Copper Storage Diseases in Dogs by J. Rothuizen - Proceedings of the Wsava 2006 Congress

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 confi rmed 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 infl ammation do not or not signifi cantly 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 traffi cking pathways, oxidative stress, and cholestasis. Copper is intracellularly bound to specifi c proteins. Small copper-binding proteins, denoted copper chaperones, distribute copper to specifi c 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 H