of copper as well as the excretion into bile. The ATPases ATP7A and ATP7B transport copper to the cuproenzymes and ameliorate excretion of excess copper. Ceruloplasmin (CP) is a metalloprotein which binds copper during synthesis and is secreted into serum. Metallothionein 1A (MT1A) is a small intracellular protein capable of chelating several metal ions, including copper. XIAP is an X-linked inhibitor of apoptosis recently associated with MURR1.
Excess copper can induce oxidative stress which could lead to cell death and chronic inflammation. The enzymatic defense against oxidative stress consists of several tightly regulated proteins such as superoxide dismutase (SOD1) and Catalase (CAT). Non-enzymatic defenses are exerted by molecules such as alpha-tocopherol, betacarotene, ascorbate, and a ubiquitous low molecular thiol component, Glutathione. The synthesis of Glutathione from glutamate, cysteine, and glycine is catalyzed by two cytosolic enzymes, γ-glutamylcysteine synthetase (GCS) and GSH synthetase (GSS). The redox status of GSH depends on the relative amounts of the reduced and oxidized forms of glutathione (GSH/GSSG).
We have investigated the presence of copper and its possible role in inflammatory and cholestatic chronic liver diseases. To study the effect of cholestasis, we examined dogs with the chronic extrahepatic cholestasis. In comparison we analysed idiopathic chronic hepatitis (CH) in breeds not associated with copper accumulation, and the only proven inherited form of copper toxicosis in dogs, CT in Bedlington terriers.
Observations on the histological grading of copper showed a marked diffuse copper accumulation in the hepatocytes and focally in macrophages in Bedlington terriers with CT, in agreement with earlier reports. On the other hand, in extrahepatic cholestasis and chronic idiopathic hepatitis there were no copper granules detectable in 33% and 50% of the cases, respectively. In the other cases there was only a slight to moderate degree of copper staining. This implies that copper accumulation is not a consistent feature and never exceeds slightly increased levels of copper in dogs with cholestasis and idiopathic hepatitis. Copper may thus be of minor importance in these diseases.
Comparing the differential mRNA expression profiles showed only significant differences between the three diseases for ATP7A and XIAP. The large increase in XIAP and ATP7A (which is the extracellular transport of copper through the trans-Golgi network) in Bedlington terriers lacking functional MURR1 is most likely a compensatory effect to overcome complete absence of the MURR1 pathway. In the EC and CH-group no significant changes were found in ATP7A, CP, and MURR1. Perhaps only the decrease in mRNA for ATP7B has had an effect to produce slight accumulation of copper in the EC and CH-group.
With respect to the defense against oxidative stress the dogs with EC and CH were similar. Cholestasis and inflammation caused reduced expression of mRNA for SOD1 and CAT. However, however, these reductions were greater in the CT-group. We conclude that, although copper is a major trigger for oxidative stress, diseases with primary copper accumulation cannot be distinguished from primary cholestatic or inflammatory diseases based on their reaction profile to exposure to ROS.
GSH/GSSG ratios were decreased in all diseases with the highest reduction in the CT-group.
The use of anti-oxidants or GSH esters may be effective in treating these liver pathologies. Furthermore, the use of SAM-e in a cirrhotic rat model has shown to have an inhibiting effect on Collagen-I production which could ameliorate liver fibrosis. Because of the decrease in oxidative stress defenses (enzymatic and non-enzymatic), the use of SAM-e could be considered in dogs with CT, but also in other inflammatory and/or cholestatic liver diseases.
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