Ann. N.Y. Acad. Sci. ISSN 0077-8923
A N N A L S O F T H E N E W Y O R K A C A D E M Y O F SC I E N C E S Issue: Human Disorders of Copper Metabolism I
New canine models of copper toxicosis: diagnosis, treatment, and genetics Hille Fieten, Louis C. Penning, Peter A.J. Leegwater, and Jan Rothuizen Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands Address for correspondence: Hille Fieten, Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 108, 3584 CM Utrecht, the Netherlands. [email protected]
The One Health principle recognizes that human health, animal health, and environmental health are inextricably linked. An excellent example is the study of naturally occurring copper toxicosis in dogs to help understand human disorders of copper metabolism. Besides the Bedlington terrier, where copper toxicosis is caused by a mutation in the COMMD1 gene, more complex hereditary forms of copper-associated hepatitis were recognized recently in other dog breeds. The Labrador retriever is one such breed, where an interplay between genetic susceptibility and exposure to copper lead to clinical copper toxicosis. Purebred dog populations are ideal for gene mapping studies, and because genes involved in copper metabolism are highly conserved across species, newly identified gene mutations in the dog may help unravel the genetic complexity of different human forms of copper toxicosis. Furthermore, increasing knowledge with respect to diagnosis and treatment strategies will benefit both species. Keywords: copper; dog; liver; genetics; One Health
Introduction Copper is an essential micronutrient for many organisms, but can be toxic when present in excessive amounts. For this reason, copper metabolism is tightly regulated and proteins involved in copper homeostasis are highly conserved across species, including humans and dogs. The liver is pivotal in copper metabolism and provides for the redistribution of copper after intestinal uptake and the excretion of excess copper via the bile. The importance of appropriate regulation of copper metabolism is illustrated by the autosomal recessive disorder Wilson’s disease, which is caused by mutations in the copper transporter ATP7B1,2 (Table 1). Clinical signs of Wilson’s disease are related to copper accumulation in the liver and brain and include chronic hepatitis and neurological or psychiatric symptoms.3 Wide ranges in age of onset and clinical presentation are recognized and there is often a lack of genotype–phenotype correlation,4 which may implicate environmental influence or an effect of modifier genes that influence clinical presentation.
Ecogenetic disorders of copper metabolism, in which genetically predisposed individuals exposed to high levels of exogenous copper develop clinical disease, include endemic Tyrolean infantile cirrhosis (ETIC),5 Indian childhood cirrhosis (ICC),6 and idiopathic copper toxicosis (Table 1).7 In these disorders, copper accumulation is restricted to the liver, and the manifestation of clinical symptoms is likely influenced by the level of copper uptake through the diet or drinking water. The genetic background of these diseases is currently unresolved. The wide variability of the human genome, the rarity of the diseases, and the variation in clinical phenotypes hamper genetic mapping studies of the diseases in humans. Several animal models for human copper toxicosis exist, such as rodent models,8–11 North Ronaldsay sheep,12 and dogs. Since their domestication, dogs have undergone artificial selection leading to the development of isolated populations of dog breeds. By inbreeding and selection on specific phenotypic characteristics, recessive diseases can be present at high frequencies in purebred dog populations, and many breeds are doi: 10.1111/nyas.12442
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Table 1. Copper toxicosis phenotypes recognized in humans and dogs
Gender predisposition Autosomal recessive copper toxicosis Wilson’s disease No
COMMD1-deficient dogs Ecogenetic copper toxicosis ICC
Age of onset
Childhood, adult
Clinical signs
Involved genes
Dietary influence
Neurologic and hepatic Hepatic
ATP7B
Possibly
COMMD1
Minor
Genetic susceptibility Genetic susceptibility Unknown Complex inheritance pattern
Suspected
No
Young to middle-aged dogs
Male
Infants
Hepatic
ETIC
No
Infants
Hepatic
ICT Labrador retriever
No Female
Childhood, adult Young to middle-aged dogs
Hepatic Hepatic
Suspected Unknown Major
ICC, Indian childhood cirrhosis; ETIC, endemic Tyrolean infantile cirrhosis; ICT, idiopathic copper toxicosis.
at increased risk for specific disorders with complex inheritance patterns.13 The limited genetic variability within inbred dog populations works as a magnifying glass, instrumental to discovering crucial and modifier genes involved in complex genetic diseases that are of interest for both dogs and humans. Furthermore, dogs with copper toxicosis can be a valuable large animal model for evaluation of new treatment strategies beneficial to both the human and canine patients. Canine copper toxicosis With respect to copper metabolism disorders, copper toxicosis in the Bedlington terrier is a good example in which gene mapping studies in the dog led to the identification of a gene that was previously unknown to be involved in copper metabolism14 (Table 1). A chromosomal deletion of 39.7 kb encompassing exon 2 of the originally named MURR1 gene caused a complete absence of the protein product,15 leading to extreme accumulation of hepatic copper. MURR1, later renamed COMMD1, was found to be a versatile gene, with many cellular functions including roles in sodium metabolism,16–18 regulation of NF-B,19,20 and HIF1␣-mediated transcription.21–23 COMMD1 plays a role in the functioning and stability of the human Wilson’s
disease gene ATP7B,24 providing a clue to how COMMD1 absence leads to copper accumulation within hepatocytes. The prevalence of Bedlington terrier copper toxicosis was very high, and estimates within different populations ranged from 25% to 40%.25,26 This prevalence dropped dramatically after the implementation of a DNA test in breeding strategies, which led to an almost complete eradication of the disease. Within the facilities of the Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, we keep a colony of COMMD1-deficient dogs for studies into the progression of chronic hepatitis27 and for the development of stem cell therapy. Increased levels of hepatic copper with or without hepatic pathology have been recognized in other breeds as well.28–30 Different forms of copper-associated hepatitis were recognized in a number of other dog breeds. Pedigree studies executed in breeds with different forms of copper-associated hepatitis including the Labrador retriever,31,32 Doberman,29,33 West Highland White terrier,34 and Dalmatian35 confirmed a hereditary background. In these breeds, a complex form of copper-associated hepatitis is present, where the susceptibility to copper is genetically determined and the expression of the disease phenotype relies
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on environmental factors such as dietary copper intake. In a retrospective review of primary hepatitis cases in our department, approximately one-third of the cases were copper related.36 Parallels can be made between these canine forms of complex copper-associated hepatitis and the human ecogenetic disorders of copper metabolism ETIC, ICC, and idiopathic copper toxicosis (Table 1). Clinical presentation The age at which dogs are presented to the veterinarian with clinical signs is variable and may depend on the breed, exposure to copper in the diet and drinking water, and gestation and lactation in bitches. In Bedlington terriers, hepatic copper accumulation is severe and levels of 2000 mg/kg dry weight liver (dwl) can be observed in dogs of 1 year of age,37 which is five times higher than the upper reference of 400 mg/kg dwl of hepatic copper in dogs.38 Initially, hepatic copper accumulation occurs without overt histological signs of hepatic inflammation; however, when hepatic copper levels rise up to 10,000 mg/kg dwl at around 3 years of age, affected Bedlington terriers may express nonspecific clinical signs including lethargy, anorexia, vomiting, and polyuria/polydipsia. In more advanced cases, icterus, ascites, and hepatic encephalopathy may be recognized. Affected Bedlington terriers may have severe acute hepatitis, resulting in a massive release of copper into the circulation, leading to a hemolytic crisis. In the dog breeds with complex copper-associated hepatitis, hepatic copper accumulation may also start early in life, but is less severe and mostly does not reach hepatic copper levels >5000 mg/kg dwl. However, more severe histological inflammatory lesions are often recognized at lower levels of hepatic copper, compared to the Bedlington terrier. In the Labrador retriever and Doberman, a strong female predisposition for copper-associated hepatitis has been noted.31,39 Comparable to humans with Wilson’s disease, Fanconi’s syndrome was recognized in a Clumber spaniel,40 a West Highland White terrier,41 and a case series of nine Labrador retrievers42 with copperassociated hepatitis. The dogs showed a marked increase in copper concentration in the kidney with accumulation of copper granules in the proximal renal tubules leading to proximal renal tubular dysfunction. Serum biochemical analysis and urinalysis 44
resolved spontaneously with appropriate chelation therapy.40–42 In contrast, Kaiser–Fleischer rings and neurological symptoms other than hepatic encephalopathy are not recognized in dogs with copper toxicosis. Laboratory diagnosis The first indicators for hepatocyte damage are an increase in alanine aminotransferase (ALT) and a slight decrease in serum albumin. In advanced disease, a more pronounced decrease in albumin and increases in ALT, alkaline phosphatase (AP), bile acids, and ammonia can be present. These abnormalities do not distinguish copper-associated liver disease from any other parenchymal liver diseases. Currently, a DNA test is only available for the Bedlington terrier. For all other dogs, a definitive diagnosis relies on histological evaluation of a liver biopsy. Ultrasonography of the liver can be used to evaluate the size, echo density, and structure of the liver and is useful for guiding percutaneous liver biopsies under local anesthesia with or without light sedation. A more invasive way to obtain liver biopsies is through laparotomy or laparoscopy, requiring general anesthesia, which enables visible inspection of the liver and the opportunity to obtain larger specimens. Approximately one-third of first-line relatives of affected Labrador retrievers have increased hepatic copper levels;31 therefore, it is recommended to evaluate liver biopsies from relatives of affected dogs. When treatment is started in the subclinical phase, treatment outcome is far more favorable compared to dogs that are admitted in end-stage liver disease. Evaluation of formalin-fixed liver biopsies includes hematoxylin and eosin,43 reticulin,44 and copper stains, such as rubeanic acid45 or rhodanin,46 for respective grading of the inflammatory activity and routine evaluation, for staging of fibrosis, and for semiquantitative determination of copper level. Localization of copper in the centrolobular hepatocytes of the liver lobule is characteristic for canine primary copper toxicosis. An inflammatory mononuclear or mixed infiltrate accompanies the copper-loaded hepatocytes, and in more advanced stages of the disease apoptosis/necrosis of affected hepatocytes and copper-loaded macrophages (Kupffer cells) can be recognized in association with the centrizonal hepatocytic copper accumulation. In advanced disease, apoptosis, necrosis,
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regeneration, and typical centrocentral bridging fibrosis are seen, eventually leading to end-stage liver micro- or macronodular liver cirrhosis. A separate biopsy is used for quantitative copper measurement by instrumental neutron activation analysis47 or by spectrophotometric methods. Parallel to the use of urine copper excretion in therapeutic monitoring in Wilson’s disease, urinary copper and zinc excretion in relation to hepatic copper concentration was evaluated for use as a noninvasive screening tool for copper.48 In Labrador retrievers, the urinary copper/zinc ratio in a single urine sample was significantly associated with hepatic copper concentration. However, due to overlap in urine copper/zinc ratio between dogs with normal and high hepatic copper, the diagnostic value of this parameter is limited.48 Copper in serum does not correlate with hepatic copper concentration, and, currently, other possible biomarkers for copper status in the dog, including ceruloplasmin, are under investigation in our lab. Toward an optimal treatment protocol The aim of treatment is to create a negative copper balance, by restriction of copper uptake or by promotion of copper excretion. As in humans, the copper chelator d-penicillamine is most commonly used in dogs to promote copper excretion; in addition, the use of tetramine cupruretic agents for copper chelation was described in dogs.49,50 Side effects of d-penicillamine therapy, including nausea and vomiting, are often encountered in dogs; however, the more severe side effects described in humans, including nephropathy, bone marrow depression, and drug-induced systemic lupus erythromatosis, are not described in dogs. In affected Bedlington terriers, lifelong continuous d-penicillamine therapy is often recommended. However, in dog breeds with a complex form of copper-associated hepatitis, continuous d-penicillamine administration may lead to copper and zinc deficiency by excretion of these metals in urine.48,51 A study to determine the optimal treatment duration with d-penicillamine in Labrador retrievers showed that the necessary treatment duration was dependent on the hepatic copper concentration at the start of chelation therapy and that the decline in hepatic copper was not linear.51 In dogs, intake of food and water can be very well standardized and controlled, which makes diet an interesting intervention strategy in copper toxico-
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sis. Dog food can be easily standardized in kibble or canned food on which the animal can be maintained for a long period. Such standardized dietary intervention would not be possible or tolerated in human patients. However, lessons learned in the canine models may well be advantageous for management of humans with copper storage diseases. Dietary intake of copper and zinc were positively and negatively associated, respectively, with hepatic copper concentrations in Labrador retrievers.52 This prompted the investigation of a low-copper, highzinc diet in the prevention of reaccumulation of copper in affected Labrador retrievers that were previously successfully treated with d-penicillamine. In approximately one-third of these Labrador retrievers, the diet alone was able to maintain hepatic copper at normal concentrations for 2 years without a significant reaccumulation. In the remaining dogs, reaccumulation occurred, and for these dogs, intermittent courses of d-penicillamine treatment or continuous d-penicillamine in a tapered dose could be useful for long-term management. In dogs, decision making in treatment protocol relies on the evaluation of a liver biopsy, which should be obtained at least once a year.53 Genome-wide association study in Labrador retrievers for unraveling the genetic background of complex forms of copper toxicosis Labrador retrievers with increased hepatic copper concentrations and histological hepatic changes have been recognized both in Europe31 and the United states.54–56 Copper accumulates over time, and overt clinical signs can be recognized from 2.5 years of age onward, but the median age at which dogs are presented to the veterinarian is at 7 years of age (approximate lifespan of a Labrador retriever is 14 years).31 Clinical signs that can be observed are lethargy, decreased appetite, nausea, icteric sclera and mucous membranes, and ascites. Centrolobular copper accumulation may be present without histological changes for a period of time; however, when copper levels exceed 1500 mg/kg dwl, inflammatory changes occur. Progressive fibrosis eventually leads to micro- or macronodular liver cirrhosis, and overall hepatic copper concentrations can decrease in the end stage of disease by replacement of copper-loaded hepatocytes by massive collagen deposits (Fig. 1).
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Figure 1. Case example of a Labrador retriever with copper toxicosis. (A) A female Labrador retriever was admitted to the veterinary clinic 4 weeks after giving birth to a litter of pups. The dog displayed clinical signs of lethargy and inappetence. (B) Physical examination revealed icteric sclerae. (C) The dog was euthanized due to clinical deterioration. The liver displayed severe macronodular cirrhosis. (D) Centrolobular area of a histological liver specimen stained with rubeanic acid showed large amounts of copper granules (black) within hepatocytes and Kuppfer cells. An inflammatory infiltrate with a predominance of lymphocytes was present in the centrolobular areas. The quantitative copper measurement was 2210 mg/kg dwl.
One of the main aims of our research in the Labrador retrievers was to elucidate the genetic background of copper-associated hepatitis, which has a high heritability of up to 0.85 in this breed.32 For mapping a complex disease, a genome-wide association study is the preferred approach.57 Mapping of disease genes in inbred dog populations has several advantages compared to mapping studies in humans. Dog breeds are closed populations and display limited locus and disease heterogeneity,58 which reduces the number of dogs and single nucleotide polymorphism (SNP) markers needed for successful mapping of a genetic disease.59 The hepatic copper level was used as the underlying quantitative trait for copper-associated hepatitis in the Labrador retriever. By using 235 Labrador retrievers and 100,000 informative SNP markers, we were successful in identifying two quantitative trait loci for hepatic copper. Next-generation sequencing, replication, and functional studies are currently being performed. Newly identified gene mutations in the Labrador retrievers may help in the understanding of phenotypic variation in human Wilson’s disease patients and in unraveling the genetic
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background of ETIC, ICC, and idiopathic copper toxicosis. Epilogue Companion animal dogs share their environments with humans, and they resemble their caretakers in the development of naturally occurring diseases similar to human diseases. Copper toxicosis in dogs phenotypically resembles copper-storage disease in humans with respect to hepatic copper accumulation and the development of liver cirrhosis. In dogs with autosomal recessive COMMD1 deficiency, copper accumulation and associated liver fibrosis is severe and follows a fixed course in time,22 making them an excellent model for cell transplantation studies and for preclinical studies of new, promising copper-chelating agents such as methanobactin.60 Other dog breeds, including the Labrador retriever, suffer from a complex form of copperassociated hepatitis, which is strongly influenced by oral intake of copper. The disease resembles ecogenetic forms of copper toxicosis in humans, and dietary studies in Labrador retrievers may aid in
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determining dietary components that may benefit human patients. As genetic studies in Bedlington terriers with copper toxicosis led to the discovery of COMMD1, elucidation of the genetic background in Labrador retrievers will probably lead to a better understanding of the disease pathogenesis of complex copper metabolism disorders. New findings in the Labrador retriever may have implications for understanding phenotypic variance in people with Wilson’s disease and in elucidating the genetic background in patients with ICC, ETIC, and idiopathic copper toxicosis. At the same time, dogs with naturally occurring copper toxicosis will benefit from joint efforts between veterinarians, geneticists, and medical professionals in the development and evaluation of new diagnostic methods and treatment strategies for copper metabolism disorders. In this way, copper metabolism disorders are an excellent example of how the One Health approach will lead to an improvement of the well-being of humans and dogs as their best companions. Conflicts of interest
New canine models of copper toxicosis
11.
12.
13.
14.
15.
16.
17.
18.
The authors declare no conflicts of interest. 19.
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A N N A L S O F T H E N E W Y O R K A C A D E M Y O F SC I E N C E S Issue: Human Disorders of Copper Metabolism I
New canine models of copper toxicosis: diagnosis, treatment, and genetics Hille Fieten, Louis C. Penning, Peter A.J. Leegwater, and Jan Rothuizen Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands Address for correspondence: Hille Fieten, Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 108, 3584 CM Utrecht, the Netherlands. [email protected]
The One Health principle recognizes that human health, animal health, and environmental health are inextricably linked. An excellent example is the study of naturally occurring copper toxicosis in dogs to help understand human disorders of copper metabolism. Besides the Bedlington terrier, where copper toxicosis is caused by a mutation in the COMMD1 gene, more complex hereditary forms of copper-associated hepatitis were recognized recently in other dog breeds. The Labrador retriever is one such breed, where an interplay between genetic susceptibility and exposure to copper lead to clinical copper toxicosis. Purebred dog populations are ideal for gene mapping studies, and because genes involved in copper metabolism are highly conserved across species, newly identified gene mutations in the dog may help unravel the genetic complexity of different human forms of copper toxicosis. Furthermore, increasing knowledge with respect to diagnosis and treatment strategies will benefit both species. Keywords: copper; dog; liver; genetics; One Health
Introduction Copper is an essential micronutrient for many organisms, but can be toxic when present in excessive amounts. For this reason, copper metabolism is tightly regulated and proteins involved in copper homeostasis are highly conserved across species, including humans and dogs. The liver is pivotal in copper metabolism and provides for the redistribution of copper after intestinal uptake and the excretion of excess copper via the bile. The importance of appropriate regulation of copper metabolism is illustrated by the autosomal recessive disorder Wilson’s disease, which is caused by mutations in the copper transporter ATP7B1,2 (Table 1). Clinical signs of Wilson’s disease are related to copper accumulation in the liver and brain and include chronic hepatitis and neurological or psychiatric symptoms.3 Wide ranges in age of onset and clinical presentation are recognized and there is often a lack of genotype–phenotype correlation,4 which may implicate environmental influence or an effect of modifier genes that influence clinical presentation.
Ecogenetic disorders of copper metabolism, in which genetically predisposed individuals exposed to high levels of exogenous copper develop clinical disease, include endemic Tyrolean infantile cirrhosis (ETIC),5 Indian childhood cirrhosis (ICC),6 and idiopathic copper toxicosis (Table 1).7 In these disorders, copper accumulation is restricted to the liver, and the manifestation of clinical symptoms is likely influenced by the level of copper uptake through the diet or drinking water. The genetic background of these diseases is currently unresolved. The wide variability of the human genome, the rarity of the diseases, and the variation in clinical phenotypes hamper genetic mapping studies of the diseases in humans. Several animal models for human copper toxicosis exist, such as rodent models,8–11 North Ronaldsay sheep,12 and dogs. Since their domestication, dogs have undergone artificial selection leading to the development of isolated populations of dog breeds. By inbreeding and selection on specific phenotypic characteristics, recessive diseases can be present at high frequencies in purebred dog populations, and many breeds are doi: 10.1111/nyas.12442
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Table 1. Copper toxicosis phenotypes recognized in humans and dogs
Gender predisposition Autosomal recessive copper toxicosis Wilson’s disease No
COMMD1-deficient dogs Ecogenetic copper toxicosis ICC
Age of onset
Childhood, adult
Clinical signs
Involved genes
Dietary influence
Neurologic and hepatic Hepatic
ATP7B
Possibly
COMMD1
Minor
Genetic susceptibility Genetic susceptibility Unknown Complex inheritance pattern
Suspected
No
Young to middle-aged dogs
Male
Infants
Hepatic
ETIC
No
Infants
Hepatic
ICT Labrador retriever
No Female
Childhood, adult Young to middle-aged dogs
Hepatic Hepatic
Suspected Unknown Major
ICC, Indian childhood cirrhosis; ETIC, endemic Tyrolean infantile cirrhosis; ICT, idiopathic copper toxicosis.
at increased risk for specific disorders with complex inheritance patterns.13 The limited genetic variability within inbred dog populations works as a magnifying glass, instrumental to discovering crucial and modifier genes involved in complex genetic diseases that are of interest for both dogs and humans. Furthermore, dogs with copper toxicosis can be a valuable large animal model for evaluation of new treatment strategies beneficial to both the human and canine patients. Canine copper toxicosis With respect to copper metabolism disorders, copper toxicosis in the Bedlington terrier is a good example in which gene mapping studies in the dog led to the identification of a gene that was previously unknown to be involved in copper metabolism14 (Table 1). A chromosomal deletion of 39.7 kb encompassing exon 2 of the originally named MURR1 gene caused a complete absence of the protein product,15 leading to extreme accumulation of hepatic copper. MURR1, later renamed COMMD1, was found to be a versatile gene, with many cellular functions including roles in sodium metabolism,16–18 regulation of NF-B,19,20 and HIF1␣-mediated transcription.21–23 COMMD1 plays a role in the functioning and stability of the human Wilson’s
disease gene ATP7B,24 providing a clue to how COMMD1 absence leads to copper accumulation within hepatocytes. The prevalence of Bedlington terrier copper toxicosis was very high, and estimates within different populations ranged from 25% to 40%.25,26 This prevalence dropped dramatically after the implementation of a DNA test in breeding strategies, which led to an almost complete eradication of the disease. Within the facilities of the Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, we keep a colony of COMMD1-deficient dogs for studies into the progression of chronic hepatitis27 and for the development of stem cell therapy. Increased levels of hepatic copper with or without hepatic pathology have been recognized in other breeds as well.28–30 Different forms of copper-associated hepatitis were recognized in a number of other dog breeds. Pedigree studies executed in breeds with different forms of copper-associated hepatitis including the Labrador retriever,31,32 Doberman,29,33 West Highland White terrier,34 and Dalmatian35 confirmed a hereditary background. In these breeds, a complex form of copper-associated hepatitis is present, where the susceptibility to copper is genetically determined and the expression of the disease phenotype relies
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on environmental factors such as dietary copper intake. In a retrospective review of primary hepatitis cases in our department, approximately one-third of the cases were copper related.36 Parallels can be made between these canine forms of complex copper-associated hepatitis and the human ecogenetic disorders of copper metabolism ETIC, ICC, and idiopathic copper toxicosis (Table 1). Clinical presentation The age at which dogs are presented to the veterinarian with clinical signs is variable and may depend on the breed, exposure to copper in the diet and drinking water, and gestation and lactation in bitches. In Bedlington terriers, hepatic copper accumulation is severe and levels of 2000 mg/kg dry weight liver (dwl) can be observed in dogs of 1 year of age,37 which is five times higher than the upper reference of 400 mg/kg dwl of hepatic copper in dogs.38 Initially, hepatic copper accumulation occurs without overt histological signs of hepatic inflammation; however, when hepatic copper levels rise up to 10,000 mg/kg dwl at around 3 years of age, affected Bedlington terriers may express nonspecific clinical signs including lethargy, anorexia, vomiting, and polyuria/polydipsia. In more advanced cases, icterus, ascites, and hepatic encephalopathy may be recognized. Affected Bedlington terriers may have severe acute hepatitis, resulting in a massive release of copper into the circulation, leading to a hemolytic crisis. In the dog breeds with complex copper-associated hepatitis, hepatic copper accumulation may also start early in life, but is less severe and mostly does not reach hepatic copper levels >5000 mg/kg dwl. However, more severe histological inflammatory lesions are often recognized at lower levels of hepatic copper, compared to the Bedlington terrier. In the Labrador retriever and Doberman, a strong female predisposition for copper-associated hepatitis has been noted.31,39 Comparable to humans with Wilson’s disease, Fanconi’s syndrome was recognized in a Clumber spaniel,40 a West Highland White terrier,41 and a case series of nine Labrador retrievers42 with copperassociated hepatitis. The dogs showed a marked increase in copper concentration in the kidney with accumulation of copper granules in the proximal renal tubules leading to proximal renal tubular dysfunction. Serum biochemical analysis and urinalysis 44
resolved spontaneously with appropriate chelation therapy.40–42 In contrast, Kaiser–Fleischer rings and neurological symptoms other than hepatic encephalopathy are not recognized in dogs with copper toxicosis. Laboratory diagnosis The first indicators for hepatocyte damage are an increase in alanine aminotransferase (ALT) and a slight decrease in serum albumin. In advanced disease, a more pronounced decrease in albumin and increases in ALT, alkaline phosphatase (AP), bile acids, and ammonia can be present. These abnormalities do not distinguish copper-associated liver disease from any other parenchymal liver diseases. Currently, a DNA test is only available for the Bedlington terrier. For all other dogs, a definitive diagnosis relies on histological evaluation of a liver biopsy. Ultrasonography of the liver can be used to evaluate the size, echo density, and structure of the liver and is useful for guiding percutaneous liver biopsies under local anesthesia with or without light sedation. A more invasive way to obtain liver biopsies is through laparotomy or laparoscopy, requiring general anesthesia, which enables visible inspection of the liver and the opportunity to obtain larger specimens. Approximately one-third of first-line relatives of affected Labrador retrievers have increased hepatic copper levels;31 therefore, it is recommended to evaluate liver biopsies from relatives of affected dogs. When treatment is started in the subclinical phase, treatment outcome is far more favorable compared to dogs that are admitted in end-stage liver disease. Evaluation of formalin-fixed liver biopsies includes hematoxylin and eosin,43 reticulin,44 and copper stains, such as rubeanic acid45 or rhodanin,46 for respective grading of the inflammatory activity and routine evaluation, for staging of fibrosis, and for semiquantitative determination of copper level. Localization of copper in the centrolobular hepatocytes of the liver lobule is characteristic for canine primary copper toxicosis. An inflammatory mononuclear or mixed infiltrate accompanies the copper-loaded hepatocytes, and in more advanced stages of the disease apoptosis/necrosis of affected hepatocytes and copper-loaded macrophages (Kupffer cells) can be recognized in association with the centrizonal hepatocytic copper accumulation. In advanced disease, apoptosis, necrosis,
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regeneration, and typical centrocentral bridging fibrosis are seen, eventually leading to end-stage liver micro- or macronodular liver cirrhosis. A separate biopsy is used for quantitative copper measurement by instrumental neutron activation analysis47 or by spectrophotometric methods. Parallel to the use of urine copper excretion in therapeutic monitoring in Wilson’s disease, urinary copper and zinc excretion in relation to hepatic copper concentration was evaluated for use as a noninvasive screening tool for copper.48 In Labrador retrievers, the urinary copper/zinc ratio in a single urine sample was significantly associated with hepatic copper concentration. However, due to overlap in urine copper/zinc ratio between dogs with normal and high hepatic copper, the diagnostic value of this parameter is limited.48 Copper in serum does not correlate with hepatic copper concentration, and, currently, other possible biomarkers for copper status in the dog, including ceruloplasmin, are under investigation in our lab. Toward an optimal treatment protocol The aim of treatment is to create a negative copper balance, by restriction of copper uptake or by promotion of copper excretion. As in humans, the copper chelator d-penicillamine is most commonly used in dogs to promote copper excretion; in addition, the use of tetramine cupruretic agents for copper chelation was described in dogs.49,50 Side effects of d-penicillamine therapy, including nausea and vomiting, are often encountered in dogs; however, the more severe side effects described in humans, including nephropathy, bone marrow depression, and drug-induced systemic lupus erythromatosis, are not described in dogs. In affected Bedlington terriers, lifelong continuous d-penicillamine therapy is often recommended. However, in dog breeds with a complex form of copper-associated hepatitis, continuous d-penicillamine administration may lead to copper and zinc deficiency by excretion of these metals in urine.48,51 A study to determine the optimal treatment duration with d-penicillamine in Labrador retrievers showed that the necessary treatment duration was dependent on the hepatic copper concentration at the start of chelation therapy and that the decline in hepatic copper was not linear.51 In dogs, intake of food and water can be very well standardized and controlled, which makes diet an interesting intervention strategy in copper toxico-
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sis. Dog food can be easily standardized in kibble or canned food on which the animal can be maintained for a long period. Such standardized dietary intervention would not be possible or tolerated in human patients. However, lessons learned in the canine models may well be advantageous for management of humans with copper storage diseases. Dietary intake of copper and zinc were positively and negatively associated, respectively, with hepatic copper concentrations in Labrador retrievers.52 This prompted the investigation of a low-copper, highzinc diet in the prevention of reaccumulation of copper in affected Labrador retrievers that were previously successfully treated with d-penicillamine. In approximately one-third of these Labrador retrievers, the diet alone was able to maintain hepatic copper at normal concentrations for 2 years without a significant reaccumulation. In the remaining dogs, reaccumulation occurred, and for these dogs, intermittent courses of d-penicillamine treatment or continuous d-penicillamine in a tapered dose could be useful for long-term management. In dogs, decision making in treatment protocol relies on the evaluation of a liver biopsy, which should be obtained at least once a year.53 Genome-wide association study in Labrador retrievers for unraveling the genetic background of complex forms of copper toxicosis Labrador retrievers with increased hepatic copper concentrations and histological hepatic changes have been recognized both in Europe31 and the United states.54–56 Copper accumulates over time, and overt clinical signs can be recognized from 2.5 years of age onward, but the median age at which dogs are presented to the veterinarian is at 7 years of age (approximate lifespan of a Labrador retriever is 14 years).31 Clinical signs that can be observed are lethargy, decreased appetite, nausea, icteric sclera and mucous membranes, and ascites. Centrolobular copper accumulation may be present without histological changes for a period of time; however, when copper levels exceed 1500 mg/kg dwl, inflammatory changes occur. Progressive fibrosis eventually leads to micro- or macronodular liver cirrhosis, and overall hepatic copper concentrations can decrease in the end stage of disease by replacement of copper-loaded hepatocytes by massive collagen deposits (Fig. 1).
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Figure 1. Case example of a Labrador retriever with copper toxicosis. (A) A female Labrador retriever was admitted to the veterinary clinic 4 weeks after giving birth to a litter of pups. The dog displayed clinical signs of lethargy and inappetence. (B) Physical examination revealed icteric sclerae. (C) The dog was euthanized due to clinical deterioration. The liver displayed severe macronodular cirrhosis. (D) Centrolobular area of a histological liver specimen stained with rubeanic acid showed large amounts of copper granules (black) within hepatocytes and Kuppfer cells. An inflammatory infiltrate with a predominance of lymphocytes was present in the centrolobular areas. The quantitative copper measurement was 2210 mg/kg dwl.
One of the main aims of our research in the Labrador retrievers was to elucidate the genetic background of copper-associated hepatitis, which has a high heritability of up to 0.85 in this breed.32 For mapping a complex disease, a genome-wide association study is the preferred approach.57 Mapping of disease genes in inbred dog populations has several advantages compared to mapping studies in humans. Dog breeds are closed populations and display limited locus and disease heterogeneity,58 which reduces the number of dogs and single nucleotide polymorphism (SNP) markers needed for successful mapping of a genetic disease.59 The hepatic copper level was used as the underlying quantitative trait for copper-associated hepatitis in the Labrador retriever. By using 235 Labrador retrievers and 100,000 informative SNP markers, we were successful in identifying two quantitative trait loci for hepatic copper. Next-generation sequencing, replication, and functional studies are currently being performed. Newly identified gene mutations in the Labrador retrievers may help in the understanding of phenotypic variation in human Wilson’s disease patients and in unraveling the genetic
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background of ETIC, ICC, and idiopathic copper toxicosis. Epilogue Companion animal dogs share their environments with humans, and they resemble their caretakers in the development of naturally occurring diseases similar to human diseases. Copper toxicosis in dogs phenotypically resembles copper-storage disease in humans with respect to hepatic copper accumulation and the development of liver cirrhosis. In dogs with autosomal recessive COMMD1 deficiency, copper accumulation and associated liver fibrosis is severe and follows a fixed course in time,22 making them an excellent model for cell transplantation studies and for preclinical studies of new, promising copper-chelating agents such as methanobactin.60 Other dog breeds, including the Labrador retriever, suffer from a complex form of copperassociated hepatitis, which is strongly influenced by oral intake of copper. The disease resembles ecogenetic forms of copper toxicosis in humans, and dietary studies in Labrador retrievers may aid in
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determining dietary components that may benefit human patients. As genetic studies in Bedlington terriers with copper toxicosis led to the discovery of COMMD1, elucidation of the genetic background in Labrador retrievers will probably lead to a better understanding of the disease pathogenesis of complex copper metabolism disorders. New findings in the Labrador retriever may have implications for understanding phenotypic variance in people with Wilson’s disease and in elucidating the genetic background in patients with ICC, ETIC, and idiopathic copper toxicosis. At the same time, dogs with naturally occurring copper toxicosis will benefit from joint efforts between veterinarians, geneticists, and medical professionals in the development and evaluation of new diagnostic methods and treatment strategies for copper metabolism disorders. In this way, copper metabolism disorders are an excellent example of how the One Health approach will lead to an improvement of the well-being of humans and dogs as their best companions. Conflicts of interest
New canine models of copper toxicosis
11.
12.
13.
14.
15.
16.
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The authors declare no conflicts of interest. 19.
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