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14 Hepatobiliary System

ANATOMY AND PHYSIOLOGY

The liver is situated in the proximal - most area of the peri-toneal cavity immediately distal to the diaphragm and is divided into grossly distinguishable lobes. Closely associ-ated with the ventral aspect of the liver in the dog and cat is the gall bladder. The liver plays a critical role in a variety of functions including nutrient homeostasis, fi ltration of particulates, protein synthesis, bioactivation, and detoxifi -cation, formation of bile and biliary secretion (Jaeschke 2008 ). Hepatic injury due to chemicals and drugs is the most common cause of acute liver failure in humans in the United States and is the leading cause of regulatory action against drugs (Watkins and Seef 2006 ). Epidemiologic studies into the incidence of drug - and chemical - induced liver injury in animals is lacking, but as the number of pharmaceuticals, neutraceuticals, and “ natural ” remedies for our companion animals increases, it is likely that the incidence of drug - induced liver injuries in companion animals will also increase.

Understanding the toxicologic changes that can occur in the liver requires understanding of its structural and functional anatomy. When viewed microscopically, the liver appears to be composed of hexagonal lobules sur-rounding a central blood vessel ( central vein ) and bordered by six portal triads at the outer edge of each lobule; each portal triad consists of a branch from each of the hepatic artery , portal vein , and bile duct (Figure 14.1 ). Between the portal triads and central vein are single - cell – wide cords of hepatocytes arranged radially around the central vein. Between the hepatocellular cords, sinusoids carry blood from the portal triads to the central vein. In this lobule

model of the liver, the lobule is divided into three areas: the centrilobular region surrounding the central vein, the periportal region at the periphery of the lobule that incor-porates the portal triads, and the midzonal region between the centrilobular and periportal regions. Pathology reports on liver histology frequently use these terms associated with the lobular model of the liver.

From a functional point of view, the hepatic lobule can be divided differently based on the fl ow of blood as it passes through the liver. Blood enters the liver lobule through branches of the hepatic artery (carrying oxygen) and the portal vein (carrying nutrients and other absorbed compounds, including toxicants, from the gastrointestinal tract) of the portal triads. The blood then fi lters within the sinusoids along the cords of hepatocytes until emptying into the central vein, giving up oxygen and picking up wastes as it passes from triad to central vein. Using the acinar model of the liver, the acinus is centered on a line from portal triad to portal triad. The area immediately around this line is termed Zone 1 and is equivalent to the periportal region. Zone 2 is equivalent to the midzonal region and Zone 3 is equivalent to the centrilobular region. Zone 1 receives blood with the highest oxygen and nutri-ent content, and Zone 3 is relatively oxygen and nutrient depleted. For instance, blood in Zone 1 contains approxi-mately 9% – 13% oxygen compared to 4% – 5% in Zone 3 (Bischoff and Ramaiah 2007 ).

From a toxicological perspective, the acinar model tends to be more relevant because it can help predict where in the liver toxicant - induced damage may occur (Table 14.1 ). Toxicants that are directly injurious to cells (e.g.,

Small Animal Toxicology Essentials, First Edition. Edited by Robert H. Poppenga, Sharon Gwaltney-Brant.© 2011 John Wiley and Sons, Inc. Published 2011 by John Wiley and Sons, Inc.

Sharon Gwaltney - Brant

100 Section 2 / A Systems-Affected Approach to Toxicology

complex mixture of bile acids, phospholipids, proteins, and other compounds that is important in the uptake of lipids from the small intestine, protection of the small intestine from oxidative injury, and the excretion of endog-enous and xenobiotic compounds (Jaeschke 2008 ). Biliary excretion is an important route of elimination for certain xenobiotics (e.g., ivermectin, naproxen in dogs) that are poorly excreted through the urine. Compounds excreted through the bile may undergo enterohepatic recirculation whereby the compound that is excreted into the duodenum from the bile duct is then reabsorbed and reenters the portal and systemic circulations. Compounds that undergo enterohepatic recirculation tend to have half - lives that are measured in days rather than hours. For example, naproxen in humans is eliminated primarily through the kidneys and has a half - life of about 6 hours. In dogs, naproxen is elimi-

iron salts, white phosphorus) will cause damage to the fi rst hepatocytes they encounter, thus injury from these types of toxicants appears primarily in Zone 1 (periportal). Zone 3 contains a higher level of cytochrome P450 metabolic enzymes, making this area most prone to injury from com-pounds that are bioactivated by these enzymes (e.g., acet-aminophen). Some toxicants can produce hepatocellular injury so severe that zonal patterns are lost as the entire lobule undergoes massive necrosis , with necrosis extend-ing from portal triads to central veins.

In contrast to blood fl ow through the hepatic acinus, bile fl ows from the canaliculi between hepatocytes into chan-nels that empty into the bile ducts within the portal triad; these bile ducts in turn empty into the gall bladder, which stores and concentrates bile prior to emptying the bile into the duodenum (Bischoff and Ramaiah 2007 ). Bile is a

Figure 14.1. Schematic illustration of the microanatomy of the liver. Each hexagon represents a hepatic lobule centered around a central vein with portal triads at the periphery. The portal triads are each composed of a portal vein, hepatic artery, and bile duct. The central lobule is divided into the three anatomic and functional zones: Zone 1 or periportal region, Zone 2 or midzonal region, and Zone 3 or centrilobular region.

Portal Triad:

Hepatic Artery

Portal Vein

Bile Duct

Central Vein

Zone 3(Centrilobular)

Zone 2 (Midzonal)

Zone 1(Periportal)

Chapter 14 / Hepatobiliary System 101

injury is a dose - dependent, predictable reaction to a toxi-cant and it is the most common type of liver injury in animals (Bischoff and Ramaiah 2007 ). There is frequently a predictable latent period between exposure to the toxi-cant and the development of clinical signs of liver insuf-fi ciency. Intrinsic liver injury is often caused by highly reactive by - products of xenobiotic metabolism, especially free radicals and electrophiles. Idiosyncratic liver injury is an unpredictable response to a xenobiotic. Idiosyncratic reactions are rare and non – dose - dependent and may be associated with extrahepatic lesions. Idiosyncratic reac-tions frequently occur following unremarkable initial exposure (sensitization) and manifest upon reexposure to the xenobiotic. With idiosyncratic drug reactions, it is not uncommon for there to be a delay of a few weeks to several months between the time of initial exposure and development of liver injury. Extrahepatic changes may include fever and dermal erythema or rash.

Free Radical – Induced Injury

The generation of free radicals within hepatocytes can occur during xenobiotic biotransformation, normal meta-bolic processes involving oxidation/reduction reactions, infl ammatory states (mediated by nitric oxide (NO)), and exposure to ionizing radiation (Bischoff and Ramaiah 2007 ). Free radicals have unpaired electrons, which make them highly susceptible to binding to other macromole-cules, including lipoproteins of cell membranes, enzy-matic proteins, and DNA. Binding of free radicals to cellular macromolecules generates even more free radical production, resulting in an escalating effect. Normally cells have scavenger molecules (e.g., glutathione) to react with and detoxify free radicals, but excessive free radical production can result in depletion of scavenger stores, resulting in increased binding of free radicals to vital cel-lular structures. Free radical damage can cause alterations of cell membrane permeability, inactivation of membrane - associated proteins, and loss of polarity to mitochondrial membranes. Inactivation of enzymatic proteins can inter-fere with normal cellular metabolism, leading to cellular degeneration or necrosis. Free radical - induced DNA damage can result in interference with transcription or translation, resulting in decreased protein synthesis.

Calcium Homeostasis Disruption

Within the cell, calcium is sequestered within organelles such as mitochondria and endoplasmic reticulum, which maintain a relatively low level of calcium within the cytosol. Xenobiotics that interfere with the pumps that maintain calcium balance can result in increased release

nated through the bile, undergoes enterohepatic recycling and has a half - life of 74 hours (Frey and Rieh 1981 ; Talcott 2006 ).

MECHANISMS OF TOXICOLOGIC INJURY

The mechanisms behind hepatic injury can be divided into two categories: intrinsic and idiosyncratic. Intrinsic liver

Table 14.1. Patterns of hepatotoxicity

Causes of Nonzonal Hepatic Injury NSAIDs Cyclophosphamide Phenobarbital Phenytoin Sulfonamides Ketoconazole

Causes of Zone 1 (Periportal) Hepatic Injury Afl atoxin Iron salts Organic arsenic White phosphorus

Causes of Zone 2 (Midzonal) Hepatic Injury Cisplatin Hexachlorophene (cats)

Causes of Zone 3 (Centrilobular) Hepatic Injury Acetaminophen Afl atoxin Blue - green algae Castor bean ( Ricinus ) Chloroform Cycad palms ( Cycas, Zamia ) Diazepam (cats) Hepatotoxic mushrooms (e.g., Amanita phalloides ) Naphthaene NSAIDs (e.g., carprofen, ibuprofen) Phenols Xylitol

Causes of Massive Hepatic Necrosis Acetaminophen Cisplatin Cycad palms ( Cycas, Zamia ) Diazepam (cats) Iron Mebendazole (dogs) Microcystin LR ( Microcystis aeruginosa )

Sources : Bischoff and Ramaiah 2007 , Haschek et al. 2007 .

102 Section 2 / A Systems-Affected Approach to Toxicology

This mechanism has been postulated, but not proven, as the mechanism by which the nonsteroidal anti - infl ammatory drug carprofen causes idiosyncratic liver injury in dogs (McPhail et al. 1998 ). The exact mechanisms of other types of idiosyncratic liver injury await further study to be elucidated.

PATTERNS OF TOXICOLOGIC INJURY

Toxic hepatic injury can manifest as hepatocellular degen-eration, hepatocellular necrosis, immune - mediated injury, biliary disorders, and sinusoidal disorders.

Hepatocellular Degeneration

Hepatocellular degeneration is a common, nonspecifi c response of the liver to toxic insult. Hepatocellular degeneration represents a sublethal insult to the liver that can often resolve if the inciting cause is removed. Although some cellular processes may be altered, the degenerative hepatocyte still retains some degree of functional integrity. Degenerating hepatocytes are gener-ally swollen and may accumulate various compounds, including metals, pigments, water, and lipids. It is impor-tant to realize that in most situations the accumulation of these compounds is a result of some form of hepa-tocellular dysfunction rather than the primary cause of hepatocellular degeneration.

Hepatic Lipidosis

Hepatic lipidosis (steatosis, fatty liver) is a form of hepa-tocellular degeneration characterized by accumulation of lipid within the hepatocyte cytosol. Lipidosis can be the result of transient disruptions in lipid metabolism, or it can refl ect a more serious metabolic condition. Severe lipid accumulation can result in suffi cient hepatocellular swell-ing to cause cholestasis.

Hepatocellular Necrosis or Apoptosis

Both hepatocellular necrosis and apoptosis represent death of hepatocytes with the difference being that apoptosis is a controlled, orderly “ shutdown ” of cellular processes leading to death of the cell. Necrosis occurs when cells are subject to insurmountable insults resulting in loss of cellular membrane integrity and disruption of cellular machinery. What follows is cellular swelling, leakage of cellular contents into the adjacent environment, dis-integration of nuclear material and an infl ux of infl am-matory cells to remove cellular debris (Jaeschke 2008 ). Because the necrotizing insult is generally locally intense, whole groups of hepatocytes, as well as support struc-tures, may be affected. In contrast, apoptosis results

of calcium into the cytosol. The released calcium ions impede normal cytoskeletal function and activate a variety of cytosolic enzymes including ATPases, phospholipases and proteases. Subsequently, mitochondrial membrane permeability increases, which causes induction of necrosis and apoptosis (programmed cell death) (Bischoff and Ramaiah 2007 ).

Mitochondrial Injury

Mitochondria are the source of energy production within the cell, and toxicants that disrupt mitochondrial function can result in cell death. Toxicants induce mitochondrial injury by altering mitochondrial DNA, interfering with oxidative phosphorylation, damaging mitochondrial mem-branes, or inactivating mitochondrial enzymes.

Cytoskeletal Disruption

The cytoskeleton of the cell is vital to maintaining normal shape and position of hepatocytes. Agents that damage cytoskeletal structure result in cellular deformation and detachment. The blue - green algae toxin microcystin - LR, produced by Microcystis aeruginosa causes hepatocellular cytoskeletal injury and severe liver damage (Haschek et al. 2007 ).

Cholestasis

Toxicants that interfere with bile transport can result in cholestasis, leading to injury to biliary ducts and hepato-cytes. Bile duct hyperplasia and biliary fi brosis may result. Cholestasis can also occur secondary to hepatocellular swelling due to degeneration or lipidosis. Clinically, cho-lestatic disorders can result in dermal photosensitization, whereby areas of skin exposed to the sun develop burns similar to sunburn. Epidermal and dermal burns from pho-tosensitization are rapid in onset and occur following exposure to wavelengths of light that would cause no injury in normal animals. Animals with white areas on their haircoats and upigmented skin are most susceptible to photosensitization. In veterinary medicine, photosensi-tization is more commonly seen in large animals grazing plants that cause bile stasis, but it has occasionally been reported in dogs with hepatic injury.

Idiosyncratic Reactions

Many idiosyncratic drug reactions are considered to be immune - mediated, triggered by the formation of adducts of reactive drug metabolites with cellular macromolecules. These adducts are identifi ed by the immune system as foreign, and an immune response is mounted, leading to death of individual hepatocytes ( “ piecemeal necrosis ” ).

Chapter 14 / Hepatobiliary System 103

erate suffi cient hepatic parenchyma to have normal liver function. As with most organs that have regenerative ability, the capacity to regenerate is dependent upon the retention of the basic tissue scaffold. Conditions that result in loss of this scaffold can result in hepatic healing via fi brosis instead. Fibrosis occurs most commonly along the terminal plates at the edges of the hepatic lobule and sur-rounding the central vein. Fibrous tissue that bridges between portal triad and central vein can result in loss of functional integrity of the acinus. Regenerative nodules of hepatocytes within bands of fi brous tissue are often dis-connected from access to central veins and are minimally functional. Cirrhosis is the term in human medicine for the end - stage form of liver injury, with irregular patches of fi brous tissue surrounding rounded nodules of hepatocytes.

Although the liver has a high regenerative capacity, massive acute loss of hepatocytes can result in death from liver failure if insuffi cient hepatocytes remain to allow the patient to survive long enough for regenerated hepatocytes to begin to function.

when enzymes termed caspases trigger the activation of various cytosolic and nuclear enzymes that compart-mentalize cellular structures and proceed to shut down cellular activity. Apoptotic cells are shrunken, with con-densed cytosol and fragmented nuclei that pinch off along with the cell membrane (which has remained intact) to form apoptotic bodies . Apoptotic bodies are then phagocytized by hepatic macrophages (Kuppfer cells) or are taken up into adjacent hepatocytes without incit-ing an infl ammatory response. Apoptosis is the means by which normal tissues eliminate unneeded or senescent cells, and it generally affects single cells. Apoptosis can be triggered by a variety of external stimuli, including ionizing radiation, drugs (NSAIDs, corticosteroids), and physiologic conditions such as stress.

HEALING AND REPAIR

The liver has a tremendous capacity for regeneration — so much so that human liver transplants can be done from liver lobes donated by living donors. Following successful transplant, both recipient and donor will eventually regen-

1. Milo, a 2 - year - old, 135 - pound, neutered male St. Bernard dog has been successfully treated for acute hepatic insuffi ciency. A liver biopsy had been taken and the pathology report indicated that there was sub-stantial centrilobular necrosis. All of the following toxicants are potential causes for Milo ’ s liver injury except a. Iron b. Blue - green algae c. Acetaminophen d. Hepatotoxic mushrooms e. Xylitol

2. In what way does enterohepatic recirculation alter the kinetics of a xenobiotic? a. Increases elimination b. Increases absorption c. Shortens the half - life d. Lengthens the half - life e. Enhances metabolism

3. Intrinsic liver injury is characterized by all of the fol-lowing except a. Dose - dependent b. Predictable latent period

c. Rare in animals d. Often caused by free radicals and electrophiles e. Predictable clinical course

4. Blue - green algae induce liver injury by which of the following mechanisms? a. Free radical formation b. Disruption of calcium homeostasis c. Cytoskeletal damage d. Immune - mediated mechanisms e. Cholestasis

CHAPTER 14 STUDY QUESTIONS

ANSWERS

1.a. Iron causes primarily periportal or Zone 1 injury in the liver.

2.d. Enterohepatic recirculation increases the half - life of a xenobiotic.

3.c. Intrinsic liver injury is the most common type of liver injury identifi ed in animals.

4.c. Blue - green algae disrupt the structural support of hepatocytes resulting in cytoskeletal failure.

104 Section 2 / A Systems-Affected Approach to Toxicology

toxicosis associated with administration of carprofen in 21 dogs . Journal of the American Veterinary Medical Associa-tion 212 ( 12 ): 1895 – 1901 .

Moeller , Robert B. 2004 . Hepatobiliary system: Toxic response of the hepatobiliary system . In Veterinary Clinical Toxicology , edited by Konnie H. Plumlee , pp. 61 – 68 . St. Louis : Mosby .

Plumlee , Konnie H. 2004 . Hepatobiliary system: differential diagnosis . In Veterinary Clinical Toxicology , edited by Konnie H. Plumlee , p. 61 . St. Louis : Mosby .

Talcott , Patricia A. 2006 . Nonsteroidal antiinfl ammatories . In Small Animal Toxicology , 2nd ed. , edited by Michael E. Peterson and Patricia A. Talcott , pp. 902 – 933 . St. Louis : Saunders .

Watkins , P.B. , Seef , L.B. 2006 . Drug - induced liver injury: summary of a single topic clinical research conference . Hepatology 43 : 618 – 631 .

REFERENCES

Bischoff , Karen and Ramaiah , Sashi K. 2007 . Liver toxicity . In Veterinary Toxicology: Basic and Clinical Principles , edited by Ramesh C. Gupta , pp. 145 – 160 . New York : Elsevier .

Frey , H.H. and Rieh , B. 1981 . Pharmacokinetics of naproxen in the dog . American Journal of Veterinary Research 42 ( 9 ): 1615 – 1617 .

Haschek , Wanda M. , Rousseaux , Colin G. , and Wallig , Matthew A. 2007 . The liver . In Fundamentals of Toxico-logic Pathology , 2nd ed. , pp. 197 – 235 . San Diego : Aca-demic Press - Elsevier .

Jaeschke , Hartmut. 2008 . Toxic responses of the liver . In Casarett and Doull ’ s Toxicology, the Basic Science of Poisons , 6th ed. , edited by Curtis D. Klaasen , pp. 557 – 582 . New York : McGraw - Hill Medical .

McPhail , C.M. , Lappin , M.R. , Meyer , D.J. , Smith , S.G. , Webster , C.R. , and Armstrong , P.J. 1998 . Hepatocellular