The liver plays an essential role in metabolic homeostasis. Hepatic functions include the synthesis, storage, and breakdown of glycogen. In addition, the liver is important in the metabolism of lipids; the synthesis of albumin, clotting factors, and other important proteins; the synthesis of the bile acids necessary for absorption of lipids and lipid-soluble vitamins; and the metabolism of cholesterol. Hepatocytes facilitate the excretion of metals, most importantly iron, copper, zinc, manganese, mercury, and aluminum, as well as the detoxification of products of metabolism, such as bilirubin and ammonia.^27,59 Generalized disruption of these important functions results in manifestations of liver failure: hyperbilirubinemia, coagulopathy, hypoalbuminemia, hyperammonemia, and hypoglycemia. Disturbances of more specific functions result in accumulation of lipids, metals, and bilirubin, and the development of lipid-soluble vitamin deficiencies.¹²⁵

The liver is also the primary site of biotransformation and detoxification of xenobiotics. The interposition of the liver between the gastrointestinal tract and systemic circulation makes it the first-pass recipient of ingested xenobiotics. The liver receives blood from the systemic circulation and participates in the detoxification and elimination of xenobiotics that reach the bloodstream through other routes, such as inhalation or cutaneous absorption.^112,125

Many xenobiotics are lipophilic and inert, requiring chemical modification followed by conjugation to make them sufficiently water-soluble to be eliminated. The liver is the primary organ responsible for this biotransformation, and contains the highest concentration of cytochrome P450 (CYP) enzymes involved in the first stage of detoxification for many lipophilic xenobiotics (Chap. 11). Although many of the xenobiotics that are detoxified in the liver are subsequently excreted in the urine, the biliary tract provides an additional route for their elimination.⁴³ Although cytochrome P450 phase I activation of xenobiotics usually leads to detoxification, in some cases it can produce xenobiotics with increased toxicity and hepatocyte injury at the site of synthesis.¹¹²

Two pathologic concepts are used to describe the appearance and function of the liver: a structural one represented by the hepatic lobule, and a functional one represented by the acinus. The basic structural unit of the liver as characterized by light microscopy is the hepatic lobule, a hexagon with the hepatic vein at the center and the portal triads at the angles. The portal triad consists of the portal vein, the common bile duct, and the hepatic artery. Cords of hepatocytes are oriented radially around the central hepatic vein, forming sinusoids. In contrast, the acinus, or “metabolic lobule” is the functional unit of the liver. Located between 2 central hepatic veins, it is bisected by terminal branches of the hepatic artery and portal vein that extend from the bases of the acini toward hepatic venules at the apices. The acinus is subdivided into 3 metabolically distinct zones: Zone 1 lies near the portal triad, zone 3 lies near the central hepatic vein, and zone 2 is the intermediate area.¹²⁵ Figure 21–1 illustrates the relationship of the structural and functional concepts of the liver.

Approximately 75% of the blood supply to the liver is derived from the portal vein, which drains the gastrointestinal tract, spleen, and pancreas. This blood is enriched with nutrients and other absorbed xenobiotics and is poor in oxygen. The remainder of the hepatic blood flow comes from the hepatic artery, which delivers well-oxygenated blood from the systemic circulation. Blood from the hepatic artery and portal vein mixes in the sinusoids, coming in close contact with cords of hepatocytes before it exits through small fenestrations in the wall of the vein.¹³⁷ Oxygen content diminishes severalfold as blood flows from the portal area to the central hepatic vein.⁵³

There are 6 types of cells in the liver. Hepatocytes and bile duct epithelia make up the parenchyma. Cells found near the sinusoids include endothelial cells, fixed macrophages (Kupffer cells), hepatic stellate cells (so-called Ito cells), and a large population of lymphocytes that roam the sinusoids. The sinusoidal lining formed by endothelial cells is thin and fenestrated, allowing transfer of fluid, chylomicrons, and proteins across the space of Disse, an extrasinusoidal space filled with microvilli.¹³⁷ Kupffer cells remove particles and cell debris that include bacteria and endotoxins from the portal circulation. They also clear many biologically active substances from the systemic circulation.¹² When immunologically activated by xenobiotics, Kupffer cells contribute to the generation of oxygen free radicals¹¹⁰ and also participate in the production of autoimmune injury to hepatocytes, including activation of hepatic stellate cells.^58,110 Hepatic stellate cells are primary sites for the storage of fat and vitamin A.^36,45 In a quiescent state, they spread out between the sinusoidal endothelium and hepatic parenchymal cells. Filled with microtubules and microfilaments, they project cytoplasmic extensions that contact several cell types.^36,45 Activated stellate cells produce collagen, proteoglycans, and adhesive glycoproteins, which are crucial to the development of hepatic fibrosis.³⁴ The liver lymphocyte population is enriched with natural killer (NK) or pit cells, which play a key role in host defense by actively lysing tumor cells and virally infected cells^18,35 (Fig. 21–2). Evidence from animal models suggests that NK cells selectively kill activated stellate cells, inhibiting the development of liver fibrosis.^34,35

Bile acids, organic anions, bilirubin, phospholipids, xenobiotics, and other molecules excreted in bile are actively transported across the hepatocyte plasma membrane into the bile canaliculi at sites that have specificity for acids, bases, and neutral xenobiotics.⁹⁴ Tight junctions separate the contents of the bile canaliculi from the sinusoids and hepatocytes, maintaining a rigid and functionally necessary compartmentalization. Bile acids use 3 active transport systems: a sodium-dependent bile salt transporter in the sinusoidal membrane, an adenosine triphosphate (ATP)-dependent bile salt carrier in the canalicular membrane, and a canalicular membrane transport site driven by the membrane proton gradient.⁹⁴ Xenobiotics bound to glucuronide are substrates for the bile acid transport systems and are actively secreted into bile. Xenobiotics with molecular weights greater than 500 Da are also preferentially secreted into bile. Like the transport and concentration of constituents from the sinusoids and hepatocytes, the flow of bile through the canaliculi is also an active process facilitated by ATP-dependent contractions of actin filaments that encircle the canaliculi.^94,129

The enterohepatic recirculation of bile acids and certain vitamins plays a crucial role in their conservation. Unfortunately, this recirculation also impedes the fecal elimination of some xenobiotics by reabsorbing and returning them back into the systemic circulation, prolonging their apparent half-lives and toxicity. Xenobiotics that have low molecular weights and are not ionized at intestinal pH (ie, methylmercury, phencyclidine, and nortriptyline) are most likely to be reabsorbed.¹⁰⁹

Owing to its location at the end of the portal system and its substantial complement of biotransformation enzymes, the liver is especially vulnerable to toxic injury. The pathologic spectrum of liver injury includes combinations of hepatocellular necrosis with focal or generalized lysis of hepatocytes and elevations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT); hepatitis associated with inflammatory cellular infiltrates and varied elevations of hepatocellular enzymes; cholestasis with pruritus, jaundice, and insignificant elevations of hepatocellular enzymes; steatosis caused by intracellular deposits of fat; apoptosis, the formation of shrunken, nonfunctioning, eosinophilic bodies; and fibrosis.¹²⁵

Hepatocellular necrosis that occurs near the portal vein is called periportal, or zone 1 necrosis. The term centrilobular or zone 3 necrosis refers to injury that surrounds the central hepatic vein. Figure 21–3 shows centrilobular necrosis caused by exposure to acetaminophen. Metabolic characteristics of the zones of the acinus have important relevance to the anatomic distribution of toxic liver injury. Because of its location in the periportal area, zone 1 has a 2-fold higher oxygen content than zone 3. Hepatic injury that results from the metabolic production of oxygen free radicals predominates in zone 1. Allyl alcohol, an industrial chemical that is metabolized to a highly reactive aldehyde, is associated with oxygen-dependent lipid peroxidation injury to hepatocytes in zone 1.³ The tendency for centrilobular or zone 3 accumulation of fat in patients with alcoholic steatosis is attributed to the effect of relative hypoxia in the central vein area on the oxidation potential of the hepatocyte.⁶ The availability of substrates for detoxification and the localization of enzymes involved in biotransformation also affect the site of injury. Zone 1 has a higher concentration of glutathione, whereas zone 3 has a greater capacity for glucuronidation and sulfation.¹³⁰ Zone 3 has higher concentrations of alcohol dehydrogenase, which leads to increased production of its toxic metabolite acetaldehyde at centrilobular sites.^26,78 Zone 3 also has high concentrations of CYP2E1, which converts many xenobiotics including acetaminophen (APAP), nitrosamines, benzene, and carbon tetrachloride (CCl₄) to reactive intermediates that cause centrilobular injury. Although CCl₄ is metabolized to a highly reactive oxygen free radical in zone 1, it primarily injures zone 3 for the following reasons: CCl₄ is metabolized by CYP2E1 in zone 3 to a trichloromethyl free radical (·CCl₃) that forms covalent bonds with cellular proteins, causes lipid peroxidation, or spontaneously reacts with oxygen to form the more highly reactive trichloromethyl peroxy radical (CCl₃OO·).^9,73 Higher oxygen tension in zone 1 fosters the formation of CCl₃OO·, which is rapidly detoxified by glutathione. Because the less reactive ·CCl₃ that predominates in zone 3 is not readily detoxified by glutathione, zone 3 incurs the greater amount of injury. Hyperbaric oxygen increases the oxygen tension throughout the liver and decreases liver injury caused by CCl₄, possibly by increasing the formation of CCl₃OO· in zone 3, which is then efficiently detoxified by glutathione.¹⁴ The observed effects of isoniazid (an inhibitor of CYP2E1) and chronic ethanol intake (an inducer of the CYP2E1) on injury in cell cultures from periportal and centrilobular areas exposed to CCl₄ support the association of CCl₄ injury with the localization of CYP2E1 activity. Acute exposure to isoniazid significantly decreases the injury associated with exposure of zone 3 cells to CCl₄, whereas chronic treatment with ethanol significantly enhances injury.⁷³

Xenobiotics that produce liver damage in all humans in a predictable and dose-dependent manner are called intrinsic hepatotoxins. They include APAP, CCl₄, and yellow phosphorus, to name a few. Those that cause liver damage in a small number of individuals and whose effect is not apparently predictable or dose dependent are called idiosyncratic hepatotoxins.³⁸

Prediction of Drug-Induced Liver Injury

Drug-induced liver injury (DILI) refers to the production of liver injury by pharmaceuticals. It is the most common cause of acute liver failure in the United States, and is the most frequent adverse event responsible for the termination of clinical trials of new drugs, as well as postmarketing withdrawals of approved drugs.⁹⁸ LiverTox (https://livertox-nih-gov.easyaccess1.lib.cuhk.edu.hk) is an up-to-date informational website about the incidence, diagnosis, and management of xenobiotic-associated liver injury.⁸

Prediction of DILI in any individual patient based on clinical trials is difficult because the incidence is rare. Drug-induced liver injury depends largely on factors that affect host susceptibility. These include age, gender, diet, underlying diseases, and concurrent exposure to other xenobiotics.⁹⁸ Genetic factors are increasingly elucidated. Many enzymes involved in biotransformation show genetic polymorphism. Inherited variations in CYP enzymes affect susceptibility to DILI.² For example, perhexiline, an antianginal marketed in Europe in the 1980s, caused severe liver injury and peripheral neuropathy in persons with an inability to metabolize debrisoquine.¹¹⁸ The congenital disorder that results in Gilbert syndrome is characterized by decreased glucuronyltransferase. These patients demonstrate decreased glucuronidation and increased bioactivation of APAP during chronic therapeutic dosing, suggesting an increased risk of hepatic injury following ingestion of APAP.²³ Gastrointestinal toxicity due to irinotecan, an antineoplastic used in the treatment of colon cancer, is associated with deficiency of glucuronyltransferase.⁸⁷

Studies that compare the genomes of patients who develop hypersensitivity reactions to various drugs with those who do not show significant associations with human leukocyte antigen (HLA) alleles.⁴¹ Hypersensitivity reactions to abacavir are significantly associated with HLA-B*5701, such that the Food and Drug Administration now recommends HLA screening prior to the initiation of abacivir.⁸¹ Human leukocyte antigen-B*5701 is also associated with cholestatic DILI in patients taking flucloxacillin, an antibiotic marketed in Europe.²¹ Similarly, HLA-B27 predisposes to the development of agranulocytosis in levamisole-adulterated crack-cocaine exposure.¹⁵ Severe hypersensitivity reactions to carbamazepine are associated with the HLA allele HLA-A*3101.⁸² Specific HLA phenotypes are implicated in the development of cholestatic liver injury in patients exposed to amoxicillin-clavulanate.⁷⁶ Drug-induced liver injury in the form of elevation of the ALT occurred in 15% of patients exposed to the first-generation direct thrombin inhibitor ximelagatran, with some developing clinically significant hepatitis. This was associated with 2 specific HLA phenotypes-DRB1*07 and DQA1*02.⁵⁷

Effects of Xenobiotics on Enzyme Function

Some xenobiotic combinations increase the possibility of hepatotoxic reactions because one xenobiotic alters the metabolism of the other, leading to the production of toxic metabolites. This is the case with combinations of rifampin and isoniazid,¹³² amoxicillin and clavulanic acid,²⁴ and trimethoprim and sulfamethoxazole.¹⁹ Changes in the activities of biotransformation enzymes that result in increased formation of hepatotoxic metabolites suggest increased susceptibility to hepatic injury. For example, chronic ethanol exposure causes proliferation of the smooth endoplasmic reticulum in the centrilobular areas resulting in increased CYP2E1 activity.⁷⁵ When bromobenzene, a xenobiotic whose metabolism and hepatotoxicity are similar to that of APAP (the toxic metabolite 3,4-epoxide is formed via CYP2E pathway), was administered to rats chronically exposed to ethanol, it caused a more rapid onset of hepatotoxicity compared to the controls. In addition, the dose of bromobenzene required for hepatic injury was not altered by immediate pretreatment with ethanol.⁴⁷ This means that hepatic toxicity caused by solvents such as CCl₄, dimethylformamide, and bromobenzene is likely exacerbated by chronic exposure to ethanol.^73,102

Availability of Substrates

The availability of substrates for detoxification significantly affects both the likelihood and the localization of hepatic injury. The metabolism of APAP illustrates the effect of glutathione concentration on the delicate balance between detoxification and the production of injurious metabolites. In healthy adults taking therapeutic amounts of APAP, approximately 90% of hepatic metabolism results in formation of the glucuronide or sulfate metabolites. Most of the remainder undergoes oxidative metabolism to the toxic electrophile N-acetyl-p-benzoquinone imine (NAPQI) and is rapidly detoxified by conjugation with glutathione.¹¹¹ Glutathione is depleted during the course of metabolism of APAP by otherwise normal livers, but it is also decreased by inadequate nutrition or liver disease.¹¹¹ Excessive amounts of APAP result in increased synthesis of NAPQI, which, in the absence of glutathione, reacts avidly with hepatocellular macromolecules. The cellular concentration of glutathione correlates inversely with the demonstrable covalent binding of NAPQI to liver cells.¹⁶ Centrilobular (zone 3) necrosis predominates in APAP-induced hepatic injury, likely related both to the centrilobular localization of CYP2E1 and to the relatively low glutathione concentrations in zone 3 compared to the periportal areas (zone 1).⁴⁸

There are several mechanisms of xenobiotic-induced hepatotoxicity.

Immune-Mediated Liver Injury

Immune-mediated liver injury is an idiosyncratic and host-dependent hypersensitivity response xenobiotic exposure.⁷ Damage to the ­hepatocytes is mediated by complement- or antibody-directed lysis, by specific cell-­mediated cytotoxicity, or by an inflammatory response stimulated by immune complexes and complement.¹²³ The antibody or cell-mediated response is precipitated by a covalently bound xenobiotic-cell protein adduct that acts as a hapten. The resultant inflammatory response provokes secondary cytokine release that promotes further cell injury by neutrophils.⁴³ Subsequent apoptosis appears to be partly mediated by tumor necrosis factor (TNF).¹²³ Immune-mediated toxic hepatitis is differentiated from liver injury caused by other autoimmune disorders by the absence of self-perpetuation; that is, there is a need for continuous exposure to the xenobiotic to perpetuate the injury.⁷ It is also less likely to recur following withdrawal of immune suppression therapy.⁷

Hypersensitivity reactions result in hepatitis, cholestasis, and mixed disorders. It is not clear whether all the autoantibodies stimulated by the xenobiotic–protein adducts are the actual mediators of cell injury.⁷ In cases where the metabolite is highly unstable, an electrophilic attack is directed against the CYP enzyme at the site of formation of the metabolite.⁷⁴ The most severe form of idiosyncratic halothane liver injury is manifested as fulminant hepatic necrosis associated with the formation of adducts of its trifluoroacetyl chloride (TFA) metabolite with numerous hepatoproteins including CYP2E1 and pyruvate dehydrogenase.^28,55 Autoantibodies specifically directed against CYP enzymes are also demonstrated for dihydralazine¹⁰ and phenytoin.⁶⁸

Cell-mediated autoimmune mechanisms are also implicated in the idiosyncratic type of halothane hepatitis, and are now recognized in an increasing number of experimental xenobiotic-mediated liver injury models.⁶³Polymorphonucleocyte (PMN) activation and infiltration appear to be an important factor in the production of cholangitis in a rat model of α-naphthyl-isothiocyanate (ANIT) liver injury. α-Naphthyl-isothiocyanate stimulates the release of cytotoxic lysosomal enzymes and oxygen free radicals by activated PMNs.⁸⁶ Antibodies directed against circulating neutrophils decrease the extent of liver damage caused by ANIT.²⁰ Natural Killer (NK) T cells are ubiquitous in the liver, and also serve an important role in the cell-mediated autoimmune liver injury.³⁵

Drugs most commonly involved in autoimmune injury are nitrofurantoin and minocycline.⁷ Drugs with hypersensitivity reactions that typically present with hepatitis include halothane,⁶³ trimethoprim-sulfamethoxazole,⁸⁰ antiepileptics,¹³⁹ and allopurinol.¹²² Drugs associated with hypersensitivity that typically present with cholestatic signs include chlorpromazine, erythromycin estolate, penicillins, rifampin, and sulfonamides.⁹⁴ Signs of injury typically begin 1 to 8 weeks following the initiation of the medication, although it takes as long as 20 weeks for drugs such as isoniazid or dantrolene. In all cases, the onset is earlier when the patient is rechallenged with the drug. Antinuclear antibodies and smooth muscle antibodies are frequently present, as are eosinophilia, atypical lymphocytosis, fever, and rash. However, their absence does not exclude an autoimmune mechanism of drug-associated liver injury.^7,74 Liver injury characterized by the formation of hepatic granulomas are also a consequence of hypersensitivity reactions.⁶⁷

Xenobiotics That Target the Biliary Tract

Xenobiotics that undergo biliary excretion are most commonly associated with jaundice in patients with hepatoxicity. Xenobiotics induce cholestasis by targeting specific mechanisms of bile synthesis and flow or by damaging canalicular cells. Elucidations of various bile transport proteins have led to better understanding of mechanisms of cholestasis in toxic liver injury.⁹⁴ Cholestasis occurs with or without associated hepatitis. The development of jaundice following hepatic necrosis is a manifestation of general failure of liver function. More discrete mechanisms that result in intrahepatic cholestasis include (a) impairment of the integrity of tight membrane junctions that functionally isolate the canaliculus from the hepatocyte and sinusoids, (b) failure of transport of bile components across the hepatocytes, (c) blockade of specific membrane active transport sites, (d) decreased membrane fluidity resulting in altered transport, and (e) decreased canalicular contractility resulting in decreased bile flow.^11,61,94,115 Xenobiotics that specifically target bile canaliculi can lead to irreversible injury, the so-called vanishing bile duct syndrome.¹²⁷ Estrogens cause intrahepatic cholestasis by altering the composition of the lipid membrane and inhibiting the rate of secretion of bile into the canaliculi.¹¹⁵ Rifampin impedes the uptake of bilirubin into hepatocytes. Methyltestosterone and C-17 alkylated anabolic steroids impair the secretion of bilirubin into canaliculi.⁷⁰ Cyclosporine inhibits sodium-dependent uptake of bile salts across the sinusoidal membrane and blocks ATP-dependent bile salt transport across the canalicular membrane.¹¹ Floxacillin causes cholestasis with minimal inflammation or evidence of hepatocellular injury.¹³¹ Exposure of rats to ANIT causes a specific injury localized to the tight junctions that separate the hepatocytes from the canaliculi. This results in reflux of bile constituents into the sinusoidal spaces and increased access of sinusoidal molecules to the biliary tree.¹³⁸

Mitochondrial Injury

Direct mitochondrial injury impairs cellular respiration and is associated with fat accumulation, diminution of ATP production, and metabolic acidosis with elevated lactate.⁶⁴ This involves attacks by xenobiotics on structural components of the mitochondria such as DNA, respiratory chain enzymes, and membranes. Nucleoside analogs that inhibit viral reverse transcriptase also inhibit mitochondrial DNA synthesis, leading to depletion of mitochondria.¹⁷

Other Targets

Stimulation of Kupffer cells enhances hepatic injury. When immunologically activated by xenobiotics, Kupffer cells contribute to the generation of oxygen free radicals and participate in the production of autoimmune injury to hepatocytes, including activation of hepatic stellate cells.¹¹⁰ Activated stellate cells produce collagen, proteoglycans, and adhesive glycoproteins, which are crucial to the development of hepatic fibrosis.³⁴ Hepatic venoocclusive disease is caused by xenobiotics that injure the endothelium of terminal hepatic venules, resulting in intimal thickening, edema, and nonthrombotic obstruction.⁶²

The liver responds to injury in a limited number of ways. Cells may swell and accumulate fat or biliary material. They may necrose and lyse or undergo the slower process of apoptosis, forming shrunken, nonfunctioning, eosinophilic bodies. Necrosis can be focal or bridging, linking the periportal or centrilobular areas; zonal or panacinar; or it can be massive.⁴³ An autoimmune type of injury is characterized by hepatitis with a prominent plasma cell infiltrate.⁷ Injury to the bile ducts results in cholestasis. Xenobiotics that target canalicular transport proteins can cause cholestasis in the absence of injury to hepatocytes.⁹⁴ Direct mitochondrial injury impairs cellular respiration and is associated with fat accumulation, diminution of ATP production, and metabolic acidosis.⁹⁵ Injuries to the intima of postsinusoidal veins cause obstruction to venous flow.¹⁴⁰ Table 21–1 lists characteristic morphologies of hepatic injury and associated xenobiotics.