s4 ch13 toxic_responsesoftheliver

CHAPTER 13

TOXIC RESPONSESOF THE LIVER

Mary Treinen-Moslen

FACTORS IN LIVER INJURY

Uptake and Concentration Bioactivation and DetoxificationActivation of Sinusoidal CellsInflammatory and Immune Responses

MECHANISMS OF LIVER INJURY

Disruption of the CytoskeletonCholestasisMitochondrial Damage

FUTURE DIRECTIONS

INTRODUCTION

PHYSIOLOGY AND PATHOPHYSIOLOGY

Hepatic FunctionsStructural OrganizationBile FormationTypes of Injury and Toxic Chemicals

Fatty LiverCell DeathCanalicular CholestasisBile Duct DamageSinusoidal DamageCirrhosisTumors

INTRODUCTION

Numerous industrial compounds and therapeutic agents have beenfound to injure the liver. Consequently, the use of such chemicalshas been eliminated or restricted. For example, carbon tetrachlo-ride was commonly used in unventilated garages for degreasing au-tomobile engines. Plastic industry workers without any protectiveequipment once crawled down into giant vats coated with residuecontaining vinyl chloride (Kramer, 1974). Now exposures to thepotent hepatotoxins carbon tetrachloride and vinyl chloride aretightly regulated. However, each year new chemicals are found todamage the liver, such as the drugs Rezulin (troglitazone), pre-scribed for type 2 diabetes, and Rimadyl (carprofen), prescribedfor dogs with arthritis. Usage of Rezulin in clinical medicine andRimadyl in veterinary medicine was recently withdrawn or re-stricted based on reports of hepatic damage in more than 100 hu-mans and over 8000 dogs, respectively (Kohlroser et al., 2000;Adams, 2000). During the 3-year period before the serious andsometimes fatal hepatotoxicity associated with their use was gen-erally recognized, these two drugs were widely prescribed to over800,000 humans and more than 4 million dogs. Initially promis-ing drugs have been withdrawn during clinical trials when their he-patotoxicity became manifest after weeks or months of exposure.The 1993 clinical trial of fialuridine as a therapy for chronic viralhepatitis was suddenly terminated when some of the participatingpatients died of liver failure (McKenzie et al., 1995). Humans andanimals continue to ingest hepatotoxins in foods, teas, and con-taminated water. The serious problem of chemically induced liverdamage has inspired excellent monographs (Zimmerman, 1978;Farrell, 1994; McCuskey and Earnest, 1997).

Observations on hepatotoxicants have advanced the under-standing of hepatic functions and cell injury. Factors are knownthat determine why the liver, as opposed to other organs, is thedominant target site of specific toxins. Scientists have identifiedmechanisms by which chemicals injure specific populations of livercells. New techniques in molecular biology, immunochemical

probes, and the availability of transgenic animals provide new in-sights into basic physiologic and pathologic processes. Yet manyquestions remain. Why does end-stage liver disease occur in only10 to 15 percent of those who chronically consume excess alco-hol? How do genetic and acquired factors enhance vulnerability toalcohol and other toxicants?

Toxicologists regard the phrase “produces liver injury” asvague, since liver cells respond in many different ways to acuteand chronic insults by chemicals. A basic understanding of chem-ical hepatotoxicity requires some appreciation of the physiologyand anatomy of the liver. Key aspects for such appreciation are (1)major functions of the liver, (2) structural organization of the liver,and (3) processes involved in the excretory function of the liver,namely bile formation. These aspects contribute to the vulnerabil-ity of hepatic cells to chemical insults.

PHYSIOLOGY ANDPATHOPHYSIOLOGY

Hepatic Functions

The liver’s strategic location between intestinal tract and the restof the body facilitates the performance of its enormous task ofmaintaining metabolic homeostasis of the body (Table 13-1). Ve-nous blood from the stomach and intestines flows into the portalvein and then through the liver before entering the systemic cir-culation. Thus the liver is the first organ to encounter ingested nu-trients, vitamins, metals, drugs, and environmental toxicants as wellas waste products of bacteria that enter portal blood. Efficient scav-enging or uptake processes extract these absorbed materials fromthe blood for catabolism, storage, and/or excretion into bile.

All of the major functions of the liver can be detrimentallyaltered by acute or chronic exposure to toxicants (Table 13-1).When toxicants inhibit or otherwise impede hepatic transport andsynthetic processes, dysfunction can occur without appreciable cell

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472 UNIT 4 TARGET ORGAN TOXICITY

damage (Fig. 13-1). Loss of function also occurs when toxicantskill an appreciable number of cells and when chronic insult leadsto replacement of cell mass by nonfunctional scar tissue. Alcoholabuse is the major cause of liver disease in most western countries(Crawford, 1999); thus ethanol provides a highly relevant exampleof a toxin with multiple functional consequences (Lieber, 1994).Early stages of ethanol abuse are characterized by lipid accumu-lation (fatty liver) due to diminished use of lipids as fuels and im-paired ability to synthesize the lipoproteins that transport lipids outof the liver. As alcohol-induced liver disease progresses, apprecia-ble cell death occurs, the functioning mass of the liver is replacedby scar tissue, and hepatic capacity for biotransformation of cer-tain drugs progressively declines. People with hepatic cirrhosis dueto chronic alcohol abuse frequently become deficient at detoxify-ing both the ammonia formed by catabolism of amino acids andthe bilirubin derived from breakdown of hemoglobin. Uncontrol-lable hemorrhage due to inadequate synthesis of clotting factors isa common fatal complication of alcoholic cirrhosis. A consequenceof liver injury that merits emphasis is that loss of liver functionscan lead to aberrations in other organ systems and to death.

Structural Organization

Two concepts exist for organization of the liver into operationalunits, namely the lobule and the acinus. Classically, the liver wasdivided into hexagonal lobules oriented around terminal hepaticvenules (also known as central veins). At the corners of the lobuleare the portal triads (or portal tracts), containing a branch of theportal vein, a hepatic arteriole, and a bile duct (Fig. 13-2). Bloodentering the portal tract via the portal vein and hepatic artery ismixed in the penetrating vessels, enters the sinusoids, and perco-lates along the cords of parenchymal cells (hepatocytes), eventu-

ally flows into terminal hepatic venules, and exits the liver via thehepatic vein. The lobule is divided into three regions known as cen-trolobular, midzonal, and periportal. Preferred as a concept of afunctional hepatic unit is the acinus. The base of the acinus isformed by the terminal branches of the portal vein and hepatic ar-tery, which extend out from the portal tracts. The acinus has threezones: zone 1 is closest to the entry of blood, zone 3 abuts the ter-minal hepatic vein, and zone 2 is intermediate. Despite the utilityof the acinar concept, lobular terminology is still used to describeregions of pathologic lesions of hepatic parenchyma. Fortunately,the three zones of the acinus roughly coincide with the three re-gions of the lobule (Fig. 13-2).

Acinar zonation is of considerable functional consequence re-garding gradients of components both in blood and in hepatocytes(Jungermann and Kietzmann, 2000). Blood entering the acinus con-sists of oxygen-depleted blood from the portal vein (60 to 70 per-cent of hepatic blood flow) plus oxygenated blood from the he-patic artery (30 to 40 percent). Enroute to the terminal hepaticvenule, oxygen rapidly leaves the blood to meet the high metabolicdemands of the parenchymal cells. Approximate oxygen concen-trations in zone 1 are 9 to 13 percent, compared with only 4 to 5percent in zone 3. Therefore hepatocytes in zone 3 are exposed tosubstantially lower concentrations of oxygen than hepatocytes inzone 1. In comparison to other tissues, zone 3 is hypoxic. Anotherwell-documented acinar gradient is that of bile salts (Groothuis etal., 1982). Physiologic concentrations of bile salts are efficientlyextracted by zone 1 hepatocytes with little bile salt left in the bloodthat flows past zone 3 hepatocytes (Fig. 13-3).

Heterogeneities in protein levels of hepatocytes along the ac-inus generate gradients of metabolic functions. Hepatocytes in themitochondria-rich zone 1 are predominant in fatty acid oxidation,gluconeogenesis, and ammonia detoxification to urea. Gradients of

Table 13-1Major Functions of Liver and Consequences of Impaired Hepatic Functions

CONSEQUENCES OF

TYPE OF FUNCTION EXAMPLES IMPAIRED FUNCTIONS

Nutrient homeostasis Glucose storage and synthesis Hypoglycemia, confusionCholesterol uptake Hypercholesterolemia

Filtration of particulates Products of intestinal bacteria Endotoxemia(e.g., endotoxin)

Protein synthesis Clotting factors Excess bleedingAlbumin Hypoalbuminemia, ascitesTransport proteins (e.g., very Fatty liver

low density lipoproteins)

Bioactivation and Bilirubin and ammonia Jaundice, hyperammonemia-detoxification related coma

Steroid hormones Loss of secondary male sexcharacteristics

Xenobiotics Diminished drug metabolismInadequate detoxification

Formation of bile and Bile acid–dependent uptake of Fatty diarrhea, malnutrition,biliary secretion dietary lipids and vitamins Vitamin E deficiency

Bilirubin and cholesterol Jaundice, gallstones,hypercholesterolemia

Metals (e.g., Cu and Mn) Mn-induced neurotoxicityXenobiotics Delayed drug clearance

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CHAPTER 13 TOXIC RESPONSES OF THE LIVER 473

enzymes involved in the bioactivation and detoxification of xeno-biotics have been observed along the acinus by immunohisto-chemistry (Jungermann and Katz, 1989). Notable gradients for he-patotoxins are the higher levels of glutathione in zone 1 and thegreater amounts of cytochrome P450 proteins in zone 3, particu-larly the CYP2E1 isozyme inducible by ethanol (Tsutsumi, 1989).

Hepatic sinusoids are the channels between cords of hepato-cytes where blood percolates on its way to the terminal hepaticvein. Sinusoids are larger and more irregular than normal capil-laries. The three major types of cells in the sinusoids are endothe-lial cells, Kupffer cells, and Ito cells (Fig. 13-4). In addition, thereare rare pit cells, a lymphocyte-type cell with anti-tumor activity.Sinusoids are lined by thin, discontinuous endothelial cells withnumerous fenestrae (or pores) that allow molecules smaller than

250 kDa to cross the interstitial space (known as the space of Disse)between the endothelium and hepatocytes. Very little if any base-ment membrane separates the endothelial cells from the hepato-cytes. The numerous fenestrae and the lack of basement membranefacilitate exchanges of fluids and molecules, such as albumin, be-tween the sinusoid and hepatocytes but hinder movement of parti-cles larger than chylomicron remnants. Endothelial cells are im-portant in the scavenging of lipoproteins and denatured proteins.Hepatic endothelial cells also secrete cytokines.

Kupffer cells are the resident macrophages of the liver andconstitute approximately 80 percent of the fixed macrophages inthe body. Kupffer cells are situated within the lumen of the sinu-soid. The primary function of Kupffer cells is to ingest and de-grade particulate matter. Also, Kupffer cells are a source of cy-

Figure 13-1. Cartoon depicting toxicant-mediated events that lead to loss of representative functions of he-patocytes, such as A, albumin secretion; B, bilirubin uptake and export into bile; C, clotting factor secretion;H and M, hormone uptake and bioactivation to metabolites.

Dysfunction without cell damage can occur when toxicants inhibit uptake and secretion or excessively stimu-late bioactivation. The dysfunction can be selective when a toxicant impedes secretion of only some compounds.Acute damage and chronic damage produce a loss of function in the cell population that dies or is replaced byscar tissue.

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tokines and can act as antigen-presenting cells (Laskin, 1990). Itocells (also known by the more descriptive terms of fat-storing cellsand stellate cells) are located between endothelial cells and hepa-tocytes. Ito cells synthesize collagen and are the major site for vi-tamin A storage in the body.

Bile Formation

Bile is a yellow fluid containing bile salts, glutathione, phospho-lipids, cholesterol, bilirubin and other organic anions, proteins,metals, ions, and xenobiotics (Klaassen and Watkins, 1984). For-

mation of this fluid is a specialized function of the liver. Adequatebile formation is essential for uptake of lipid nutrients from thesmall intestine (Table 13-1), for protection of the small intestinefrom oxidative insults (Aw, 1994), and for excretion of endogenousand xenobiotic compounds. Hepatocytes begin the process by trans-porting bile salts, glutathione, and other solutes into the canalicu-lar lumen, which is a space formed by specialized regions of theplasma membrane between adjacent hepatocytes (Fig. 13-4). Tightjunctions seal the canalicular lumen from materials in the sinusoid.The structure of the biliary tract is analogous to the roots and trunkof a tree, where the tips of the roots equate to the canalicularlumens. Canaliculi form channels between hepatocytes that con-nect to a series of larger and larger channels or ducts within theliver. The large extrahepatic bile ducts merge into the common bileduct. Bile can be stored and concentrated in the gallbladder beforeits release into the duodenum. However, the gallbladder is not es-sential to life and is absent in several species, including the horse,whale, and rat.

Our understanding of bile formation has evolved from a de-scriptive orientation toward identification of specific cellular andsubcellular processes (Trauner et al., 1998). The major drivingforce is the active transport of bile salts and other osmolytes intothe canalicular lumen. Transporters on the sinusoidal and canalic-ular membranes of hepatocytes are responsible for the uptake of

Figure 13-2. Schematic of liver operational units, the classic lobule and the acinus.

The lobule is centered around the terminal hepatic vein (central vein), where the blood drains out of the lobule.The acinus has as its base the penetrating vessels, where blood supplied by the portal vein and hepatic arteryflows down the acinus past the cords of hepatocytes. Zones 1, 2, and 3 of the acinus represent metabolic re-gions that are increasingly distant from the blood supply.

Figure 13-3. Schematic of the acinar gradient of bile salts.

Efficient uptake of bile salts by zone 1 hepatocytes results in very low lev-els of bile salts in the blood that flows past zone 3 hepatocytes. A less steepgradient exists for the uptake of bilirubin and other organic anions.

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bile salts and bilirubin from blood and then the secretion of thesesolutes into the canalicular lumen (Fig. 13-5). Similarly, biliary se-cretion of drugs, hormones, and xenobiotics involves an extractionfrom the blood, transcytosis across hepatocytes, and then transportacross the canalicular membrane by ATP-dependent exporters.Lipophilic cationic drugs, estrogens, and lipids are exported by thecanalicular MDR (multiple-drug resistance) p-glycoproteins, oneof which is exclusive for phospholipids (Gosland et al., 1993;Kusuhara et al., 1998). Conjugates of glutathione, glucuronide, andsulfate are exported by the canalicular multiple organic anion trans-porter (cMOAT), which is also somewhat confusingly known asMRP2, based on similarities of cMOAT with the product of themultidrug-resistance gene.

Metals are excreted into bile by a series of partially under-stood processes that include (1) uptake across the sinusoidal mem-brane by facilitated diffusion or receptor-mediated endocytosis; (2)storage in binding proteins or lysosomes; and (3) canalicular se-cretion via lysosomes, a glutathione-coupled event, or a specificcanalicular membrane transporter (Ballatori, 1991). Biliary excre-

tion is important in the homeostasis of multiple metals, notablycopper, manganese, cadmium, selenium, gold, silver, and arsenic(Klaassen, 1976; Gregus and Klaassen, 1986). Species differencesare known for biliary excretion of several toxic metals; for exam-ple, dogs excrete arsenic into bile much more slowly than rats. In-ability to export Cu into bile is a central problem in Wilson’s dis-ease, a rare genetic disorder characterized by accumulation of Cuin the liver and then in other tissues. The exact nature of the de-fect in Cu export is uncertain, since the product of the Wilson’sdisease gene does not localize to the canalicular membrane(Nagano et al., 1998).

Canalicular lumen bile is propelled forward into larger chan-nels by dynamic, ATP-dependent contractions of the pericanalicu-lar cytoskeleton (Watanabe et al., 1991). Bile ducts, once regardedas passive conduits, modify bile by absorption and secretion ofsolutes (Lira et al., 1992). Biliary epithelial cells also express a va-riety of phase I and phase II enzymes, which may contribute to thebiotransformation of chemical toxicants present in bile (Lakehal etal., 1999).

Figure 13-4. Schematic of liver sinusoidal cells.

Note that the Kupffer cell resides within the sinusoidal lumen. The Ito cell is located in the space of Disse be-tween the thin, fenestrated endothelial cells and the cord of hepatocytes.

Figure 13-5. Processes involved in hepatocyte uptake and biliary secretion of endogenous solutes and toxi-cants.

Transporters localized to the sinusoidal membrane extract solutes from the blood. Exporters localized to canalic-ular membrane move solutes into the lumen of the canaliculus. Exporters of particular relevance to canalicularsecretion of toxic chemicals and their metabolites are the canalicular multiple organic anion transporter (MOAT)system and the family of multiple-drug resistant (MDR) P-glycoproteins.

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Secretion into biliary ducts is usually but not always a pre-lude to toxicant clearance by excretion in feces or urine. Excep-tions occur when compounds such as arsenic are repeatedly deliv-ered into the intestinal lumen via bile, efficiently absorbed fromthe intestinal lumen, and then redirected to the liver via portalblood, a processes known as enterohepatic cycling. A few com-pounds, such as methyl mercury, are absorbed from the biliary tract;the extensive reabsorption of methyl mercury from the gallbladderis thought to contribute to the long biological half-life and toxic-ity of this toxin (Dutczak et al., 1991). Alternatively, secretion intobile of toxicant metabolites can be a critical prelude to the devel-opment of injury in extrahepatic tissues. A clinically relevant ex-ample of bile as an important delivery route for a proximate toxi-cant is that of diclofenac, a widely prescribed nonsteroidalanti-inflammatory drug (NSAID) that causes small intestinal ul-ceration. Convincing experiments with mutant rats lacking a func-tional canalicular MOAT exporter (Fig. 13-5) have shown that thesemutants secrete little of the presumptive proximate toxicantmetabolite into bile and are resistant to the intestinal toxicity of di-clofenac (Seitz and Boelsterli, 1998).

Toxicant-related impairments of bile formation are more likelyto have detrimental consequences in populations with other con-ditions where biliary secretion is marginal. For example, neonatesexhibit delayed development of multiple aspects of bile formation,including synthesis of bile salts and the expression of sinusoidaland canalicular transporters (Arrese et al., 1998). Neonates aremore prone to develop jaundice when treated with drugs that com-pete with bilirubin for biliary clearance. Populations with sepsisare also of concern based on animal studies indicating that sepsisis associated with down-regulation of multiple canalicular ex-porters (Trauner et al., 1998).

Types of Injury and Toxic Chemicals

Hepatic response to insults by chemicals depends upon the inten-sity of the insult, the population of cells affected, and whether theexposure is acute or chronic (Fig. 13-1). Acute poisoning with car-bon tetrachloride causes rapid lipid accumulation, before necrosis

becomes evident. Some chemicals produce a very specific type ofdamage; others, notably ethanol, produce sequential types of dam-age or combinations of damage (Table 13-2). Note that the repre-sentative hepatotoxins listed in Table 13-2 include pharmaceuticals(valproic acid, cyclosporin A, diclofenac), recreational drugs(ethanol, ecstasy), a vitamin (vitamin A), metals (Fe, Cu, Mn),hormones (estrogens, androgens), industrial chemicals (dimethyl-formamide, methylene dianiline), compounds found in teas(germander) or foods (phalloidin, pyrrolidine alkaloids), and tox-ins produced by fungi (sporidesmin) and algae (microcystin). SeeFig. 13-6 for the structures of representative hepatotoxic chemi-cals.

Fatty Liver This change, also known as steatosis, is defined bio-chemically as an appreciable increase in the hepatic lipid content,which is �5 percent by weight in normal human liver. Histologi-cally, in standard paraffin-embedded and solvent-extracted sec-tions, hepatocytes containing excess fat appear to have multipleround, empty vacuoles that displace the nucleus to the peripheryof the cell. Use of frozen sections and special stains is needed todocument the contents of the vesicles as fat. Fatty liver can stemfrom one or more of the following events: oversupply of free fattyacids to the liver, interference with the triglyceride cycle, increasesin synthesis or esterification of fatty acids, decreased fatty acid ox-idation, decreased apoprotein synthesis, and decreased synthesis orsecretion of very low density lipoproteins.

Steatosis is a common response to acute exposure to manybut not all hepatotoxins (Farrell, 1994). An exception is acetamin-ophen. Compounds that produce prominent steatosis associatedwith lethality include the antiepileptic drug valproic acid (Hall,1994) and the antiviral agent fialuridine (Honkoop et al., 1997).Often, toxin-induced steatosis is reversible and does not lead to death of hepatocytes. The metabolic inhibitors ethionine,puromycin, and cycloheximide cause fat accumulation withoutcausing death of cells. Many other conditions besides toxin expo-sure, such as obesity, are associated with marked fat accumulationin the liver. Therefore assumptions about cause-effect relationshipsin regard to toxins and steatosis need to be made judiciously.

Table 13-2Types of Hepatobiliary Injury

TYPE OF INJURY

OR DAMAGE REPRESENTATIVE TOXINS

Fatty liver CCl4, ethanol, fialuridine, valproic acid

Hepatocyte death Acetaminophen, Cu, dimethylformamide, ethanol, Ecstasy

Immune-mediated response Diclofenac, ethanol, halothane, tienilic acid

Canalicular cholestasis Chlorpromazine, cyclosporin A, 1,1-dichloroethylene,estrogens, Mn, phalloidin

Bile duct damage Amoxicillin, ANIT, methylene dianiline,sporidesmin

Sinusoidal disorders Anabolic steroids, cyclophosphamide, microcystin,pyrrolidine alkaloids

Fibrosis and Arsenic, ethanol, vitamin A, vinyl chloridecirrhosis

Tumors Aflatoxin, androgens, thorium dioxide, vinyl chloride

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Cell Death Liver cells can die by two different modes, necrosisand apoptosis. Necrosis is characterized by cell swelling, leakage,nuclear disintegration, and an influx of inflammatory cells. Apop-tosis is characterized by cell shrinkage, nuclear fragmentation, for-mation of apoptotic bodies, and a lack of inflammation. Apopto-sis is more difficult to detect histologically because of the rapidremoval of affected cells (Corcoran et al., 1994). Lysed debris ofnecrotic cells can persist for days when large numbers of cells die.When necrosis occurs in hepatocytes, the associated plasma mem-brane leakage can be detected biochemically by assaying plasma(or serum) for liver cytosol-derived enzymes. Particularly inform-ative are the activity levels of alanine aminotransferase (ALT), apredominantly hepatocyte enzyme, unlike lactate dehydrogenase(LDH), which is found in many tissues. Biochemical assays pro-vide a relatively simple way to screen populations for potential he-patocyte necrosis due to occupational or environmental toxins. Acareful occupational health study by Redlich et al. (1988; 1990) ina New Haven, Connecticut, plant with primitive systems for workerprotection found that exposure to dimethylformamide was associ-ated with liver damage. Serum ALT levels were appreciably ele-vated in most of the exposed workers; however, liver biopsies in-dicated that a substantial cause of the liver damage in one of theworkers was an infectious agent. Thus a limitation of biochemicalindices of hepatocyte necrosis is the inability to distinguish be-

tween chemically induced effects and other causes such as hepati-tis virus.

Hepatocyte death can occur in a focal, zonal, or panacinar(panlobular) pattern. Focal cell death is characterized by the ran-domly distributed death of single hepatocytes or small clusters ofhepatocytes. Zonal necrosis is death to hepatocytes predominantlyin zone 1 (periportal) or zone 3 (centrolobular). Many toxins causezone 3 necrosis, while fewer agents are known to specifically dam-age cells in zone 1 or zone 2. Information about the zonal locationof injury by a given chemical helps identify a sensitive, noninva-sive index of functional change. For example, serum levels of bilesalts are more likely to be elevated after damage to zone 1 than tozone 3 due to the direction of the gradient for bile salt uptake fromblood (Fig. 13-3).

Some reasons why chemical toxins preferentially damage he-patocytes in specific zones are discussed further on, under “Fac-tors in Liver Injury.” Zone 3 necrosis can affect just a narrow rimof cells around the central vein, or it may extend into zone 2.Panacinar necrosis is massive death of hepatocytes with only a fewor no remaining survivors. An intermediate form of substantialnecrosis is called bridging necrosis because the extensive zones ofcell lysis become confluent with each other. Mechanisms of toxin-induced injury to liver cells include lipid peroxidation, binding tocell macromolecules, mitochondrial damage, disruption of the cy-toskeleton, and massive calcium influx. Progression from injury todeath may involve activation of sinusoidal cells and, with repeatedtoxicant exposure, may lead to an antibody-mediated immune at-tack.

Canalicular Cholestasis This form of liver injury is definedphysiologically as a decrease in the volume of bile formed or animpaired secretion of specific solutes into bile. Cholestasis is char-acterized biochemically by elevated serum levels of compoundsnormally concentrated in bile, particularly bile salts and bilirubin.When biliary excretion of the yellowish bilirubin pigment is im-paired, this pigment accumulates in the skin and eyes, producingjaundice, and spills into urine, which becomes bright yellow ordark brown. Dyes that are excreted in bile, such as bromsulphalein(BSP), have been used to assess biliary function. The histologicfeatures of cholestasis can be very subtle and difficult to detectwithout ultrastructural studies. Structural changes include dilationof the bile canaliculus and the presence of bile plugs in bile ductsand canaliculi. Toxin-induced cholestasis can be transient or chronic;when substantial, it is associated with cell swelling, cell death, andinflammation. Many different types of chemicals—including met-als, hormones and drugs—cause cholestasis (Table 13-2).

Bile Duct Damage Another name for damage to the intrahepaticbile ducts is cholangiodestructive cholestasis (Cullen and Ruebner,1991). A useful biochemical index of bile duct damage is a sharpelevation in serum activities of enzymes localized to bile ducts,particularly alkaline phosphatase. In addition, serum levels of bilesalts and bilirubin are elevated, as observed with canalicularcholestasis. Initial lesions following a single dose of cholangiode-structive agents include swollen biliary epithelium, debris of dam-aged cells within ductal lumens, and inflammatory cell infiltrationof portal tracts. Chronic administration of toxins that cause bileduct destruction can lead to biliary proliferation and fibrosis re-sembling biliary cirrhosis. Another response is the loss of bileducts, a condition known as vanishing bile duct syndrome. This

Figure 13-6. Structures of representative hepatotoxic chemicals.

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persisting problem has been reported in patients receiving antibi-otics (Davies et al., 1994).

Methylene dianiline, a compound used to make epoxy resins,is a noteworthy cause of bile duct damage. Small doses of meth-ylene dianiline produce selective bile duct injury and thus providean experimental model to study mechanisms of chemically inducedbile duct damage (Kanz et al., 1992). The potent toxicity of meth-ylene dianiline was first recognized in 1966, when an epidemio-logic study established this compound as the causal agent of“Epping jaundice”—an outbreak of jaundice and severe hepato-biliary disease in more than 80 residents of the English village ofEpping. The affected villagers had eaten bread made from flourcontaminated with this compound (Kopelman et al., 1966). A morerecent episode of methylene dianiline poisoning occurred at a“technoparty” where six young people ingested this agent due toconfusion between its MDA abbreviation and the MDMA abbre-viation for the synthetic amphetamine popularly known as Ecstasy(Tillman et al., 1997). All six individuals developed the jaundice,dark urine, abdominal pain, and nausea consistent with bile ductdamage. If these young people had ingested the intended drug Ec-stasy, they might also have developed liver problems. AlthoughEcstasy does not target bile ducts, numerous cases of severe liverdamage have been reported after single and repeated exposure tothis recreational drug (Andreu et al., 1998).

Sinusoidal Damage The sinusoid is, in effect, a specialized cap-illary with numerous fenestrae for high permeability. The func-tional integrity of the sinusoid can be compromised by dilation orblockade of its lumen or by progressive destruction of its en-dothelial cell wall. Dilation of the sinusoid will occur wheneverefflux of hepatic blood is impeded. The rare condition of primarydilation, known as peliosis hepatis, has been associated with ex-posure to anabolic steroids and the drug danazol. Blockade willoccur when the fenestrae enlarge to such an extent that red bloodcells become caught in them or pass through with entrapment inthe interstitial space of Disse. Such changes have been illustratedby scanning electron microscopy after large doses of the drug acet-aminophen (Walker et al., 1983). A consequence of extensive si-nusoidal blockade is that the liver becomes engorged with bloodcells while the rest of the body goes into shock. Microcystin pro-duces this effect within hours in rodents (Hooser et al., 1989). Mi-crocystin dramatically deforms hepatocytes by altering cytoskele-ton actin filaments, but it does not affect sinusoidal cells (Hooseret al., 1991). Thus the deformities that microcystin produces on thecytoskeleton of hepatocytes likely produce a secondary change inthe structural integrity of the sinusoid owing to the close proxim-ity of hepatocytes and sinusoidal endothelial cells (Fig. 13-4).

Progressive destruction of the endothelial wall of the sinusoidwill lead to gaps and then ruptures of its barrier integrity, with en-trapment of red blood cells. These disruptions of the sinusoid areconsidered the early structural features of the vascular disorderknown as veno-occlusive disease (DeLeve et al., 1999). Well es-tablished as a cause of veno-occulsive disease are the pyrrolizidinealkaloids (e.g., monocrotaline, retrorsine, and seneciphylline)found in some plants used for herbal teas and in some seeds thatcontaminate food grains. Numerous episodes of human and animalpoisoning by pyrrolizidine alkaloids have been reported around theworld, including massive problems affecting thousands of peoplein Afghanistan in 1976 and 1993 (Huxtable, 1997). Veno-occlusivedisease is also a serious complication in about 15 percent of thepatients given high doses of chemotherapy (e.g., cyclophos-

phamide) as part of bone-marrow transplantation regimens(DeLeve et al., 1999). Experimental studies indicate that toxicant-induced killing of sinusoidal endothelial cells can occur withoutbioactivation by hepatocytes (DeLeve and Huybrechts, 1996). De-pletion of glutathione within sinusoidal endothelial cells precedesthe preferential injury to this type of hepatic cell (Wang et al.,2000).

Cirrhosis This form of injury is the end, often fatal, stage ofchronic progressive liver injury. Cirrhosis is characterized by theaccumulation of extensive amounts of fibrous tissue, specificallycollagen fibers, in response to direct injury or to inflammation. Fi-brosis can develop around central veins and portal tracts or withinthe space of Disse, which limits diffusion of material from the si-nusoid. With repeated chemical insults, destroyed hepatic cells arereplaced by fibrotic scars. With continuing collagen deposition, thearchitecture of the liver is disrupted by interconnecting fibrousscars. When the fibrous scars subdivide the remaining liver massinto nodules of regenerating hepatocytes, fibrosis has progressedto cirrhosis and the liver has meager residual capacity to performits essential functions.

Cirrhosis is not reversible, has a poor prognosis for survival,and is usually the result of repeated exposure to chemical toxins.For example, cirrhosis associated with vitamin A has been reportedin patients with dermatologic problems on high-dose therapy(�100,000 IU) for an average of 7 years (Geubel et al., 1991). Therisk for cirrhosis in alcoholics increases dramatically in males whoconsume �80 g/day and in females who consume �20 g day for10 years. Note that the amount of ethanol in 8 beers, 8 glasses ofwine, or 7 oz of 80-proof liquor is approximately equivalent to 80g. The greater vulnerability of females to alcohol can be explainedonly partially by their smaller body size and lower capacity forethanol metabolism in the stomach.

Tumors Chemically induced neoplasia can involve tumors thatare derived from hepatocytes, bile duct cells, or the rare, highlymalignant angiosarcomas derived from sinusoidal lining cells. He-patocellular cancer has been linked to abuse of androgens and ahigh prevalence of aflatoxin-contaminated diets. The synergisticeffect of co-exposure to aflatoxin and hepatitis virus B was clearlydocumented by a recent prospective study where the risk for he-patocellular carcinoma was found to be increased threefold in afla-toxin-exposed men with chronic hepatitis B infection (Sun et al.,1999). The investigators monitored urine specimens from the sub-jects for a metabolite of aflatoxin in order to verify dietary afla-toxin exposure. Angiosarcomas have been tightly associated withoccupational exposure to vinyl chloride and arsenic (Farrell, 1994).

Exposure to Thorotrast has been linked to tumors derived fromhepatocytes, sinusoidal cells, and bile duct cells (cholangiocarci-noma). The history of Thorotrast (radioactive thorium dioxide) isa sad tale of a useful agent with an unanticipated toxicity due toprolonged retention within the body. Between 1920 and 1950, anestimated 2.5 million people were injected with suspensions ofThorotrast as a contrast medium for radiologic procedures. Thecompound accumulates in Kupffer cells, the resident macrophageof the sinusoid, and emits radioactivity throughout its very extendedhalf-life. Thus, it is not surprising that multiple types of liver tu-mors are linked to thorium dioxide exposure. One study of Dan-ish patients exposed to Thorotrast found that the risk for bile ductand gallbladder cancers was increased 14-fold and that for livercancers more than 100-fold (Andersson and Storm, 1992).

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FACTORS IN LIVER INJURY

Why is the liver the target site for so many chemicals of diversestructure? Why do many hepatotoxicants preferentially damage onetype of liver cell? Our understanding of these fundamental ques-tions is incomplete. Influences of several factors are of obvious im-portance (Table 13-3). Location and specialized processes for up-take and biliary secretion produce higher exposure levels in theliver than in other tissues of the body and strikingly high levelswithin certain types of liver cells. Then the abundant capacity forbioactivation reactions influences the rate of exposure to proximatetoxicants. Subsequent events in the pathogenesis appear to be crit-ically influenced by responses of sinusoidal cells and the immunesystem. Discussion of the evidence for the contributions of thesefactors to the hepatotoxicity of representative compounds requirescommentary about mechanistic events; therefore this section of thechapter is closely related to the next one, entitled “Mechanisms ofLiver Injury.”

Table 13-4 lists experimental systems useful for defining fac-tors and mechanisms of liver injury. In vitro systems using the iso-lated perfused liver, isolated liver cells, and cell fraction allow ob-servations at various levels of complexity without the confoundinginfluences of other systems. Models using co-cultures or agentsthat inactivate a given cell type can document the contributions andinteractions between cell types. Whole-animal models are essen-tial for assessment of the progression of injury and responses tochronic insult. Use of agents that induce, inhibit, deplete, or inac-tivate can define roles of specific processes, although potential in-fluences of nonspecific actions can confound interpretations. Ap-plication of molecular biology techniques for gene transfection orrepression attenuates some of these interpretive problems. Knock-out rodents provided extremely useful models for complex aspectsof hepatotoxicity. The reason for the persuasiveness of observa-

tions from experiments with knockout rodents is that the gene prod-uct of interest is not present and therefore not just inhibited by anonspecific agent with potential confounding effects on otherprocesses.

Uptake and Concentration

Hepatic “first pass” uptake of ingested toxic chemicals is facili-tated by the location of the liver downstream of the portal bloodflow from the gastrointestinal tract. Lipophilic compounds, partic-ularly drugs and environmental pollutants, readily diffuse into he-patocytes because the fenestrated epithelium of the sinusoid en-ables close contact between circulating molecules and hepatocytes.Thus, the membrane-rich liver concentrates lipophilic compounds.Other toxins are rapidly extracted from blood because they are sub-strates for sinusoidal transporters present exclusively or predomi-nantly in the liver.

Phalloidin and microcystin are illustrative examples ofhepatotoxins that target the liver as a consequence of extensive up-take into hepatocytes by sinusoidal transporters (Frimmer, 1982;Runnegar et al., 1995a,b). Ingestion of the mushroom Amanitaphalloides is a common cause of severe, acute hepatotoxicity incontinental Europe and North America. Microcystin has producednumerous outbreaks of hepatotoxicity in sheep and cattle whodrank pond water containing the blue-green alga Microcystis aerug-inosa (Farrell, 1994). An episode of microcystin contamination ofthe water source used by a hemodialysis center in Brazil led toacute liver injury in 81 percent of the 124 exposed patients and thesubsequent death of 50 of these (Jochimsen et al., 1998). Micro-cystin contamination was verified by analysis of samples from thewater-holding tank at the dialysis center and from the livers of pa-tients who died. This episode indicates the vulnerability of the liverto toxicants regardless of the route of administration. Because of

Table 13-3Factors in the Site-Specific Injury of Representative Hepatotoxicants

REPRESENTATIVE POTENTIAL EXPLANATION FOR

SITE TOXICANTS SITE-SPECIFICITY

Zone 1 hepatocytes Fe (overload) Preferential uptake and high (versus zone 3) oxygen levels

Allyl alcohol Higher oxygen levels for oxygen-dependent bioactivation

Zone 3 hepatocytes CCl4 More P450 isozyme for bioactivation(versus zone 1) Acetaminophen More P450 isozyme for bioactivation and

less GSH for detoxificationEthanol More hypoxic and greater imbalance in

bioactivation/detoxification reactions

Bile duct cells Methylene dianiline, Exposure to the high concentration ofSporidesmin reactive metabolites in bile

Sinusoidal Cyclophosphamide, Greater vulnerability to toxicendothelium Monocrotaline metabolites and less ability to (versus hepatocytes) maintain glutathione levels

Kupffer cells Endotoxin, GdCl3 Preferential uptake and then activation

Ito cells Vitamin A Preferential site for storage and thenengorgement

Ethanol (chronic) Activation and transformation to collagen-synthesizing cell

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its dual blood supply from both the portal vein and the hepatic ar-tery, the liver is presented with appreciable amounts of all toxi-cants in the systemic circulation.

An early clue to preferential uptake as a factor in phalloidin’starget-organ specificity was the observation that bile duct ligation,which elevates systemic bile salt levels, protects rats against phal-loidin-induced hepatotoxicity in association with an 85 percent de-crease in hepatic uptake of phalloidin (Walli et al., 1981). Subse-quent studies found that co-treatment with substrates (e.g.,cyclosporin A, rifampicin) known to prevent the in vivo hepato-toxicity of phalloidin or microcystin would also inhibit their up-take into hepatocytes by sinusoidal transporters for bile acids ororganic anions (Ziegler and Frimmer 1984; Runnegar et al., 1995a).

Accumulation within liver cells, by processes that facilitateuptake and storage, is a determining factor in the hepatotoxicity ofvitamin A and several metals. Vitamin A hepatotoxicity initially af-fects the sinusoidal Ito cells, which actively extract and store thisvitamin. Early responses to high-dose vitamin A therapy are Itocell engorgement, activation, increase in number, and protrusioninto the sinusoid (Geubel et al., 1991). Cadmium hepatotoxicitybecomes manifest when the cells exceed their capacity to sequestercadmium as a complex with the metal-binding protein metallo-thionein. This protective role for metallothionein was definitivelydocumented by observations with transgenic mice. Specifically,high expression of this metal-binding protein in the transgenic micerendered them more resistant than wild-type mice to the hepato-toxicity and lethality of cadmium poisoning (Liu et al., 1995).

Iron poisoning will produce severe liver damage. Hepatocytescontribute to the homeostasis of Fe by extracting this essentialmetal from the sinusoid by a receptor-mediated process and main-taining a reserve of Fe within the storage protein ferritin. Acute Fetoxicity is most commonly observed in young children who acci-dentally ingest iron tablets. The cytotoxicity of free Fe is attrib-uted to its function as an electron donor for the formation of re-active oxygen species, which initiate destructive oxidative stressreactions. Accumulation of excess Fe beyond the capacity for itssafe storage in ferritin is initially evident in the zone 1 hepatocytes,which are closest to the blood entering the sinusoid. Thus the zone1 pattern of hepatocyte damage after iron poisoning is attributableto location for (1) the preferential uptake of Fe and (2) the higheroxygen concentrations that facilitate the injurious process of lipidperoxidation (Table 13-3). Chronic hepatic accumulation of excessiron in cases of hemochromatosis is associated with a spectrum ofhepatic disease including a greater than 200-fold increased risk forliver cancer.

Bioactivation and Detoxification

Hepatocytes have very high constitutive activities of the phase Ienzymes that often convert xenobiotics to reactive electrophilicmetabolites. Also, hepatocytes have a rich collection of phase IIenzymes that add a polar group to a molecule and thereby enhanceits removal from the body. Phase II reactions usually yield stable,nonreactive metabolites. In general, the balance between phase Iand phase II reactions determines whether a reactive metabolitewill initiate liver cell injury or be safely detoxified. The balancecan be shifted towards liver injury by acquired or genetic condi-tions that enhance bioactivation processes or impair detoxificationprocesses. Notable acquired conditions for such a shift in balanceare drug or pollutant induction of phase I enzymes and/or deple-tion of antioxidants.

Ethanol Genetic conditions of high clinical relevance to thebioactivation/detoxification balance are the polymorphisms in theenzymes that control the two-step metabolism of ethanol. Specif-ically, ethanol is bioactivated by alcohol dehydrogenase to ac-etaldehyde, a reactive aldehyde, which is subsequently detoxifiedto acetate by aldehyde dehydrogenase. Both enzymes exhibit ge-netic polymorphisms that result in higher concentrations ofacetaldehyde—a “fast” activity isozyme of alcohol dehydrogenase[ALD2*2] and a physiologically very “slow” mitochondrialisozyme of aldehyde dehydrogenase [ALDH2*2]. Approximately50 percent of Asian populations but virtually no Caucasians havethe slow aldehyde dehydrogenase; alcohol consumption by peoplewith this slow polymorphism leads to uncomfortable symptoms offlushing and nausea due to high systemic levels of acetaldehyde.Thus this slow detoxification polymorphism serves as a strong de-terrent for alcoholism. The alcohol dehydrogenase fast polymor-phism, which occurs in about 20 percent of Asian and less than 5percent of Caucasian populations, has also been linked to a lowerrate of alcoholism. An extremely low risk for alcoholism has beenfound in Asians with both polymorphisms that lead to higher lev-els of acetaldehyde (Chen et al., 1999a).

Allyl alcohol toxicity is also influenced by a balance betweenthe formation and detoxification of its reactive aldehyde metabo-lite, acrolein, by sequential actions of alcohol dehydrogenase andaldehyde dehydrogenase enzymes. Age and gender differences inallyl alcohol hepatotoxicity can be explained by variations in thebalance between these two enzymes (Rikans and Moore, 1987).Allyl alcohol is used in the production of resins, plastics, and fireretardants. The preferential occurrence of allyl alcohol injury inzone 1 hepatocytes (Table 13-3) is due to oxygen-dependent bioac-tivation. This aspect of its mechanism of hepatotoxicity wasdemonstrated by creative experiments where the typical decline inthe oxygen gradient from zone 1 to zone 3 was reversed by a ret-rograde (backward) perfusion of isolated perfused livers (Badr etal., 1986).Cytochrome P450 The importance of cytochrome P450-dependent bioactivation as a mechanism of hepatotoxicity must beemphasized. This common theme can be a factor even forassumedly safe compounds since some P450 isozymes generate re-active oxygen species during biotransformation reactions (Albanoet al., 1996). Concern about CYP2E1 generation of reactive oxy-gen species and other free radicals has come largely from effortsto determine why only a fraction of chronic heavy drinkers developserious, end-stage liver damage. Dietary manipulations that lead tomore severe alcohol-associated liver damage in the animal model

Table 13-4Experimental Systems

In vitro systemsCell fractionsPrimary cell culturesPrimary cell co-culturesTransfected cell systemsIsolated perfused livers

In vivo systemsMultiple species and strainsTransgenic and knockout rodents

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show a linkage between dietary induction of CYP2E1 and hepa-totoxicity (French et al., 1995; Korourian et al., 1999). These ma-nipulation have involved the amount of dietary lipid, the type offatty acid in the lipid component of the diet, diets deficient in car-bohydrates, and co-exposure to inhibitors of CYP2E1. The issueis complex because ethanol is an inducer of P450; heavy drinkersexhibit approximately threefold higher activities of CYP2E1 thannondrinkers. However, the observed modulation of liver injury bydietary manipulations does indicate a role for nutrients in the he-patotoxicity of alcohol.

CYP2E1 is not the only cytochrome P450 isozyme with animpact on hepatotoxicity. An appropriately germane example is thefolk medicine plant germander (Teucrium chamaedrys L.), whichwas considered a safe component of herbal teas until widespreadconsumption of capsules containing large amounts of germanderfor weight control in France was linked to multiple cases of he-patic damage (Larrey et al., 1992; Loeper et al., 1994). Systematicexperimental studies have identified the specific toxic constituentin germander plants, demonstrated a predominant role for theCYP3A isozyme in germander bioactivation to reactive elec-trophiles, and determined that toxicity can be attenuated by main-tenance of glutathione levels (Loeper et al., 1994; Lekehal et al.,1996). This kind of information has practical applications for iden-tifying populations at enhanced risk for germander toxicity due toacquired conditions—such as obesity, sudden weight loss, or con-current ingestion of other folk medications or pharmaceuticals—that might shift the balance between CYP3A bioactivation and glu-tathione-dependent detoxification.Carbon Tetrachloride Cytochrome P450-dependent conversionof CCl4 to •CCl3 and then to CCl3OO• is the classic example ofxenobiotic bioactivation to a free radical that initiates lipid perox-idation by abstracting a hydrogen atom from the polyunsaturatedfatty acid of a phospholipid (Rechnagel, 1967). Experimental stud-ies have identified numerous treatments and conditions that mod-ulate the extent of liver damage produced by CCl4. Protective sit-uations include baby animals with little cytochrome P450;treatments with compounds that inhibit cytochrome P450; and pre-treatment with a small dose of the same toxin that diminishes cy-tochrome P450 levels (Reynolds and Moslen, 1980). Augmentingsituations include hypoxia, diabetes, diets low in vitamin E (sincethis antioxidant scavenges lipid peroxide radicals), and pretreat-ment with other chemicals, notably ethanol and acetone, which in-duce CYP2E1, the isozyme most effective in the activation of CCl4.Anecdotal reports of human exposures to this toxicant suggest agreater vulnerabilty in individuals who should have high levels ofhepatic CYP2E1 due to a history of alcohol abuse (Manno et al.,1996). Uncertainties about the importance of other P450 isozymeshave been squelched by documentation of virtual resistance to thehepatotoxicity of CCl4 in CYP2E1 knockout mice (Wong et al.,1998). Investigations about events in the pathogenesis of carbontetrachloride hepatotoxicity paved the way for identification of fac-tors important in the toxicity of other insults that also cause lipidperoxidation.Acetaminophen The hepatotoxicity of this extensively used anal-gesic is a clinically important problem and an exemplary instanceof how acquired factors (e.g., diet, drugs, diabetes, obesity) can en-hance hepatotoxicity. Typical therapeutic doses of acetaminophenare not hepatotoxic, since the dominant pathways of biotransfor-mation are conjugation with glucuronide or sulfate with little drugbioactivation. Injury after large doses of acetaminophen is en-

hanced by fasting and other conditions that deplete glutathione andis minimized by treatments that enhance hepatocyte synthesis ofglutathione, particularly cysteine, the rate-limiting amino acid inglutathione synthesis. Introduction of interventive therapy with N-acetylcysteine, a well- tolerated source of intracellular cysteine, hassaved the lives of many who took overdoses of acetaminophen,usually in suicide attempts (Smilkstein et al., 1988).

Alcoholics are vulnerable to the hepatotoxic effects of aceta-minophen at dosages within the high therapeutic range (Lieber,1994). This acquired enhancement has widely been attributed toaccelerated bioactivation of acetaminophen to the electrophilic N-acetyl-p-benzoquinone imine (NAPQI) intermediate by ethanol in-duction of CYP2E1 (Fig. 13-7). However, alcoholic beverages alsocontain higher-chain alcohols, such as isopentanol, in appreciableamounts up to 0.5% (w/v), and alcohol consumption has many ef-fects on the liver besides induction of CYP2E1 (Lieber, 1994). Theassumed exclusive role for CYP2E1 bioactivation is controversial,in part because agents used to inhibit this isozyme also inhibit otherisozymes of cytochrome P450. Evidence for roles of other alco-hols besides ethanol and for other isozymes of P450 is availablein the literature. For example, CYP2E1 knockout mice are not de-void of a toxic response to acetaminophen (Lee et al., 1996). Par-ticularly convincing evidence is provided by a new report showing(1) synergistic enhancement of acetaminophen hepatotoxicity inrats pretreated with liquid diets containing ethanol plus isopentanoland (2) attenuation of acetaminophen hepatotoxicity when the al-cohol-pretreated animals were given a specific inhibitor of CYP3A(Sinclair et al., 2000). This report should heighten suspicion aboutthe potential influences of other CYP3A inducers on acetamino-phen toxicity, since hepatic activities of this isozyme are increasedby many drugs and by dietary chemicals such as caffeine.

Covalent binding, or adduction, of the reactive NAPQI inter-mediate of acetaminophen to hepatic proteins is a widely acceptedmechanism for the hepatotoxicity of this drug. Adduction of amacromolecule could alter its functional integrity and thus consti-tute a detrimental molecular change. Early acceptance of the ad-duction mechanism for acetaminophen stemmed from close paral-lels found between the magnitude of injury and the extent ofcovalent binding in livers of animals given labeled acetaminophen(Mitchell et al., 1973). A key variable in these seminal experimentswas the availability of glutathione for detoxification. Developmentof antibodies that recognize adducts of acetaminophen to macro-molecules allowed demonstration of adduct formation before celldamage, predominantly in zone 3, where histologic injury occurs(Roberts et al., 1991). Thus adduct formation as a mechanism ofinjury is plausible temporally and locationally. One difficulty thatresearchers encountered in looking for a dose-dependent relation-ship between acetaminophen adducts and liver damage was an ap-propriate time to measure adduct formation, since damaged cellsleak adducted proteins as well as enzymes indicative of tissue-specific injury (e.g., ALT).

A persisting concern about the adduction theory for aceta-minophen toxicity is whether adduct formation is the critical eventin acetaminophen toxicity or a biomarker of exposure to elec-trophilic metabolites. Prompting this concern are questions abouthow adduction to hepatocyte proteins could explain the importanceof macrophages to the toxicity of this drug. A series of studies(Laskin et al., 1990; Gardner et al., 1998) have demonstrated pro-tection by inactivation of hepatic macrophages (Kupffer cells) andcontributions of reactive nitrogen species. Pretreatments that inac-

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tivate macrophages and attenuate toxicity by acetaminophen didnot, however, diminish the extent of acetaminophen adduct for-mation (Michael et al., 1999). Yet the pretreatments did diminishthe extent of hepatocyte adduction by reactive nitrogen species, anintriguing observation that fits with the reported contributions ofreactive nitrogens to acetaminophen toxicity. Michael et al. (1999)have proposed an attractive “two hit” type of revised theory for thehepatotoxicity of acetaminophen. Perhaps adduction by a reactivedrug metabolite “primes” the hepatocytes for destructive insults byreactive nitrogen species (e.g., peroxynitrite) (Fig. 13-7).

Activation of Sinusoidal Cells

Four kinds of observations, collectively, indicate roles for sinu-soidal cell activation as primary or secondary factors in toxin-induced injury to the liver:

1. Kupffer cells and Ito cells exhibit an activated morphology—enlarged and ruffled—after acute and chronic exposure to he-patotoxicants (Laskin et al., 1990).

2. Pretreatments that activate or inactivate Kupffer cells appro-priately modulate the extent of damage produced by classic

toxicants (e.g., acetaminophen, carbon tetrachloride, andalcohol). For example, a series of studies by Sipes and col-leagues demonstrated that Kupffer cell activation by vitaminA profoundly enhances the acute toxicity of carbon tetrachlo-ride; this enhancement did not occur when animals were alsogiven an inactivator of Kupffer cells (ElSisi et al., 1993).

3. Activated Kupffer cells secrete appreciable amounts of solu-ble cytotoxins, including reactive oxygen and nitrogen species.Administration of agents that scavenge or inhibit these solu-ble cytotoxins attenuates the effects of sinusoidal cellenhancement (Gardener et al., 1998; Thurman et al., 1997).

4. Acute and chronic exposure to alcohol directly or indirectlyaffects sinusoidal cells (Thurman et al., 1997).

Kupffer cells are rapidly activated when the liver is perfusedwith solutions containing ethanol. Alcoholics have elevated sys-temic levels of endogenous Kupffer cell activators, notably tumornecrosis factor and the bacterial product endotoxin. The extent ofliver injury in animal models of alcoholic hepatitis is consistentlydiminished by treating animals with antibiotics that lower endo-toxin levels or with antibodies to tumor necrosis factor. This an-tibiotic protection disappears when the animals also receive endo-

Figure 13-7. Schematic of key events in the bioactivation and hepatotoxicity of acetaminophen.

Bioactivation of acetaminophen by cytochrome P450 isozymes leads to the formation of the reactive interme-diate N-acetyl-p-benzoquinone (NAPQI), which can deplete glutathione or form covalent adducts with hepaticproteins. Experimental observations suggest that such effects “prime” hepatocytes for cytokines released by ac-tivated Kupffer cells. Progression to cell death is thought to involve activation of iNOS and other processes thatproduce reactive nitrogen species and oxidative stress. Agents that activate Kupffer cells exacerbate the toxic-ity. Exchange of signals between toxicant-primed and activated Kupffer cells is likely a factor in the acute he-patotoxicity produced by many compounds that damage hepatocytes.

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toxin. New animal experiments with knockout mice provide strongevidence for an essential role of tumor necrosis factor in alcohol-induced liver injury (Yin et al., 1999). The knockout mice used inthese experiments lacked a receptor for tumor necrosis factor. Ob-servations during the latter phases of chronic alcohol-induced liverdisease, when there is an abundance of collagen scarring, show ac-tivated, transformed Ito cells; transformed Ito cells are thought tobe little factories for collagen synthesis.

Figure 13-8 summarizes information presented in this and ear-lier sections of this chapter about the multiplicity of toxin-inducedinteractions with and between various liver cells. The effect on agiven cell type can be direct or may result from a cascade of sig-nals and responses between cell types.

Inflammatory and Immune Responses

Migration of neutrophils, lymphocytes, and other inflammatorycells into regions of damaged liver is a well-recognized feature ofthe hepatotoxicity produced by many chemicals. In fact, the po-tentially confusing term hepatitis refers to hepatocyte damage byany insult where hepatocyte death is associated with an influx ofinflammatory cells. The progressive phase of alcohol-induced liverdisease (between simple fatty liver and cirrhosis) is called alco-holic hepatitis. The liver damage occasionally observed after mul-tiple exposures to the anesthetic halothane is known as halothanehepatitis.

A relevant question for chemically induced acute damage tothe liver is: Does the influx of inflammatory cells facilitate bene-ficial removal of debris from damaged liver cells or does the in-flux contribute in a detrimental way to the extent of liver damageafter chemical injury? Detrimental effects are plausible, since ac-tivated neutrophils release cytotoxic proteases and reactive oxygenspecies. Pretreatments with prostaglandins and other compoundswith anti-inflammatory activity reduce the acute hepatotoxicity of�-naphthyl-isothiocyanate (ANIT), an extensively studied com-

pound that causes histologic damage to hepatocytes and bile ducts(Dahm et al., 1991); CCl4; and other compounds (Farrell, 1994).More direct evidence for a detrimental role of neutrophils has comefrom experiments where depletion of neutrophils diminished thehepatotoxic and cholestatic effects of ANIT. Further insight intothe roles of neutrophils in ANIT toxicity has been obtained by useof co-culture techniques for isolated primary hepatocytes, bile ductcells, and neutrophils (Hill et al., 1999). Placement of differenttypes of cells on opposite sides of a permeable chamber indicateda chain reaction of events whereby ANIT-treated bile duct cells re-lease a factor that attracts neutrophils and stimulates them to dam-age hepatocytes.

Immune responses are considered factors in the hepatotoxicityoccasionally observed after repeated exposure to chemicals, usu-ally drugs. Individuals who develop infrequent, unpredictable re-sponse are considered to be hypersensitive. An immune-mediatedresponse is considered plausible when the problem subsides aftertherapy is halted and then recurs on drug challenge or restorationof therapy. Although the concept is generally accepted, compellingevidence for immune-mediated responses is available only forethanol, halothane, and a few other hepatotoxicants (Pohl, 1990).Some kind of chemical-related molecular change is needed to stim-ulate an immunological attack, such as the formation of adductsbetween a reactive metabolite of the drug and hepatocyte proteins.Figure 13-9 depicts key features of the assumed scenario wherebyhepatic protein adducts could become antigenic and stimulate theproduction of antibodies. If on reexposure, more drug-proteinadducts are formed, cells with such adducts could be attacked bysystemic antibodies. Bioactivation to reactive species capable offorming protein adducts is a commonality for many drugs thoughtto produce immune-mediated hepatic injury (Selim and Kaplowitz,1999).

Reports about sporadic instances of apparent immune-mediated injury in individuals taking the widely prescribed NSAIDdiclofenac have spurred extensive research on this popular and ef-

Figure 13-8. Schematic depicting the complex cascade of toxin-evoked interactions between hepatocytes andsinusoidal cells.

Sinusoidal cell responses to toxins can lead to either injury or activation. A scenario could involve (1) toxin in-jury to hepatocytes, (2) signals from the injured hepatocyte to Kupffer and Ito cells, followed by (3) Kupffercell release of cytotoxins and (4) Ito cell secretion of collagen. Activation of Kupffer cells is an important fac-tor in the progression of injury evoked by many toxicants. Stimulation of collagen production by activated Itocells is a proposed mechanism for toxicant-induced fibrosis. [Concept from Crawford JM: The liver and the bil-iary tract, in Cotran RS, Kumar V, Robbins SL (eds): Robbins: Pathologic Basis of Disease, 6th ed. Philadel-phia: Saunders, 1999, pp 845–901, with permission.]

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fective anti-arthritic drug (Sallie et al., 1991; Farrell, 1994). He-patic bioactivation of diclofenac leads to the formation of multipleadducts. Some of the diclofenac adducts localize to hepatocytemembrane proteins (Hargus et al., 1994), where recognition by an-tibodies is feasible. There is considerable evidence for immune-mediated responses as factors in ethanol-induced liver disease. Ac-etaldehyde, the reactive metabolite of ethanol, forms adducts withhepatic proteins. Circulating antibodies that recognize acetalde-hyde-adducted proteins can be found in patients with liver diseaserelated to alcohol, and in some studies the antibody titer is higherin those with more severe disease (Rolla et al., 2000). However,specific contributions of drug-protein adducts and their antibodiesto the pathogenesis of diclofenac and alcohol have yet to be defined.

MECHANISMS OF LIVER INJURY

Some aspects of the mechanistic basis for hepatotoxicity aregeneric, since liver cells are vulnerable to the same types of insultsthat injure other tissues. Preferential liver damage frequently en-sues simply from the location of the liver and/or its high capacityfor converting chemicals to reactive entities. Exceptions that meritexplanation are the toxins that target the cytoskeleton due to theirexclusive uptake by hepatocytes and the drugs that damage hepaticmitochondria due to the potentially fatal systemic consequences.This section emphasizes mechanisms that produce cholestasis, be-cause biliary secretion is a unique and vital function of the liver.

Disruption of the Cytoskeleton

Phalloidin and microcystin disrupt the integrity of hepatocyte cy-toskeleton by affecting proteins that are vital to its dynamic nature.

The detrimental effects of these two potent hepatotoxicants are in-dependent of their biotransformation and are exclusive for hepato-cytes, since there is no appreciable uptake of either toxin into othertypes of cells. Tight binding of phalloidin to actin filaments pre-vents the disassembly phase of the normally dynamic rearrange-ment of the actin filament constituent of the cytoskeleton.Phalloidin uptake into hepatocytes leads to striking alterations inthe actin-rich web of cytoskeleton adjacent to the canalicular mem-brane; the actin web becomes accentuated and the canalicular lu-men dilates (Phillips et al., 1986). Experiments using time-lapsevideo microscopy have documented dose-dependent declines in thecontraction of canalicular lumens between isolated hepatocyte cou-plets after incubation with a range of phalloidin concentrations(Watanabe and Phillips, 1986).

Microcystin uptake into hepatocytes leads to hyperphosphory-lation of cytoskeletal proteins secondary to this toxicant’s covalentbinding to the catalytic subunit of serine/threonine protein phos-phatases (Runnegar et al., 1995c). Reversible phosphorylations ofcytoskeletal structural and motor proteins are critical to the dynamicintegrity of the cytoskeleton. As depicted in Fig. 13-10, extensivehyperphosphorylation produced by large amounts of microcystinleads to marked deformation of hepatocytes due to a unique col-lapse of the microtubular actin scaffold into a spiny central aggre-gate (Hooser et al., 1991). Lower doses of microcystin, insufficientto produce the gross structural deformations, diminish uptake andsecretory functions of hepatocytes in association with preferentialhyperphosphorylation of the cytoplasmic motor protein dynein(Runnegar et al., 1999). Dynein is a mechanicochemical protein thatdrives vesicles along microtubules using energy from ATP hydrol-ysis; central to the hydrolysis of the dynein-bound ATP is a cycleof kinase phosphorylation and phosphatase dephosphorylation.

Figure 13-9. Proposed scenario of events leading to immune-mediated hepatotoxicity after repeated expo-sure to a toxicant that produces drug-protein adducts (�).

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Thus, hyperphosphorylation of dynein freezes this motor pump.New experiments that looked at effects of chronic exposure to lowlevels of microcystin have raised new concerns about the health ef-fects of this water contaminant. Specifically, low levels of micro-cystin promote liver tumors and kill hepatocytes in the zone 3 re-gion, where microcystin accumulates (Solter et al., 1998).

Information about the binding of phalloidin and microcystinto specific target molecules is valuable for two reasons. First, thelinkages of specific binding to loss of target protein functions pro-vide compelling evidence that such binding constitutes a definedmolecular mechanism of injury. Second, the demonstrations ofhigh-affinity binding to a target molecule without confounding ef-fects on other processes or tissues have translated into applicationsof these toxins as tools for cell biology research. For example, phal-loidin complexed with a fluorochrome (e.g., rhodamine phalloidinor Texas Red phalloidin) is used to visualize the actin polymer

component of the cytoskeleton in all types of permeabilized cells.The collapse of actin filaments into spiny aggregates after micro-cystin treatment was visualized by fluorescence microscopy of cellsstained with rhodamine phalloidin (Hooser et al., 1991). Low lev-els of microcystin are being used to discriminate the roles of dyneinfrom other cytoskeletal motor proteins (Runnegar et al., 1999).

Cholestasis

Bile formation is vulnerable to toxicant effects on the functionalintegrity of sinusoidal transporters, canalicular exporters,cytoskeleton-dependent processes for transcytosis, and the con-tractile closure of the canalicular lumen (Fig. 13-11). Changes thatweaken the junctions that form the structural barrier between theblood and the canalicular lumen allow solutes to leak out of thecanalicular lumen. These paracellular junctions provide a size and

Figure 13-10. Schematic of events in the mechanism by which microcystin damages the structural and func-tional integrity of hepatocytes.

Microcystin is taken up exclusively into hepatocytes by a sinusoidal transporter in a manner inhibitable by bilesalts and organic anions. Then microcystin inhibition of protein phosphatases leads to hyperphosphorylation ofcytoskeletal proteins whose dynamic functions are dependent upon reversible phosphorylations. Extensive hy-perphosphorylation of microtubular proteins leads to a collapse of the microtubular actin filament scaffold intoa spiky aggregate that produces a gross deformation of hepatocytes. More subtle changes in microtubule-medi-ated transport activities have been linked to hyperphosphorylation of dynein, a cytoskeletal motor protein. (Con-cept courtesy of Dr. Maria Runnegar, University of Southern California School of Medicine.)

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charge barrier to the diffusion of solutes between the blood and thecanalicular lumen while water and small ions diffuse across thesejunctions. One hepatotoxin that causes tight-junction leakage isANIT (Krell, 1987).

Cholestatic effects of pharmaceuticals present serious com-plications that often require restrictions in dose or termination oftherapy. Such problems are encountered with cyclosporin A, animmunosuppressive drug frequently reported to cause elevated lev-els of serum bile salts and bilirubin as well as a reductions in bileflow (Farrell, 1994). Careful studies with hepatocyte membranefractions demonstrated that cyclosporin A is a potent competitiveinhibitor of the ATP-dependent bile salt exporter on the canalicu-lar membrane (Bohme et al., 1994). Agents can also impedecanalicular export activities by stimulating the retraction of canalic-ular exporters away from the canalicular membrane or by down-regulating their expression (Trauner et al., 1998; Kubitz et al.,1999).

Compounds that produce cholestasis do not necessarily act bya single mechanism or at just one site. Chlorpromazine impairs bileacid uptake and canalicular contractility (Farrell, 1994). Multiplealterations have been well documented for estrogens, a well-knowncause of reversible canalicular cholestasis (Vore, 1991; Bossard etal., 1993). Problems occur with both synthetic estrogens andmetabolites of endogenous estrogens, particularly D-ring glu-curonides. Estrogens decrease bile salt uptake by effects at the si-nusoidal membrane including a decrease in the Na�, K�-ATPasenecessary for Na-dependent transport of bile salts across the plasmamembrane and changes in lipid component of this membrane. At the canalicular membrane, estrogens diminish the transport of

glutathione conjugates and reduce the number of bile salt trans-porters.

An additional mechanism for canalicular cholestasis is con-centration of reactive forms of chemicals in the pericanalicular area(Fig. 13-11). Most chemicals that cause canalicular cholestasis areexcreted in bile. Therefore the proteins and lipids in the canalicu-lar region must encounter a high concentration of these chemicals.Observations consistent with this concentration mechanism havebeen reported for Mn and 1,1-dicloroethylene. Manganese is aknown cholestatic agent in humans and experimental animals(Lustig et al., 1982). Treatments that modify the extent of Mn-induced cholestasis produce consonant changes in the amount ofMn recovered in the canalicular membrane fraction (Ayotte andPlaa, 1985). A postulated mechanism for the canaliculus as the tar-get site of low doses of 1,1-dichloroethylene is the congregationof its reactive thioether glutathione conjugates (Liebler et al., 1988)in the pericanalicular region (Moslen and Kanz, 1993). One strik-ing effect of 1,1-dicloroethylene is a rapid decrease in biliary se-cretion phospholipids, a function of the MDR2 exporter (Woodardand Moslen, 1998). Concentration within a confined region is alsoa plausible factor in the target site selectivity of chemicals thatdamage bile ducts, since all recognized bile duct toxins are ex-creted in bile. Sporidesmin, a fungus-derived bile duct toxin, isconcentrated in bile up to 100-fold (Farrell, 1994).

Mitochondrial Damage

Preferential injury to mitochondrial DNA, as opposed to nuclearDNA, is a plausible mechanistic basis for the structural and func-tional alterations to hepatic mitochondria associated with nucleo-side analog therapy for hepatitis B and AIDS infections and withalcohol abuse. Of particular concern is the lactic acidosis that re-sults when the liver, due to a massive deficit in hepatic mitochon-drial function, can no longer maintain systemic lactate homeostasisor even supply its own ATP needs without anaerobic glycolysis.

Mitochondrial DNA codes for several proteins in the mito-chondrial electron transport chain. Nucleoside analog drugs causemitochondrial DNA damage directly when incorporation of theanalog base leads to miscoding or early termination of polypep-tides. The severe hepatic mitochondrial injury produced by the nu-cleoside analog fialuridine is attributed to its higher affinity for thepolymerase responsible for mitochondrial DNA synthesis than forthe polymerases responsible for nuclear DNA synthesis (Honkoopet al., 1997). Mitochondrial DNA is also more vulnerable to mis-coding (mutation) due to its limited capacity for repair.

Alcohol abuse can lead to mitochondrial injury by mecha-nisms involving metabolic imbalance and/or oxidative stress. Ashift in the bioactivation/detoxification balance for ethanol can leadto an accumulation of its reactive acetaldehyde metabolite withinmitochondria, since mitochondrial aldehyde dehydrogenase is themajor enzymatic process for detoxification of acetaldehyde. Bioac-tivation of large amounts of ethanol by alcohol dehydrogenase ham-pers the detoxification reaction, since the two enzymes require acommon, depletable cofactor—namely, nicotinamide adenine di-nucleotide (NAD). Any type of ethanol-induced change that en-hances the leakiness of the mitochondrial transport chain wouldlead to an increased release of reactive oxygen species capable ofattacking nearby mitochondrial constituents. Animal experimentshave shown that high doses of ethanol lead to adduction of mito-chondrial cytochrome oxidase by reactive oxygen species and todeclines in the activity of this electron transport chain protein (Chen

Figure 13-11. Schematic of six potential mechanisms for cholestasis in-volving inhibited uptake, diminished transcytosis, impaired secretion, di-minished contractility of the canaliculus, leakiness of the junctions thatseal the canalicular lumen from the blood, and detrimental consequencesof high concentrations of toxic entities in the pericanalicular area.

Note that impaired secretion across the canalicular membrane can resultfrom inhibition of a transporter or retraction of a transporter away from thecanalicular membrane.

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et al., 1999b). Ethanol-induced damage to mitochondrial DNA bywhat appears to be oxidative stress pathway is attenuated by an-tioxidant pretreatment (Mansouri et al., 1999). Too little is knownabout antioxidant deficiencies as acquired risk factors for alcohol-induced liver damage.

FUTURE DIRECTIONS

Our understanding of mechanisms and critical factors in chemi-cally mediated hepatotoxicity will continue to improve through theapplication of model systems that allow for the observation of

events at the level of the cell, organelle, and molecule. Advancesin the area of cholestasis are possible using highly purified canalic-ular membranes, hepatocyte couplets that secrete bile, and culturesof primary bile duct cells. Consequences of damage to specificparts of the liver will be clarified through experiments with chem-icals that have defined target sites. Important interrelationships be-tween sinusoidal cells and other types of liver cells can be identi-fied using coculture systems or treatments that modify functionsof each type of sinusoidal cell. Knockout rodents and other appli-cations of molecular biology will provide insight into the roles ofbioactivation and excretion processes in hepatotoxicity.

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