JPC SYSTEMIC PATHOLOGY

DIGESTIVE SYSTEM

October 2024

D-T05

 

SLIDE A: Signalment (JPC #1783194): 3-year-old female Bedlington terrier

 

HISTORY: This dog presented with lethargy, anorexia, vomiting, and icterus.

 

HISTOPATHOLOGIC DESCRIPTION: Liver: Affecting 30% of the hepatic parenchyma and centered on the centrilobular zone, hepatocytes are either lost or are separated and surrounded by predominantly lymphohistiocytic inflammation with fewer plasma cells and neutrophils. Inflammatory cells are admixed with eosinophilic cellular and basophilic karyorrhectic debris (necrosis), moderate amounts of eosinophilic fibrillar material (fibrin), clear space with dilated lymphatics (edema), and fibrous connective tissue with scattered reactive fibroblasts (fibrosis) that bridges to other central veins (centrilobular bridging fibrosis) or less often to portal areas. Hepatocytes adjacent to the affected centrilobular zones are either shrunken (atrophic), swollen with pale eosinophilic vacuolated cytoplasm and occasionally binucleated (degeneration), or shrunken and angular with hypereosinophilic cytoplasm and pyknotic to karyorrhectic nuclei (single cell death). Macrophages within the areas of inflammation, numerous Kupffer cells, and occasional hepatocytes contain grey-brown to golden brown, granular to globular, intracytoplasmic pigment (copper, lipofuscin, hemosiderin, or bile). Multifocally and most prominent adjacent to the centrilobular zone, scattered bile canaliculi are expanded by curvilinear plugs of green-brown bile (cholestasis). There are also occasional dilated lymphatics within the portal areas (edema).

 

SLIDE B: Rhodanine: Primarily within centrilobular inflammatory foci and centrilobular hepatocytes as well as scattered throughout the remainder of the section, hepatocytes, macrophages, and Kupffer cells contain abundant, intracytoplasmic, red-brown, granular to globular pigment (copper).

 

MORPHOLOGIC DIAGNOSIS: Liver: Hepatocellular degeneration and necrosis, centrilobular, chronic, diffuse, marked, with lymphohistiocytic hepatitis, cholestasis, centrilobular bridging fibrosis, and abundant hepatocellular and histiocytic intracytoplasmic copper, Bedlington terrier, canine.

 

CONDITION: Inherited copper toxicosis of Bedlington terriers 

 

GENERAL DISCUSSION:

• Copper plays a critical role as a cofactor for the following enzymes:

• Cytochrome C oxidase - mitochondrial respiration
• Lysyl oxidase - connective-tissue maturation (collagen cross-linking)
• Superoxide dismutase - antioxidant defense
• Tyrosinase - melanin synthesis
• Ceruloplasmin - iron metabolism
• Dopamine β hydroxylase - neurotransmitter biosynthesis

• Excess copper catalyzes the formation of free-radicals which ultimately results in damage to cellular components (e.g. lipids, proteins, and nucleic acids) via oxidation once cellular protective mechanisms are exhausted (e.g. glutathione)
• Following lysosome saturation, copper accumulates in the nucleus and damages DNA with subsequent apoptosis (likely due to induction of p53 protein)
• Mechanisms of hepatic copper toxicosis:

• Primary metabolic defect in hepatic copper metabolism
• Altered hepatic biliary excretion of copper
• Excess dietary intake of copper

• In Bedlington terriers, autosomal recessive disorder of copper metabolism due to exon 2 deletion of COMMD1 gene (copper metabolism MURR1 domain protein 1) causing progressive hepatic copper accumulation with age, leading to progressive fatal hepatitis or chronic hepatitis with cirrhosis
• Only Bedlington terriers have been shown to accumulate copper continuously throughout life

 

PATHOGENESIS:

Normal copper homeostasis:

• Ingested dietary copper > absorbed by small intestine enterocytes via the divalent metal transporter (DMT1) and copper transporter 1 (Ctr1) > bound to ceruloplasmin, transcuprein, or albumin and transported via the portal blood > liver > sequestered in metallothionein or glutathione in hepatocytes > excretion into bile (major route) or blood regulated by chaperones (e.g. copper-transporting ATPase ATP7A)
  • Serum copper exists in two pools:
    • Exchangeable pool - loosely bound to transcuprein (80%), albumin, or other low molecular weight molecules en route to the liver
    • Tightly bound - copper exported from the liver in the form of ceruloplasmin

Altered copper homeostasis in Bedlington terriers with COMMD1 gene exon 2 deletion:

• Affected dogs have reduced copper excretion into the bile causing increased hepatic and renal copper
• Copper accumulates in almost all hepatocellular organelles; the majority is bound to metallothionein in lysosomes
• Copper does not cause hepatitis in Bedlington terriers until concentrations reach 2000 ppm dry weight (DW)

• Normal is <400 ppm DW
• Hepatic copper levels may reach 12,000 ppm DW in Bedlington terriers with homozygous recessive trait

• After reaching peak levels at 6 years of age, there is a trend of decreasing hepatic copper levels possibly due to: 1) cirrhosis, which dilutes copper since scar tissue does not contain copper, 2) hepatocyte adaptation, or 3) normal homeostatic regulatory mechanisms
• Chronic cholestasis may also result in elevated copper levels in the liver

 

TYPICAL CLINICAL FINDINGS:

• Ascites, CNS signs, depression, weight loss, vomiting; jaundice inconsistently
• Hemolysis is rare; usually due to an acute event on top of chronic hepatitis 
• Serum copper level is normal regardless of the hepatic copper concentration
• Hypoglycemia

 

TYPICAL GROSS FINDINGS:

 

TYPICAL LIGHT MICROSCOPIC FINDINGS:

• Lesions (necrosis and hepatitis) begin in centrilobular area (zone 3) then spread and increase in size as the disease progresses and are associated with copper-laden hepatocytes
• Progresses to portal and diffuse fibrosis and chronic-active periportal hepatitis, with macronodular regeneration (cirrhosis)
• Golden brown to amphophilic, occasionally refractile granules in hepatocytes and Kupffer cells (autophagolysosomes that contain ferric iron and copper)

 

ULTRASTRUCTURAL FINDINGS:

• Cu < 2000 ppm: Cu in electron dense lysosomes; minimal cytoplasmic changes
• Cu > 2000 ppm: Cu also in the nucleus; mild nuclear degeneration
• Cu > 7000 ppm: Cu accumulates (in descending order) in lysosomes, nucleus, and cytoplasm; shrunken hepatocytes with electron dense organelles, nuclei contracted and misshapen with chromatin condensation and fragmentation, and apoptotic bodies in sinusoids

 

ADDITIONAL DIAGNOSTIC TESTS:

• Special stains (rubeanic acid, rhodanine) can identify copper-laden lysosomes at concentrations > 400 ppm dw
• Blood or kidney copper levels in sheep with suspect chronic copper toxicity

• Quantitative copper analysis: Fresh or formalin-fixed tissues; normal is <400 ppm DW

• Hepatocytes in regenerative nodules often have lower copper levels and intervening areas of parenchymal collapse may contain high copper; because of this regional variation, small samples, (e.g. from needle biopsies), are often inaccurate, and larger samples are required

• Cytology: May suggest the presence of hepatic fibrosis (pink fibrillar extracellular matrix material and spindled mesenchymal cells); copper within hepatocytes stains as pale blue, coarse, crystalline granules with routine cytochemical stains

• Immunolabeling for markers of oxidative stress and apoptosis is positively correlated with hepatic copper accumulation (Yamkate, J Comp Pathol, 2022)

• Clinical pathology

• Elevated ALT and ALP, hyperbilirubinemia, hypoproteinemia, (hypoalbuminemia), hypergammaglobulinemia, bilirubinuria; in advanced CH, liver enzyme activities can appear normal or below reference range 
• Liver function tests: Usually abnormal; possibly prolonged coagulation times, but hemorrhagic diathesis/hemorrhage is unlikely 

 

DIFFERENTIAL DIAGNOSIS:

For chronic hepatotoxicity in dogs:

• Idiopathic chronic active hepatitis (D-M17): Periportal (zone 1) or random small foci of inflammation and piecemeal necrosis; periportal copper accumulation (<2000ppm) secondary to inflammatory disease; typically no cholestasis; micronodular regeneration; hyperglycemia; especially middle-aged, females (Doberman pinschers); the significance of increased hepatic copper concentration in this breed remains controversial
• West Highland white terriers: 

• Inherited copper storage disease; accumulates in centrilobular areas (zone 3)
• No correlation between age and hepatic copper levels (non-progressive), and hepatocellular damage is detected at lower concentrations of copper (< 2000 ppm DW)

• Other dog breeds: Excess hepatic copper causes centrilobular acute hepatic necrosis, subacute hepatitis, chronic hepatitis, and cirrhosis

• Causes may include:

• Metabolic defects (new variant inherited primary toxicosis) – Doberman pinschers and Dalmatians
• Cholestasis (secondary) – Skye terrier hepatitis (intracanalicular)
• High copper levels in commercial dog food

• Also reported in cocker spaniels, German shepherd dogs, keeshonds, Kerry blue terriers, Labrador retrievers, old English sheepdogs and Samoyeds; associated with renal tubular dysfunction in Labrador retrievers

• Drug-induced hepatopathy: 

• Anticonvulsants (primidone, phenytoin, phenobarbital); bridging portal fibrosis, biliary hyperplasia, nodular regeneration, mild inflammatory cell infiltrates Sulfonamides - hepatitis and hepatic necrosis; Doberman Pinschers especially sensitive
• Mebendazole - centrilobular necrosis

• Indospicine poisoning from ingestion of legumes: Centrilobular and piecemeal necrosis with accumulation of ceroid pigment in macrophages 

 

COMPARATIVE PATHOLOGY:

• Animals deficient in vitamin E or molybdenum are especially susceptible to acute copper poisoning
• Sheep: Chronic copper toxicosis (U-T10)

• May result from:

1. Excessive copper intake –water/pasture/feed
2. Increased availability of dietary copper often due to low levels of dietary molybdenum – molybdenum forms insoluble complexes with copper in the gut and liver, making the copper inert
3. Other hepatotoxins – most commonly pyrrolizidine alkaloids (from Heliotropium or Echium) and phomopsin from lupins

• Copper tends to accumulate in the liver of sheep and they have a low rate of excretion into bile, increasing their susceptibility to copper toxicity 

• North Ronaldsay sheep especially susceptible – they eat a diet of seaweed (low in copper and molybdenum but high in zinc), zinc can also interfere with copper uptake, and when these sheep go back on a normal diet they quickly develop copper toxicity

• Rate of hepatocellular loss exceeds capacity of the liver to phagocytize cell debris > plasma copper levels rise > damage circulating erythrocytes > intravascular hemolysis and anemia > increased hepatocellular damage > progressive intravascular hemolysis and liver failure > hemoglobinuria-associated acute tubular injury (formerly hemoglobinuric nephrosis) > death
• Blood or kidney copper levels give a truer indication of a prior hemolytic crisis caused by chronic copper poisoning than elevation of liver copper
• Gross findings in sheep that die of acute hemolytic syndrome secondary to chronic copper toxicity include: Icterus, red discoloration due to hemoglobin but may also be brown due to methemoglobin, splenomegaly, dark red to black kidneys and dark red urine, hepatomegaly with yellow discoloration or atrophic and fibrotic liver if preceded by longstanding liver injury

• Pigs and cattle: Chronic copper poisoning
  • Especially common in calves, associated with acute intravascular hemolysis
  • More hepatic evidence of chronic damage with extensive portal fibrosis and biliary hyperplasia within triads
• Acute copper poisoning:
  • Most often seen in ruminants after administration of single large doses of copper (oral or parenteral)
    • Case report of chronic toxicosis in a crossbred heifer calf (Wong, J Vet Diagn Invest, 2020); liver was diffusely yellow-orange, turgid, and exuded watery, thin blood and the cortex and medulla of both kidneys was dark brown to black; centrilobular hepatocellular degeneration and renal tubules contained intraluminal bright eosinophilic fluid and red-orange granular casts
  • Develop severe gastroenteritis, abdominal pain, diarrhea, and dehydration
  • Liver lesions vary with chronicity of exposure from acute centrilobular necrosis to cholangiohepatitis with periportal fibrosis

• Cats:  Chronic hepatitis suggestive of primary copper storage disease and acute hepatitis secondary to increased copper levels
• Invertebrates: Copper toxicity in several aquatic and semi-aquatic invertebrates; causes “distress syndrome: in freshwater pulmonate snails (immobility, muscular spasms, extension of cephalopedal mass, inability to attach foot process to substrate, swelling of tentacles, epidermal sloughing)
• Animal models: Long-Evans cinnamon rat and toxic milk mice – genetic defect similar to Wilson’s disease in humans

 

REFERENCES:

1. Cullen JM, Stalker MJ. Liver and biliary system. In: Jubb, Kennedy, and Palmer’s Pathology of Domestic Animals. Vol 2. 6th ed. St. Louis, MO: Elsevier, 2016:301-305, 342-343. 
2. Newton AL, Smolowitz R. Invertebrates. In: Terio KA, McAloose D, St. Leger J eds. Pathology of Wildlife and Zoo Animals. Cambridge, MA: Elsevier Inc. 2018:1023-4.
3. Peters LM, Meyer DJ. Chapter 9: Hepatobiliary System. In: Raskin RE, Meyer DJ, & Boes KM eds. Canine and Feline Cytopathology: A Color Atlas and Interpretation Guide. 4th ed. St. Louis, MO: Elsevier; 2022:350-352.
4. Siegel A, Wiseman MD. The Liver. In: Valenciano AC, Cowell RL, eds. Diagnostic Cytology and Hematology of the Dog and Cat. 5th ed. St. Louis, MO: Elsevier Mosby; 2014:331-333, 337-338. 
5. Stockham SL, Scott MA. Enzymes. In: Fundamentals of Veterinary Clinical Pathology. 2nd ed. Hoboken, NJ: Wiley; 2013: 651.
6. Stockham SL, Scott MA. Liver Function. In: Fundamentals of Veterinary Clinical Pathology. 2nd ed. Hoboken, NJ: Wiley; 2013: 677.
7. Wong A, Wilson-Frank CR, Hooser SB, et al. Chronic copper toxicosis in a crossbred heifer calf. J Vet Diagn Invest. 2020; 32(3):458-62.
8. Van Wettere AJ, Brown DL. Hepatobiliary System and Exocrine. In: Zachary JF, ed. Pathologic Basis of Veterinary Disease. 7th ed. St. Louis, MO: Elsevier; 2022:529, 533. 
9. Yamkate P, Lidbury JA, Steiner JM, Suchodolski JS, Giaretta PR. Immunohistochemical Expression of Oxidative Stress and Apoptosis Markers in Archived Liver Specimens from Dogs with Chronic Hepatitis. J Comp Pathol. 2022 May;193:25-36.

 

 

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