Chapter 1:                 LITERATURE REVIEW

1.1         The Basic Structure and Functions of the Liver and Its Role in Disease

The liver arises early in embryogenesis from an out-pouching of endoderm that later forms the duodenum.⁵⁹ By adulthood,  it is the largest solid organ in the body and weighs approximately 1.5 kg in humans and represents 2% of adult body weight.²⁴ In horses and other herbivores, the liver constitutes roughly 1% of body weight,⁵⁹ or about 5kg in an average 500kg equine.

The liver has classically been organized into lobules in which hepatocytes (hepatic epithelial cells) are arranged in cords around a central vein (also referred to as terminal hepatic venules). Venous sinusoids are located between hepatic cords. Along the periphery of the lobule are portal triads consisting of bile ducts, branches of the hepatic artery, portal vein, nerves and lymphatic vessels,⁵⁹ within a network of collagenous connective tissue. The liver has a dual blood supply, receiving 75% to 80% from the gastrointestinal tract (portal circulation) and 20% to 25% arterial blood through the hepatic arteries.²⁴ The blood exits the liver via the sinusoids and central and hepatic veins to the vena cava.²⁴ It is surrounded by a fibrous capsule also known as the capsule of Glisson.

Hepatocytes (hepatic parenchyma) are the most numerous cell type within the liver and occupy 78% to 80% of the total liver volume.²³ Other cell types associated with the parenchyma include intra-sinusoidal Kupffer cells, pit cells, sinusoidal endothelial cells and hepatic stellate cells within the space of Disse.³⁰⁴ The sinusoids, which carry portal blood toward the central vein, are lined by special fenestrated endothelial cells arranged in sieve plates and lack a basal lamina.^252,303 The fenestrae act as a selective sieve, allowing only molecules of certain sizes through, inhibiting the passage of chylomicrons greater than 200 to 250nm.²⁵² Species-specific differences in the number and diameter of fenestrae have been demonstrated in several animals,²⁵² though no data could be found for equines. Additionally, endothelial cells have high endocytic activity, acting as a “scavenger” mechanism for waste molecules from the blood.²⁵²  Hepatic stellate cells are mesenchymal cells that can transform from quiescent lipid and vitamin A storing cells to active myofibroblasts through extracellular signals from inflammatory cells, including Kupffer cells.²⁷⁸ Pit cells are found within the hepatic sinusoid and are frequently found adhered to endothelial cells. These cells have cytotoxic activity and act as liver-specific natural killer cells.¹⁷⁰ Kupffer cells, which are resident macrophages within the hepatic sinusoids and occasionally within the space of Disse,²⁵² have high phagocytic activity and can modulate the immune system through excretion of bioactive factors, including reactive oxygen species, inflammatory and growth control mediators.²³⁰ Their location within the sinusoid makes them the first cells of the innate immune system to come in contact with gastrointestinal-derived foreign and noxious material such as infectious agents, their products (including lipopolysaccharide, LPS) and other toxic substances.²⁵²

In general, the liver serves the following physiologic functions:

The liver plays a central role in carbohydrate and lipid metabolism. It removes ingested carbohydrates and triglycerides that arrive from the gastrointestinal tract via the portal circulation. Energy is stored in the liver as glycogen or exported to other organs as fatty acids.⁵⁹ Glycogen makes up approximately 5% of the wet weight of the liver,²⁸⁸ which would correspond to about 250g in a 500kg equine.  Energy is generated in the form of adenosine triphosphate through glycolysis, and fatty acid oxidation and approximately 28% of the total hepatocyte volume is taken up by mitochondria.²³ The liver is also the primary site of cholesterol synthesis and degradation.⁵⁹ Cholesterol synthesis is dependent on the amount of dietary cholesterol intake, and the rate of synthesis, as well the hepatic contribution thereof, varies between different species.⁷⁴

Synthesis of several plasma proteins also occurs within the liver. These proteins include albumin, ceruloplasmin, lipoproteins, proteins of the coagulation cascade (factors II, V, VII to XIII), some acute phase proteins and those of the complement system.⁵⁹ Protein catabolism is also completed here, with the conversion of toxic ammonia to non-toxic urea which is excreted by the kidneys into the urine.⁵⁹

The liver is important in immune function. For example, hepatocytes produce acute phase proteins and coagulation factors which are involved in the systemic inflammatory response.⁵⁹ Additionally, Kupffer cells and natural killer cells are crucial components of the innate immune system, are first in the line of defense against foreign and noxious materials in the liver. Due to their location within the hepatic sinusoid, Kupffer cells are in a prime position to phagocytize foreign pathogens and materials from both portal and arterial circulation, and, therefore, form a major defense against gastrointestinal immunogenic materials.⁷⁷ Phagocytosis of foreign material, including bacterial endotoxin,⁹⁷ results in the release of numerous bioactive compounds involved in the inflammatory response.^70,168 These include interleukins, interferons, arachidonic acid, leukotrienes, nitric oxide, among others.^70,168 Natural killer cells make up approximately 20 to 30% of the liver resident lymphocytes in humans.⁷⁸

One of the main functions of the liver is the excretion of bile. Bile is an aqueous solution containing cholesterol, bile salts, bilirubin, amino acids, steroid, and enzymes as well as vitamins, heavy metals and toxins.²⁹ Bile is produced by hepatocytes from cholesterol, excreted into bile canaliculi (formed by tight junction sealed apical membranes of adjacent hepatocytes) and drain into bile ducts, making the liver an exocrine organ.²⁹ Bile is modified by the bile duct epithelium by way of energy dependent transport system that creates an osmotic gradient.²⁹ The principal functions of bile are for the excretion of cholesterol and (harmful) lipophilic substances, the emulsification of dietary lipids and the absorption of fat-soluble vitamins (A, D, E, and K).⁵⁹  Bile also serves to transport immune globulin A to the mucosal surface of the gastrointestinal tract.²⁹

The chemical modification of drugs, pollutants, and other potentially harmful molecules for removal from the body is an important task of the liver. Enzymes of the smooth endoplasmic reticulum within hepatocytes are responsible for the transformation of these molecules into metabolites that are more easily excreted, often by making them more water soluble.^6,229 The modification process starts with cytochrome p450 enzymes in a process known as phase I metabolism and is followed by phase II metabolism where conjugates are added through a different set of enzymes. Reabsorption of some metabolites from the gastrointestinal tract occurs in a process known as enterohepatic circulation.⁶

The liver also serves as an endocrine organ, producing insulin-like growth factor-1 and angiotensinogen.⁹³ More often, it is involved in the metabolic transformation of sterol and thyroid hormones, for example, the conversion of T₃ to T₄ or androgens to estrogens.¹⁰

As with all other organs, the liver can be affected by primary disease. However, because of its involvement in numerous processes, as outlined above, and its unique exposure to both the portal and systemic circulation, it is also particularly susceptible to secondary disease.  Hepatic involvement in gastrointestinal tract disease is not uncommon.³⁵ The inflammatory mediators that the liver produces not only are crucial in the clearing of bacteria and other immunogenic material but can also create hepatic inflammation and damage.³⁰⁷ Despite the myriad of disease-causing agents and processes, the liver reacts in limited and predictable ways to injury. These include inflammation, fibrosis, biliary hyperplasia, and regeneration.⁵⁹ Because of this, it is often difficult to discern primary versus secondary hepatic disease on histology alone.

1.2         Reaction Patterns of the Liver in Disease

1.2.1        Hepatic inflammation

Human hepatic diseases in which inflammation is central to the disease process include: alcoholic liver disease, nonalcoholic steatohepatitis (progressive form of nonalcoholic fatty liver disease),¹² ischemia/ reperfusion injury, parasitic infection, and viral hepatitis.^12,144

Kupffer cells make up nearly 15% of all cells in the liver.²⁸ Kupffer cells are tantamount in the initiation and progression of the inflammatory response, both locally within the liver and the systemic response. The myriad of pro-inflammatory, biologically active products produced by activated Kupffer cells is reviewed extensively by Decker.⁷⁰ The pro-inflammatory mediators produced by Kupffer cells may have deleterious effects on normal hepatic constituents and therefore are important in the development of hepatitis.²¹ Blockage of Kupffer cell function by gadolinium chloride resulted in a decrease in both hepatocellular necrosis and inflammation after (hepatotoxic) cadmium exposure.^76,237 Natural killer T cells play a co-stimulatory role, along with Kupffer cells, and induce the Th-1 response. Later, during chronic inflammation, there is a shift to cytokines that induce the Th-2 response.¹²⁶ Sinusoidal endothelial cells, hepatic stellate cells, natural killer cells, and hepatocytes also produce inflammatory mediators, that coordinate with Kupffer cells to recruit neutrophils.⁷³

The liver is involved in sepsis and critical for the clearance of bacteria and toxins from the bloodstream.²²² Patients with sepsis had liver lesions that included portal inflammation, lobular inflammation, centrilobular necrosis hepatocellular necrosis, cholangitis, and steatosis.¹⁶⁰ Hepatic dysfunction occurs early in sepsis and includes decreased glucose and xenobiotic metabolism, but hepatic lesions may not be apparent at this time. Later in the septic process, and as a response to decreased hepatic function, hepatocellular damage, cholestasis and suppurative inflammation occurred.¹⁹⁷ During sepsis, the liver is responsible for several functions including production of pro-inflammatory cytokines, coagulation factors, complement and other acute phase proteins.³⁰⁷ Neutrophils are attracted to the liver by the production of chemokines secreted by Kupffer cells, and together with platelets, Kupffer cells, sinusoidal endothelial cells, and hepatic stellate cells help clear bacteria.³⁰⁵ Some B lymphocytes also display phagocytic activity.²²⁸

 

1.2.2        Hepatic fibrosis

Hepatic fibrosis may develop in response to viral hepatitis, therapeutic agents, alcohol abuse, autoimmune disease, non-alcoholic steatohepatitis, or metabolic disease in humans.¹⁵ It is considered a form of wound healing, whereby the “scar” is characterized by the exuberant accumulation of extracellular matrix (ECM). There is a shift in the composition of the ECM during fibrosis, from basement membrane matrix to matrix containing dense fibrillar collagen.⁹⁸

Liver fibrosis has traditionally been viewed as an irreversible process, even if the underlying cause is removed.⁹⁹ More recent evidence has demonstrated that fibrosis is reversible, at least in part, in certain instances. For example, there was a significant reduction in hepatic fibrosis in the majority of patients after alleviation of stenosis of the common bile duct.¹¹⁸ In addition, fibrosis can be reversed or stabilized in up to 80% of patients with chronic hepatic inflammation.⁶³ The use of anti-inflammatories also highlights the importance of inflammation in the development of hepatic fibrosis. Reversal or stabilization of hepatic fibrosis is, however, not consistent in every case and the mechanisms of this are still being worked out.

Acute hepatocellular damage results in regeneration of those cells. However, in chronic injury, regeneration fails and the liver is substituted with abundant ECM.¹⁵ Hepatic stellate cells are activated after injury and transform to myofibroblasts who are able to migrate to the area of injury and produce large quantities of ECM.^177,187 Kupffer cells are one of the major sources of TGFß1, which stimulates stellate cells to transform into myofibroblasts.¹⁵⁸ Myofibroblasts present within the portal area may also contribute to production of ECM.¹⁵³

Reversal of hepatic fibrosis may occur after removal of the insulting agent and has been observed in experimental models and human patients.^63,134 Collagenolysis occurs by interstitial matrix metalloproteinases, activated due to the decrease in the expression of tissue inhibitor of metalloproteinase-1.¹³⁴ Furthermore, the remodeling of the ECM may result in apoptosis of hepatic stellate cells.¹³⁴ Alternatively, reversion of hepatic stellate cell activation may occur by interleukin-10, which has been shown to down-regulate inflammation and increase collagenase activity.^274,291

1.2.3        Hepatic regeneration

The liver is unique in its ability to regenerate. Two-thirds of the liver can be removed without clinical recourse, and it returns to normal mass within the span of a few weeks.¹²² Unfortunately, the liver is unable to regenerate fully after chronic injury, whereby the functional hepatic parenchyma is replaced by connective tissue.²⁵ After an injury, hepatocytes are the first cell type that enter the cell cycle and undergo mitosis, followed by the proliferation of cholangiocytes, hepatic stellate cells, and Kupffer cells.²²⁶ However, hypertrophy of hepatocytes occurs before cell division in models after 70% partial hepatectomy (PHx). In models of 30% PHx, hepatocytes infrequently enter into mitosis, and organ mass is regained predominantly by hypertrophy.¹⁹² Biliary hyperplasia (discussed below) through hepatic progenitor cells, may form an additional line of regeneration. Hepatic progenitor cells, with proper growth factors produced by activated hepatic stellate cells,⁸⁹ differentiate into basophilic hepatocytes within four to five days.^89,90 In one study, in patients with severe hepatic impairment, hepatic progenitor cells were activated after 50% loss of hepatocytes.¹⁴⁹ The hepatic progenitor cell is also able to produce alpha-fetoprotein and albumin, suggesting an additional important physiologic function during hepatic dysfunction.⁹⁰ Angiogenesis is also initiated during hepatic regeneration to re-establish the hepatic vasculature.²⁵

Coordination of hepatic regeneration involves numerous growth factors and other metabolic networks.^25,173 Tumor necrosis factor-α (TNF-α) binds to Kupffer and other non-parenchymal cells, which triggers the release of nuclear factor κB, and interleukin-6 (IL-6).⁹⁵ Both can act on hepatocytes and are key in initiating hepatocellular regeneration through intracellular signaling pathways.^57,95 In turn, hepatocytes produce signals for other cell types to stimulate mitosis, including TGF-α, FGF1/2, VEGF, GM-CSF, and angiopoeitin1/2.^185,186

Hepatic regeneration is impaired by numerous therapeutic agents, including chemotherapeutic drugs, statins, ß- blockers, non-steroidal anti-inflammatories, and others.^159,239  In addition, after PHx in rodents, a period of hypoglycemia occurs, and supplementation of dextrose also impairs hepatic regeneration. This suggests that the regenerative response is coupled to the metabolic needs of the animal.²⁹⁹ Regeneration ultimately leads to and stops when there is 100% restoration of a species and age specific ratio of total body mass.^159,206,271

Several animal models have been developed to study hepatic regeneration. Zebrafish are becoming more relevant models for developmental biology in general, and have been the focus of studies of liver development and disease research.^107,300 The most cited model is the rodent model after PHx, in which the large and median lobes of the liver are surgically removed, resulting in loss of approximately two-thirds of the liver.^103,122,276 Rodent models involving chemically-mediated hepatotoxic injury include the use of carbon tetrachloride (CCL₄),¹³³ and D-galactosamine.¹⁶⁶ Hepatic injury caused by acetaminophen has also been studied in rodents.¹⁵⁵ Genetically modified animal models have also been developed in mice,¹⁴ and swine.¹²⁰

1.2.4        Biliary hyperplasia

Biliary hyperplasia, also referred to as bile duct hyperplasia or ductular reaction is a reactive process that occurs with hepatic exposure to various insults, and is not limited to biliary injury. insults. It involves proliferation of biliary epithelial cells, hepatic progenitor cells and occasionally metaplasia of mature hepatocytes.⁶⁰ In animals, there are three recognized mechanisms for biliary hyperplasia.⁶⁰ Bile duct obstruction leads to ductular multiplication of cholangiocytes within the portal stroma, and these small ducts contain no bile.¹¹⁰ Leakage of bile into the surrounding tissue may result in recruitment of neutrophils. Chronic fibrosis and eventually cirrhosis will occur if the obstruction cannot be relieved.¹¹⁰ In liver diseases in which there is severe damage to mature hepatocytes, and especially when they are unable to replicate, ductular reaction involves proliferation and differentiation of bipotent hepatic progenitor stem cells (previously referred to as oval cells).⁶⁰ These cells are located within the canals of Hering and are cytokeratin-7 and -9 positive. This corresponds to a biliary phenotype, despite a more prominent hepatocyte morphology.²⁰⁸ In contrast, mature hepatocytes are positive for cytokeratin 8 and 18.⁹¹ With ductular reaction, progenitor cells replicate and differentiate to form numerous small and tortuous ducts. A final mechanism of ductular reaction described is under conditions of hypoxia when mature hepatocytes can undergo metaplasia and proliferation with an intermediate phenotype between cholangiocytes, and hepatocytes.⁷² Ductular reactions may lead to parenchymal regeneration, fibrosis, and cirrhosis. They may also play a role in neoplasia, either through malignant transformation of the hepatic progenitor cells or by contributing to the development of a tumor microenvironment.¹¹⁰

In rats, biliary hyperplasia with hepatic progenitor cell proliferation is seen with D-galactosamine and CCl₄ induced hepatic damage.^166,249 and experimental fascioliasis.¹⁶⁹ Ductular reaction, both of the biliary replication and hepatocyte metaplasia variants, have been described in canine liver disease, including chronic hepatitis, cirrhosis and cholangiocarcinoma.^129,309 In equines, biliary hyperplasia is seen with pyrrolizidine alkaloid toxicosis, aflatoxicosis, and serum hepatitis.⁶⁰ There are species specific differences in the types of biliary reactions. Therefore discernment is required when comparing animal, and human disease models.^72,110

1.3         Metallothionein

1.3.1        Introduction

Metallothionein (MT) was first isolated from the equine kidney in 1957 by Margoshes and Vallee as a cadmium binding protein from the equine renal cortex.¹⁷⁶ It was later purified and further characterized by Kägi and Vallee in 1960.¹⁴⁵ MT is a ubiquitously expressed, low molecular-weight (500-1400 Da) cysteine rich intracellular protein.¹⁷⁶ At the time of its discovery, MT’s physical characteristics were suggestive of a homeostatic biological role, such as catalysis, detoxification, or storage.¹⁴⁵ Early research focussed on the role of metallothionein in heavy metal detoxification, particularly that of cadmium.²⁹² Research into its potential role has since grown exponentially, and a recent PubMed search for “metallothionein” resulted in over 12,900 publications, with 207 publications already in the first half of  2017. Research is focussed on a wide variety of topics such as physical chemistry, molecular biology, and environmental pollution monitoring. Much of the applied medical research has involved studies on humans and research animals (predominantly rodents), involving both biochemical and genetic studies of its role. MT’s true biological function, however, remains ill-defined, despite over 50 years of research.²¹⁰ It has been extensively reviewed in the literature.^{56,68,117,131,132,194,210,235,272,273}

Metallothionein is a low molecular weight (< 7 kDa), cysteine rich protein with a high affinity for divalent metals, such as zinc, copper, cadmium, and mercury.¹⁴⁵ Because of their unique bioinorganic structure and their ability to bind metals, they are referred to as metalloproteins.²⁷² Their unique chemical structure confers (heat) stability, a degree of specificity, and dynamic behavior.⁵⁶

 

Metallothionein is a ubiquitously expressed protein and found in a large diversity of organisms including yeast,²⁹⁴ fungi, and plants.²⁴² A review of MT in animals including mammals, vertebrates, nematodes, annelids, mollusks, and arthropods, among others is provided by Isani and Carpene.¹³² In it they describe the genetic heterogeneity between MT isoforms of different species, and that these differences do not appear to follow an evolutionary trend in that the least complex MT isoforms appear in more evolved vertebrates.¹³² In primitive life forms, MT plays a role in the sequestration of toxic environmental metals.⁵⁶ For example, MT is constitutively expressed within the pharynx of  Caenorhabditis elegans which may act to induce MT expression within intestinal cells upon heavy metal exposure.¹²³ The potential diversity of the function of the MT protein, based on its structure and biochemistry, is limited only by what is asked of it, from an evolutionary perspective. Therefore, the “true” function of MT is most likely dependant on the particular needs of that organism, even though it is frequently cited that there is difficulty in finding a unified role for MT, due to its specific and variable roles in different life-forms.^22,56

There are four major groups of MT isoforms, which are classified based on numerous factors such as their molecular weight, metal which binds, and genetics.²⁷³ The major isoforms include MT-1 and MT-2, whereas the minor isoforms include MT-3 and MT-4. MT-1 and -2 are the major isoforms and are expressed during all stages of development and in most organs.²⁰⁹ MT-3 and -4 are considered minor isoforms. MT-3 was first isolated from brain neurons, and functions as a growth inhibitory factor.²⁸³ MT-3, in addition to neurons, is also expressed in the male reproductive organs.¹⁹³ MT-4 was first isolated from differentiating stratified squamous epithelia.²²⁵ It is also found in the upper gastrointestinal tract where it functions to help regulate stomach pH, the sense of taste and texture on the tongue and protection against UV damage of the skin.²⁷³

Not only do the MT isoforms show tissue specificity, but also in the metals that they bind in those tissues. MT is bound predominantly to zinc in the human and equine liver.^34,223 However, it mostly binds cadmium and zinc in nearly equal proportion in the human kidney.²²³ Equine renal cortex has the highest levels of cadmium, in relation to other organs, and MT represents 1 to 2% of the total weight of soluble protein in the renal cortex.¹⁴⁵

Metallothionein shows a wide variation in expression in different species and tissue types, and this concentration may be dependant on the age, diet, and developmental stage of the organism.¹⁴⁶ For example, human fetuses did not show evidence of MT expression by immunohistochemistry (IHC).¹⁰⁰ MT protein synthesis in rat intestinal mucosal cells is related to high levels of zinc in the diet.¹⁸¹ In dogs, MT immunoreactivity has been demonstrated in glandular epithelia (mammary, uterine, sweat, olfactory, thyroid, and perianal), as well as fundic cells of the stomach, intestinal epithelial cells and hair follicle epithelial cells.²⁴⁷ In the equine, MT has been isolated and sequenced from in the liver, kidney and intestine.^117,156,157

Within the cell, MT is predominantly found in the cytoplasm. However, degradation of hepatic MT occurs predominantly within lysosomes, and the release of its bound metal is a prerequisite for this process.⁴⁹ It has been observed that MT accumulates bound to copper within hepatic lysosomes of Bedlington Terriers with inherited copper toxicosis.¹⁴¹ Transient translocation of MT into the nucleus has been seen in cells during proliferation and differentiation, and the elevated levels of MT in the nucleus may reflect an increase in demand for zinc (for metalloenzymes and transcription factors), or to protect from DNA damage which may lead to apoptosis.^46,50,224

In humans, there are at least ten functional MT isoforms, of the four types, encoded by a family of 14 genes on chromosome 16.²⁹⁷ In contrast, there are only four MT encoding genes on chromosome 8 in mice.²²⁵ This has implications for gene regulation, and that what we know in mouse models may not apply to other species.

Transcriptional regulation of the MT gene is achieved in a variety of ways. Within the promotor are a number of regions responsive to different molecules. Metals, bound to transcription factors, bind to specific areas of the promotor known as metal response elements (MRE). For example, binding of MRE-binding transcription factor-1 (MTF-1) is a zinc finger protein and is activated by zinc binding to DNA.⁶⁸ Free zinc can bind to the MRE via MTF-1, thus increasing MT mRNA.²⁹⁰ MT DNA transcription is also regulated by stress, through glucocorticoid response elements within the MT gene promotor.¹⁵¹ Hormones and cytokines may act on the promoter through intracellular signaling such as protein kinases.⁶⁸ For example, 1 alpha, 25-dihydroxy vitamin D3 is known to increase the level of MT mRNA in mice when orally administered.¹⁴⁷ Bacterial endotoxin (LPS) is also known to increase MT mRNA, potentially through IL-6 and other cytokines in the mouse.⁶⁹ Additionally, DNA methylation of the promoter region is associated with suppression of MT gene expression in neoplastic hepatocytes in rats, and some other types of tumors.¹⁰⁴ Regulation of MT gene expression is achieved through many signals, both endogenous and exogenously produced.

1.3.2        Metallothioneinand its role in inflammation

Metallothionein plays a very important role in inflammation and is considered to have anti-inflammatory properties. In humans, patients with diseases such as autoimmune or inflammatory bowel disease, cholestasis, and lymphoma had marked increased expression of hepatic MT by immunohistochemistry.⁷ Bacterial endotoxin (LPS) can rapidly induce the transcription of hepatic metallothionein in mice.⁶⁹ In addition, IL-6, TNF-α, and interferon-γ (IFN-γ) were also able to induce hepatic metallothionein transcription, when injected intraperitoneally.⁶⁹

The relationship of metallothionein with the acute phase response has been investigated, and MT has been considered an acute phase protein because of its rapid response to inflammatory stimuli and association with other acute phase proteins. A significant relationship between MT and fibrinogen levels was found in mice injected with paraquat, menadione, and CCl₄ subcutaneously. Furthermore, this hepatic MT was bound to zinc.¹⁹⁰

The relationship between zinc levels and acute inflammation are well established. Zinc plays an important role in both the innate and adaptive immune system.^26,243 Hypozincemia has been seen during episodes of acute stress, such as during sepsis in which patients also have increased markers of oxidative stress and inflammatory biomarkers.¹⁸⁴ However, hypozincemia is associated with concomitant increases in hepatic zinc levels.²⁶⁴ Hepatic levels of zinc increased in mice following LPS exposure, ^255,264  but this accumulation did not occur in MT knock-out mice.²²⁰  In addition, it is known that metallothionein can sequester zinc in the liver during the acute stage of infection.²⁵⁶

The liver is a major contributor of inflammatory cytokines during systemic inflammation. The liver itself is susceptible to cytokine induced pro-inflammatory injury, and this has been investigated in a mouse model of thermal skin injury.^18,44,262 MT-I/II mRNA and intranuclear protein levels was elevated after burn injury when compared to MT-knockout mice. Concurrently increased levels of zinc, copper, and iron was also noted.⁴⁸ This suggests a role of MT in the pathogenesis of hepatic damage during the systemic inflammatory response. With regard to other organs, MT knockout mice were more susceptible to lung inflammation than wild-type (WT) mice when challenged with intratracheal LPS. Histologically, MT knockout mice showed degeneration of type I pneumocytes and endothelial cells, whereas the WT mice did not.²⁶⁶

1.3.3        Metallothionein and its role in fibrosis and biliary hyperplasia

The most recent research on metallothionein has focussed on the use of MT gene therapy. Hepatic fibrosis induced by CCl₄ was reversed in mice with the adenoviral delivery of the MT-II gene.¹³⁸ Increased levels of MT was associated with increased collagenase activity, and there was increased hepatocyte regeneration after gene therapy.¹³⁸  Furthermore, MT-II gene therapy was able to reverse the phenotype of activated hepatic stellate cells in vitro, thereby reducing the mRNA and protein levels of smooth muscle actin and collagen-I from these cells.³⁰⁶ In dogs with chronic hepatitis, MT expression was negatively correlated with the amount of fibrosis.²⁵⁷ However, no such correlation was found in equines with chronic hepatic disease (in submission), suggesting a very species specific mechanism for the potential anti-fibrotic activity of MT.

MMPs are important in the degradation of the ECM and for the reversal of hepatic fibrosis, as described earlier. MMPs are a family of endopeptidases and are dependent on zinc for their activity.^80,111,152 Is it plausible that metallothionein plays a role in fibrinolysis and resolution of hepatic fibrosis? Indeed, treatment with luteolin in a CCl₄ mouse model of hepatic fibrosis resulted in the concomitant increase of MT-I/II and MMP-9.⁷⁹ Furthermore, supplementation with zinc reduced liver fibrosis in mice as levels of MMP-13 and collagenase activity increased.²⁴⁶

There is no established relationship within the liver between metallothionein and biliary hyperplasia. In one study by Schmitz et. al., metallothionein overexpression was associated with poor patient outcome in cases of cholangiosarcoma. Bile duct epithelium in normal livers was occasionally only weakly expressed.²⁴¹ No other reports were evident in the literature.

 

1.3.4        Metallothionein in hepatic regeneration and neoplasia

MT expression has been positively correlated with hepatic regeneration (growth fraction) in dogs with chronic hepatitis.²⁵⁷ In fetal and newborn rats, MT-1 and MT-2 are found in both the nucleus and the cytoplasm, which suggests a role for MT during phases of rapid growth.⁴³ Intranuclear localization of MT has also been observed in several human tumors, with more intense immunohistochemical staining of MT at the proliferating edge of malignant tumors.⁴⁵ In PHx rats, MT is highly expressed within the nucleus and is translocated from the cytoplasm rapidly after hepatectomy.^276,280 In addition, MT stained hepatocytes were also shown within the S and G2/M phases of the cell cycles and occurs most prominently within the cytoplasm during G₀ and G₁ phases.^198,279,280

MT-I/II knockout mice after PHx have significantly less hepatic proliferation than wild-type mice after, suggesting an important role for MT in hepatic regeneration.²⁰⁴ It is believed that MT functions as a storage pool and chaperone molecule for zinc, which is required by proliferating cells for transcription factors, growth factors, and metalloenzymes.^38,284,310

1.3.5        MT expression as prognostic tool and the therapeutic potential of MT

MT has been investigated as a potential biomarker in various neoplasms. MT expression has been evaluated in a variety of cancers such as breast cancer,^{16,20,139,240} ovarian cancer,²⁶³ renal cell carcinoma,¹⁹¹ acute lymphoblastic leukemia,²³⁸ lung carcinomas,⁸⁶ colorectal cancer,²⁰¹ pancreatic carcinoma,²⁰² and melanoma,^{109,234,261,295}among others. In many instances, overexpression of MT is considered of prognostic value, as higher levels of MT within tumor cells was associated with increased risk for progression, and reduced patient survival in melanoma and cholangiosarcoma patients.^241,295 However, a recent meta-analysis of MT as an IHC biomarker showed a significantly positive association between (human) tumors of the head and neck as well as ovarian tumors, but a negative association with liver tumors. Furthermore, a significant positive relationship was found between MT expression and tumor grade and patient survival.⁶ Within the veterinary research, MT has been investigated in both mammary and melanoma neoplasms of dogs and cats. Although no prognostic implication was found in this study, it highlighted the species specificity in MT expression within neoplastic lesions.⁷⁵

Interestingly, carcinogenesis and metastasis are also often correlated with an excess of metals such as copper and iron.⁹⁶ Tumor growth, angiogenesis and metastasis have been correlated to excess copper.^30,113 In turn, copper chelation was shown to inhibit copper-induced migration of neoplastic cells in prostate cancer.²¹³ Zinc chelation has been demonstrated to disrupt the conformation of the tumor suppressor protein p53 and was thereby able to modulate transcriptional activity.¹⁸³ Metallothionein, which binds heavy metals in the liver may, therefore, act as a source of cancer-promoting copper or zinc.

Although hepatic fibrosis is considered a reversible process, current therapeutic regimens against hepatic fibrosis have not provided a complete and consistent response, and therefore this is currently a “hot topic” of liver disease research.^63,296 The most recent research on metallothionein has focussed on the use of MT gene therapy for the reversal of hepatic fibrosis, as previously described.

Of course, one must be judicious in interpreting results from mouse and in vitro models and applying them to human subjects. Although the liver has a limited number of reaction patterns, for example fibrosis, the etiologies and pathways to obtain that reaction pattern may be numerous and diverse. Considering the number and type of immune cells and their inflammatory mediators, it would be foolish to consider all types of liver disease to be the same. By manipulating the expression levels of MT in the tissues of patients, we may be exposing them to increased risk of neoplasia.

1.4         Equine Liver Disease

 

1.4.1        Hepatic Disease in the Adult Equine

In general, primary hepatic disease in adult equines is not common but may result in mortality in up to 25% cases.⁸⁵ In one study evaluating the cause of death of 241 mature horses; hepatic disease was only found in six horses.¹⁸⁸ Because of the liver’s large reserve capacity, signs of hepatic disease may not become apparent until there is a loss of greater than 75% of the liver.²⁷⁰ Very little information is available on the prevalence of hepatic disease in horses, except a few reviews out of the western United States,^114,188,212 and the United Kingdom.^{81,84,180,298} Most of these reviews focus on the clinicopathologic correlation of hepatic disease with clinical signs, histopathology, or survival times, but do not relate findings to possible etiologies.

Hepatic disease in equines may result in transient or subclinical liver dysfunction or may cause more severe illness or death. Disease may follow an acute or chronic time course or may progress as a combination of the two.

Primary (bacterial) hepatitis is rare in mature equines and is more often secondary to gastrointestinal disease. The bacteria are thought to ascend the bile duct and gain access to the liver, resulting in a suppurative cholangiohepatitis.²¹⁸ Horses with proximal enteritis had significantly increased serum hepatic enzyme activity, suggestive of hepatic damage.^66,285 A recent case of necrotizing hepatitis, or black disease, caused by Clostridium novyiwas described in a 20 year old pony in Western Canada.⁶⁵

Other important causes of hepatic disease include lipidosis/ hyperlipemia, especially in ponies, donkeys, and miniature horses.^{102,128,137,195,196} Cholelithiasis with suppurative cholangiohepatitis has also been described.¹⁴² Serum sickness, also known as Theiler’s disease, has been reported in horses receiving a commercial plasma transfusion,⁴ and from the administration of tetanus antitoxin.¹¹² Hepatic lesions involve acute hepatic necrosis and hepatitis, and the loss of hepatocytes may be so severe that the liver appears markedly reduced in size.²⁶⁰ Other infrequent causes of hepatic disease include abscessation and iron toxicity.²⁰⁵

The nature of the grazing animal puts them at risk for pasture and feed contaminated toxicities. Liver damage may result from the ingestion of pyrrolizidine alkaloid toxin containing plants,^3,182 alsike clover,¹⁹⁹ and aflatoxins.^36,62,287

Granulomatous and eosinophilic lesions in the liver are most often a result of parasitic larval migration of Strongylus vulgarus, Strongylus equinus, Strongylus edentatus, Parascaris equorum and Habronema sp..^179,258,281 Multisystemic eosinophilic disease has infrequently been reported in the literature, but may be a cause of eosinophilic granulomatous lesions in the equine liver.⁴¹

1.4.2        Hepatic Disease in the Juvenile Equine

Clostridium piliforme is an important cause of hepatitis and death in foals less than 30 days of age.²⁶⁵ The bacterium was first described by E. E. Tyzzer in a colony of waltzing mice in 1917.²⁸² Pathogenesis of the disease is due to the ingestion of the bacterium in fecal matter from adult horses, and its subsequent overgrowth in the intestine of foals with high levels of nutrients.²⁶⁵

Toxic hepatopathy, described as massive necrosis and lobular collapse with mild portal fibrosis, has also been described in neonatal foals after administration of a nutritional paste containing Aspergillus sp. and an iron compound.²

Genetic diseases that can cause hepatic lesions are rare. However, a retrospective study from Berne, Switzerland identified 30 Swiss Freiberger foals with hepatic disease consistent with congenital hepatic fibrosis, which is a genetically inherited disease in other species.¹¹⁵

Septicemia may cause secondary lesions of multi-focal hepatocellular necrosis in the liver. Foal septicemia is an important cause of illness in young animals and mortality may reach 75%.¹⁶¹ E. coli is frequently cultured (blood culture) in septicemic foal cases.¹⁶¹ Infection was the leading cause of morbidity in the first year of life for Thoroughbreds in Ireland, and septicemia was diagnosed in 5.9% of foals, all occurring between 1 and 35 days of age.¹⁰¹ Unfortunately, there are no specific symptoms for septicemia, and clinical signs of liver disease often do not accompany the clinical disease.¹⁶¹

1.4.3        Hepatic Disease in the Equine Fetus

Few etiologic agents or disease processes cause significant hepatic pathology in the fetal liver. Equine herpes virus-1, and less frequently -4 (EHV-1 and -4), are causes of fetal abortion, often despite vaccination of the mare.^{106,125,174,175,301} EHV-1 was the leading cause of reproductive loss (abortion, stillbirth and early neonatal loss) in 21.3% of 103 cases in a study from central Italy,¹⁷⁴ 8.9% of 290 cases from Michigan,²⁶⁸ and 6.5% of 1252 cases in a study from the United Kingdom,¹⁰⁶ and 3.3% of 1211 cases from a study out of Kentucky, USA.¹²⁵ Occasionally, there is co-infection of EHV-1 with significant bacterial isolates such as Klebsiella pneumoniae (lungs), or Actinobacillus equuli (septicemia).¹⁷⁵

Hepatic lesions may be seen histologically in other disease processes.  Equine viral arteritis predominantly causes lesions in the mare, but when present in the fetus include perivascular lymphocytic infiltrates or severe vasculitis involving the liver, spleen, lung, brain, and allantochorion.^71,140 Infectious agents responsible for placentitis may gain access to the fetal fluids or organs, which can result in hepatic lesions such as hepatocellular degeneration and necrosis.³⁰¹ Leptospirosis, most frequently due to serovars kennewicki or bratislava, is an important cause of fetal loss and will often result in gross and microscopic lesions in the fetus.^{88,106,106,301} Hepatic lesions in the fetus include portal lymphocytic and histiocytic infiltrates with giant cells within the hepatic parenchyma.³⁰¹

1.4.4        Neoplastic Disease in Equines

Neoplastic disease of the liver is not common in equines. Nevertheless, aged equines are susceptible to neoplastic lesions, and was the cause of death or euthanasia of 18.7% of 241 equines in one study, though no hepatic neoplastic disease was found.¹⁸⁸ Primary liver tumors described in older horses include cholangiocarcinoma,^82,250 a mixed hepatocellular carcinoma and cholangiocarcinoma,¹⁴⁸ and a single case of hepatic biliary adenofibroma (a variant of hepatic biliary cystadenoma).²³⁶ Metastatic or multicentric hepatic neoplasia is more common than primary disease.⁵⁸ Multicentric hemangiosarcoma,⁹⁴ metastatic renal carcinoma,¹⁵⁴ and  multicentric lymphoma⁸³ have all been described in the equine liver. In a retrospective study of 92 horses in the western United States performed by Hackett et. al., 5 cases of hepatic neoplasia were identified and included 2 cases of lymphosarcoma, an undifferentiated neuroendocrine carcinoma, one metastatic thyroid carcinoma, and a leukemia with neoplastic lymphoid infiltrates within the hepatic sinusoids.¹¹⁴

 

Primary hepatic neoplasia is even rarer in young animals. In equines, hepatoblastoma is one of the more frequently occurring neoplasms in fetuses and foals and has been described several times in the literature.^{17,39,108,167,221,289} Hepatoblastoma is also one of the more common pediatric tumors in humans and accounts for 91% of primary hepatic tumors in children less than 5 years of age.⁶⁴ Other tumors described in equine fetuses and foals include mixed hamartoma²³³ and mesenchymal hamartoma³² in fetuses and hepatocellular carcinoma in foals and young equines.^17,136,232

1.5         Copper and Zinc in the Equine Liver

Copper and zinc play vital roles in the structure and function of the (mammalian) organism. Copper is essential for the functioning of enzymes,¹¹⁹ as a structural component of tissues, and as an antioxidant and oxidant.²⁰⁷ Similarly, zinc is important in enzyme function, immune function,²⁴³ and for DNA and protein synthesis.¹⁷²  In humans, over 10% of the proteome codes for zinc containing enzymes.⁸

Equines appear to be relatively resistant to toxicities caused by excessive dietary copper,²⁵³ unlike what is observed in dogs,^92,251 sheep,¹²¹ or other ruminants such as goats.⁵⁴ However, musculoskeletal deformities in foals and young horses have been implicated in copper deficiencies or with elevated zinc levels,^{31,37,40,87,162} though copper has not been implicated in the pathogenesis of osteochondrosis dissecans.²⁹³

The main source of zinc and copper is diet, but the absorption of copper may be hindered by elevated levels of zinc.¹⁵⁰ Dietary supplementation of copper positively affects hepatic copper concentrations in horses.²¹⁵ There appears to be no relationship between plasma and hepatic copper levels or plasma levels and dietary intake in equines.⁶¹  Therefore, determination of the hepatic copper concentration is a better representation of copper metabolism, than plasma levels.²¹⁶ In contrast, mean hepatic zinc concentrations did not differ among a group of horses fed diets with varying levels of zinc.⁶¹

A recent study (2014) by Paßlack et al.,²¹⁴ determined heavy metal concentrations of naturally occurring copper, zinc and cadmium, among others, of 21 horses in Germany.  They found that both copper and zinc were higher, and in almost equal proportions, in the liver and renal cortex compared to the renal medulla. Cadmium was the highest within the renal cortex, followed by renal medulla and liver. No gender differences were found in the amounts of copper and zinc. Copper concentrations in the liver were highest in young horses less than one year of age. In general, however, there was large individual variation in heavy metal detection between horses.