Genetic Factors

Genetic determinants predispose to drug-induced liver disease,¹³ as they do for other types of drug reaction, such as penicillin allergy. Atopic patients have been thought to have an increased risk of some types of drug hepatitis, but this increase in risk has not been proved. Genetic factors determine the activity of drug-activating and antioxidant pathways, encode pathways of canalicular bile secretion, and modulate the immune response, tissue stress responses, and cell death pathways (see Chapter 72). Documented examples of drugs associated with a familial predisposition to adverse hepatic drug reactions are few and include valproic acid and phenytoin.^5,8,13 Inherited mitochondrial diseases are a risk factor for valproic acid–induced hepatotoxicity.¹⁴ Some forms of drug-induced liver disease, particularly drug-induced hepatitis and granulomatous reactions, can be associated with the reactive metabolite syndrome (see later).

Weak associations have been reported between specific human leukocyte antigen (HLA) haplotypes and some types of drug-induced liver disease. Andrade and colleagues¹³ found positive associations between the class II HLA haplotype and cholestatic or mixed liver damage for some drugs. They suggested that no specific HLA allele predisposed to the overall risk of drug-induced liver disease but that the pattern of liver injury could be influenced by these genetic determinants. Other investigators have found stronger associations between the HLA haplotype and cholestatic reactions to amoxicillin-clavulanic acid and ticlopidine (see later).^13,15

Age

Most hepatic drug reactions are more common in adults than in children. Exceptions include valproic acid hepatotoxicity, which is most common in infants younger than three years of age and rare in adults, and Reye’s syndrome, in which salicylates play a key role.^16,17 As discussed later, both may be examples of mitochondrial toxicity.¹⁴ In adults, the risk of isoniazid-associated hepatotoxicity is greater in persons older than 40 years of age. Similar observations have been made for nitrofurantoin, halothane, etretinate, diclofenac, and troglitazone.^1,4,5,10 The increased frequency of adverse drug reactions in older subjects is largely the result of increased exposure, the use of multiple agents, and altered drug disposition.^1,4,5,10 In addition, clinical severity of hepatotoxicity increases strikingly with age, as exemplified by fatal reactions to isoniazid and halothane.

Gender

Women are particularly predisposed to drug-induced hepatitis, a difference that cannot be attributed simply to increased exposure. Examples include toxicity caused by halothane, nitrofurantoin, sulfonamides, flucloxacillin, minocycline, and troglitazone.^5,6 Drug-induced chronic hepatitis caused by nitrofurantoin, diclofenac, or minocycline has an even more pronounced female preponderance.⁶ Conversely, equal sex frequency or even male preponderance is common for some drug reactions characterized by cholestasis, as for amoxicillin-clavulanic acid. Azathioprine-induced liver disease is more likely to develop in male renal transplant recipients than in female recipients.¹⁸

Concomitant Exposure to Other Agents

Patients who are taking multiple drugs are more likely to experience an adverse reaction than those who are taking one agent.^5,9,10 The mechanisms include enhanced cytochrome P450 (CYP)-mediated metabolism of the second drug to a toxic intermediate (see later). Examples discussed later include toxicity caused by acetaminophen, isoniazid, valproic acid, other anticonvulsants, and anticancer drugs. Alternatively, drugs may alter the disposition of other agents by reducing bile flow or competing with canalicular pathways for biliary excretion (phase 3 drug elimination) (see later). This mechanism may account for apparent interactions between oral contraceptive steroids and other drugs to produce cholestasis. Drugs or their metabolites may also interact in mechanisms of cellular toxicity and cell death that involve mitochondrial injury, intracellular signaling pathways, activation of transcription factors, and regulation of hepatic genes involved in controlling the response to stress and injury that triggers pro-inflammatory and cell death processes.^19,20

Previous Drug Reactions

A history of an adverse drug reaction generally increases the risk of reactions to the same drug and also to some other agents (see later). Nevertheless, instances of cross-sensitivity to related agents in cases of drug-induced liver disease are surprisingly uncommon. Examples of cross-sensitivity between drugs (or drug classes) include the haloalkane anesthetics (see Chapter 87), erythromycins, phenothiazines and tricyclic antidepressants, isoniazid and pyrazinamide, sulfonamides and other sulfur-containing compounds (e.g., some clyclooxygenase-2 [COX-2] inhibitors), and some NSAIDs. A crucial point is that a previous reaction to the same drug is a major risk factor for an increase in the severity of drug-induced liver injury.⁶

Alcohol

Chronic excessive alcohol ingestion decreases the dose threshold for, and enhances the severity of, acetaminophen-induced hepatotoxicity and increases the risk and severity of isoniazid hepatitis, niacin (nicotinic acid, nicotinamide) hepatotoxicity, and methotrexate-induced hepatic fibrosis.

Nutritional Status

Obesity is strongly associated with the risk of halothane hepatitis (see Chapter 87) and appears to be an independent risk factor for NASH and hepatic fibrosis in persons taking methotrexate or tamoxifen. Fasting also predisposes to acetaminophen hepatotoxicity,²¹ and a role for undernutrition has been proposed in isoniazid hepatotoxicity.²²

Preexisting Liver Disease

In general, liver diseases such as alcoholic cirrhosis and cholestasis do not predispose to adverse hepatic reactions. Exceptions include toxicity to some anticancer drugs, niacin, pemoline, and hycanthone. Preexisting liver disease is a critical determinant of methotrexate-induced hepatic fibrosis (discussed later). Patients with chronic HBV infection¹² and possibly those with chronic HCV infection or HIV/AIDS appear to be at heightened risk of liver injury during anti-tuberculosis or HAART therapy,²³ after exposure to ibuprofen and possibly other NSAIDs, after myeloablative therapy in preparation for bone marrow transplantation (resulting in sinusoidal obstruction syndrome [see later]),²⁴ and possibly after taking antiandrogens, such as flutamide and cyproterone acetate.²⁵ A particularly strong association has been observed between HCV infection (present in 33% of patients with HIV/AIDS) and the risk of liver injury during HAART; the risk may be increased 2- to 10-fold.^26–30

Other Diseases

Rheumatoid arthritis appears to increase the risk of salicylate hepatotoxicity, and a curious, unexplained observation is that hepatitis associated with sulfasalazine appears to be more common in patients with rheumatoid arthritis than in those with inflammatory bowel disease.^8–10,31,32 Diabetes mellitus, obesity, and chronic kidney disease predispose to methotrexate-induced hepatic fibrosis, whereas HIV/AIDS confers a heightened risk of sulfonamide hypersensitivity.^31–33 A retrospective cohort study of five health maintenance organizations found that the age- and sex-standardized incidence of drug-induced acute liver failure in patients with diabetes mellitus was 0.08 to 0.15 per 1000 person-years, irrespective of the therapeutic agent used (the number using troglitazone was small); the incidence was highest (approximately 0.3 per 1000) during the first six months of exposure.³² Renal transplantation is a risk factor for azathioprine-associated vascular injury, whereas kidney disease predisposes to tetracycline-induced fatty liver.⁶ Finally, sinusoidal obstruction syndrome induced by anticancer drugs is more common after bone marrow transplantation²⁴ and in persons with HCV infection.^{5,6,8–10,26}

Cytochrome P450

Most type 1 reactions are catalyzed by microsomal drug oxidases, the key component of which is a hemoprotein of the CYP gene superfamily. The apparent promiscuity of drug oxidases toward drugs, environmental toxins, steroid hormones, lipids, and bile acids results from the existence of multiple closely related CYP proteins. More than 20 CYP enzymes are present in the human liver.³⁴

The reaction cycle involves binding of molecular oxygen to the iron in the heme prosthetic group, with subsequent reduction of oxygen by acceptance of an electron from nicotinamide-adenine dinucleotide phosphate (NADPH) cytochrome P450 reductase, a flavoprotein reductase. The resulting “activated oxygen” is incorporated into the drug or another lipophilic compound. Reduction of oxygen and insertion into a drug substrate (“mixed function oxidation”) can result in formation of chemically reactive intermediates, including free radicals, electrophilic “oxy-intermediates” (e.g., unstable epoxides, quinone imines), and reduced (and therefore reactive) oxygen species (ROS). The quintessential example is the CYP2E1-catalyzed metabolite of acetaminophen, N-acetyl-p-benzoquinone imine (NAPQI), an oxidizing and arylating metabolite that is responsible for liver injury associated with acetaminophen hepatotoxicity. Other quinone compounds are potential reactive metabolites of troglitazone, quinine, and methyldopa. Epoxide metabolites of diterpenoids may be hepatotoxic products of the hepatic metabolism of some plant toxins (see Chapter 87).³⁵ ROS have broad significance in the production of tissue injury, particularly by contributing to the production of oxidative stress and triggering tissue stress responses and cell death pathways, as discussed later.

The hepatic content of CYP proteins is higher in acinar zone 3 (see Chapter 71). Localization of CYP2E1 is usually confined to a narrow rim of hepatocytes 1 to 2 cells thick around the terminal hepatic venule. This finding explains in part the zonality of hepatic lesions produced by drugs and toxins, such as acetaminophen and carbon tetrachloride, which are converted to reactive metabolites.

Genetic and Environmental Determinants of Cytochrome P450 Enzymes

Other Pathways of Drug Oxidation

In addition to CYP enzymes, electron transport systems of mitochondria may lead to the generation of tissue-damaging reactive intermediates during the metabolism of some drugs. Examples of such reactive intermediates include nitroradicals from nitrofuran derivatives (nitrofurantoin, cocaine). Subsequent electron transfer by flavoprotein reductases into molecular oxygen generates superoxide and other ROS. Some anticancer drugs such as doxorubicin and the imidazole antimicrobial agents can participate in other oxidation-reduction (redox) cycling reactions that generate ROS.

Phase 2 (Conjugation) Reactions

Phase 2 reactions involve formation of ester links to the parent compound or a drug metabolite. The responsible enzymes include glucuronosyl (or glucuronyl) transferases, sulfotransferases, glutathione-S transferases, and acetyl and amino acid N-transferases. The resulting conjugates are highly water soluble and can be excreted readily in bile or urine. Conjugation reactions can be retarded by depletion of their rate-limiting cofactors, such as glucuronic acid and inorganic sulfate, and the relatively low capacity of these enzyme systems restricts the efficacy of drug elimination when substrate concentrations exceed enzyme saturation. In general, drug conjugates are nontoxic, and phase 2 reactions are considered to be detoxification reactions, with exceptions. For example, some glutathione conjugates can undergo cysteine S-conjugate β-lyase-mediated activation to highly reactive intermediates. Little is known about the regulation of such enzymes or their potential significance for drug-induced liver disease or hepatocarcinogenesis.

Phase 3 Pathways

Several transporters secrete drugs, drug metabolites, or their conjugates into bile, and this mechanism is often referred to as phase 3 of hepatic drug elimination. These pathways involve ATP-binding cassette (ABC) proteins, which derive the energy for their transport functions from hydrolysis of ATP. ABC transport proteins are widely distributed in nature and include the cystic fibrosis transmembrane conductance regulator (CFTR) (see Chapter 76) and the canalicular and intestinal copper transporters (see Chapter 75). The role of ABC transport proteins in secretion of bile has been reviewed (see Chapter 64).^37,38,41,42

Multidrug resistance protein 1 (MDR1) is highly expressed on the apical (canalicular) plasma membrane of hepatocytes, where it transports cationic drugs, particularly anticancer agents, into bile. Another family of ABC transporters, the multidrug resistance-associated proteins (MRPs), is also expressed in the liver. At least two members of this family excrete drug (and other) conjugates from hepatocytes: MRP-3 on the basolateral surface facilitates passage of drug conjugate into the sinusoidal circulation, and MRP-2, expressed on the canalicular membrane, pumps endogenous compounds (e.g., bilirubin diglucuronide, leukotriene-glutathionyl conjugates, glutathione) and drug conjugates into bile. The bile salt export pump (BSEP) and MDR3 (in humans) and Mdr2 (in mice) are other canalicular transporters concerned, respectively, with bile acid and phospholipid secretion into bile. Genetic polymorphisms of these genes are associated with human cholestatic liver diseases (see Chapters 64 and 76). BSEP interacts with several drugs.⁴²

Regulation of the membrane expression and activity of these drug elimination pathways is complex. The possibility that their altered expression or impaired activity (by competition between agents, changes in membrane lipid composition, or damage from reactive metabolites or covalent binding) could lead to drug accumulation, impairment of bile flow, or cholestatic liver injury has been demonstrated for estrogens,^43,44 troglitazone,⁴⁵ terbinafine,⁴⁶ and flucloxacillin⁴⁷ and may have wider mechanistic importance for drug-induced cholestasis and other forms of liver injury.⁴²

Direct Hepatotoxins and Reactive Metabolites

Highly hepatotoxic chemicals injure key subcellular structures, particularly mitochondria and the plasma membrane. The injury arrests energy generation, dissipates ionic gradients, and disrupts the physical integrity of the cell. This type of overwhelming cellular injury does not apply to currently relevant hepatotoxins, most of which require metabolic activation to mediate damage to liver cells. The resulting reactive metabolites can interact with critical cellular target molecules, particularly those with nucleophilic substituents such as thiol-rich proteins and nucleic acids. Together with ROS, they act as oxidizing species within the hepatocyte to establish oxidative stress, a state of imbalance between pro-oxidants and antioxidants. ROS are also key signaling molecules that mediate biological responses to stress, as discussed later. Alternatively, reactive metabolites bind irreversibly to macromolecules, particularly proteins and lipids. Such covalent binding may produce injury by inactivating key enzymes or by forming protein-drug adducts that could be targets for immunodestructive processes that cause liver injury.

Oxidative Stress and the Glutathione System

The liver is exposed to oxidative stress by the propensity of hepatocytes to reduce oxygen, particularly in mitochondria and also in microsomal electron transport systems (such as CYP2E1), and by NADPH-oxidase-catalyzed formation of ROS and nitroradicals in Kupffer cells, endothelial cells, and stimulated polymorphs and macrophages. To combat oxidative stress, the liver is well-endowed with antioxidant mechanisms, including micronutrients, such as vitamin E and vitamin C, thiol-rich proteins (e.g., metallothionein, ubiquinone), metal-sequestering proteins (e.g., ferritin), and enzymes that metabolize reactive metabolites (e.g., epoxide hydrolases), ROS (e.g., catalase, superoxide dismutase), and lipid peroxides (e.g., glutathione peroxidases). Glutathione (l-gamma-glutamyl-l-cyteine-glycine) is the most important antioxidant in the mammalian liver.¹⁹

Hepatocytes are the exclusive site of glutathione synthesis. Hepatic levels of glutathione are high (5 to 10 mmol/L) and can be increased by enhancing the supply of cysteine for glutathione synthesis; this mechanism is the cornerstone of thiol antidote therapy for acetaminophen poisoning. Hepatocyte glutathione synthesis increases in response to pro-oxidants, as occurs when CYP2E1 is overexpressed as a result of signaling via the redox-sensitive transcription factor Nrf.^19,20,48,49 Glutathione is a critical cofactor for several antioxidant pathways, including thiol-disulfide exchange reactions and glutathione peroxidase. Glutathione peroxidase has a higher affinity for hydrogen peroxide than does catalase, and it disposes of lipid peroxides, free radicals, and electrophilic drug metabolites. Reduced glutathione is a cofactor for conjugation reactions catalyzed by the glutathione S-transferases. Other reactions proceed nonenzymatically. In turn, the products include glutathione-protein mixed disulfides and oxidized glutathione. The latter can be converted back to glutathione by proton donation catalyzed by glutathione reductase.

Normally, most glutathione within the hepatocyte is in the reduced state, indicating the importance of this pathway for maintenance of the redox capacity of the cell. The reduced form of NADPH is an essential cofactor for glutathione reductase; NADPH formation requires ATP, thereby illustrating a critical link between the energy-generating capacity of the liver and its ability to withstand oxidative stress. Glutathione is also compartmentalized within the hepatocyte, with the highest concentrations found in the cytosol. Adequate levels of glutathione are essential in mitochondria, where ROS are constantly being formed as a minor by-product of oxidative respiration and in response to some drugs or metabolites that interfere with the mitochondrial respiratory chain. Mitochondrial glutathione is maintained by active uptake from the cytosol, a transport system that is altered by chronic ethanol exposure, and is, therefore, another potential target of drug toxicity.¹⁹

Biochemical Mechanisms of Cellular Injury

Mechanisms once thought to be central to hepatotoxicity, such as covalent binding to cellular enzymes and peroxidation of membrane lipids, are no longer regarded as exclusive pathways of cellular damage. Rather, oxidation of proteins, phospholipid fatty acyl side chains (lipid peroxidation), and nucleosides appear to be components of the biochemical stress that characterizes toxic liver injury. Secondary reactions also may play a role; these reactions include post-translational modification of proteins via adenosine diphosphate (ADP) ribosylation or protease activation, cleavage of DNA by activation of endogenous endonucleases, and disruption of lipid membranes by activated phospholipases.²⁰ Some of these catabolic reactions could be initiated by a rise in the cytosolic ionic calcium concentration [Ca²⁺]_i, as a result of increased Ca²⁺ entry or release from internal stores in the endoplasmic reticulum and mitochondria.^19,20

The concept that hepatotoxic chemicals cause hepatocyte cell death by a biochemical final common pathway (e.g., activation of catalytic enzymes by a rise in [Ca²⁺]_i) has proved inadequate to explain the diverse processes that can result in lethal hepatocellular injury. Rather, a variety of processes can damage key organelles, thereby causing intracellular stress that activates signaling pathways and transcription factors. In turn, the balance between these factors can trigger the onset of cell death or facilitate protection of the cell, as discussed in the following sections.

Types of Cell Death

Apoptosis

Apoptosis is an energy-dependent, genetically programmed form of cell death that typically results in controlled deletion of individual cells. In addition to its major roles in developmental biology, tissue regulation, and carcinogenesis, apoptosis is important in toxic, viral, and immune-mediated liver injury.^50–53 The ultrastructural features of apoptosis are cell and nuclear shrinkage, condensation and margination of nuclear chromatin, plasma membrane blebbing, and ultimately fragmentation of the cell into membrane-bound bodies that contain intact mitochondria and other organelles. Engulfment of these apoptotic bodies by surrounding epithelial and mesenchymal cells conserves cell fragments that contain nucleic acid and intact mitochondria. These fragments are then digested by lysosomes and recycled without release of bioactive substances. As a consequence, apoptosis in it purest form (usually found only in vitro) does not incite an inflammatory tissue reaction. The cellular processes that occur in apoptosis are often mediated by caspases, a family of proteolytic enzymes that contain a cysteine at their active site and cleave polypeptides at aspartate residues; non–caspase-mediated programmed cell death has also been described in experimental hepatotoxicity (see also Chapter 72).

Apoptosis rarely, if ever, is the sole form of cell death in common forms of liver injury, such as ischemia-reperfusion injury, cholestasis, and toxic liver injury, all of which are typically associated with a hepatic inflammatory response. Whether or not activation of pro-death signals causes cell death depends on several factors, including pro-survival signals, the rapidity of the process, the availability of glutathione and ATP, and the role of other cell types. Some of these issues are discussed briefly here and are reviewed in more detail elsewhere.^20,50–53

Hepatocytes undergo apoptosis when pro-apoptotic intracellular signaling pathways are activated, either because of toxic biochemical processes within the cell (intrinsic pathway) or because cell surface receptors are activated to transduce cell death signals (external pathway). Pro-apoptotic receptors are members of the tumor necrosis factor-α (TNF-α) receptor superfamily, which possess a so-called death domain. These receptors include Fas, for which the cognate ligand is Fas-ligand (Fas-L), TNF-R1 receptor (cognate ligand is TNF), and TNF-related apoptosis-inducing ligand (TRAIL) receptors (cognate ligand is TRAIL). In addition to model hepatotoxins such as the quinone, menadione, and hydrogen peroxide, some drugs (e.g., acetaminophen, plant diterpenoids) have been shown to be converted into pro-oxidant reactive metabolites, thereby initiating the following sequence: CYP-mediated metabolism to form reactive metabolites → glutathione depletion → mitochondrial injury with release of cytochrome c and operation of the mitochondrial membrane permeability transition (MPT) → caspase activation → apoptosis.

Mitochondria play a pivotal role in pathways that provoke or oppose apoptosis.^50,51,53 In the external pathway, activation of the death domain of pro-apoptotic receptors recruits adapter molecules—Fas-associated death domain (FADD) and TNF receptor-associated death domain (TRADD)—which bind and activate procaspase 8 to form the death-inducing signaling complex (DISC). In turn, caspase 8 cleaves Bid, a pro-apoptotic member of the B cell lymphoma/leukemia (Bcl-2) family, to tBid. tBid causes translocation of Bax to the mitochondria, where it aggregates with Bak to promote permeability of the mitochondria.⁵⁰ Release of cytochrome c and other pro-death molecules, including Smac (which binds caspase inhibitor proteins, such as inhibitor of apoptosis proteins [IAPs]) and apoptosis-inducing factor (AIF, also known as Apaf)⁵¹ allows formation of the “aptosome,” which activates caspase 9 and eventually caspase 3 to execute cell death (Fig. 86-1). Intracellular stresses in various sites release other mitochondrial permeabilizing proteins (e.g., Bmf from the cytoskeleton and Bim from the endoplasmic reticulum), whereas members of the Bcl-2 family, Bcl-2 and Bcl-X_L, antagonize apoptosis and serve as survival factors by regulating the integrity of mitochondria; the protective mechanism is poorly understood. Stress-activated protein kinases, particularly c-jun N-terminal kinase (JNK) may also be pro-apoptotic⁵² by phosphorylating and inactivating the mitochondrial protective protein Bcl-X_L.

Figure 86-1. Apoptosis and necrosis pathways in mammalian cells. See text for details. Bcl, B-cell lymphoma/leukemia family (Bax, Bid, and Bcl-xL are members); DISC, death-inducing signaling complex; FADD, Fas-associated death domain; FLIP, FLICE-inhibitory proteins; IAP, inhibitor of apoptosis proteins; MPT, mitochondrial permeability transition; RIP, receptor-interacting protein; TNF, tumor necrosis factor; TNF-R1, TNF receptor-1; TRADD, TNF receptor-associated death domain; TRAF2, TNF receptor-associated factor-2; TRAIL, TNF-related apoptosis ligand.

Execution of cell death by apoptosis usually occurs via activation of caspase 3, but more than one caspase-independent pathway of programmed cell death has been described.⁵³ Stresses to the endoplasmic reticulum can bypass mitochondrial events by activation of caspase 12, which in turn activates caspase 9 independently of the apoptosome. The final steps of programmed cell death are energy dependent. Therefore, depletion of ATP abrogates the controlled attempt at “cell suicide,” resulting instead in necrosis (see later) or an overlapping pattern that has been designated as “apoptotic necrosis” or “necraptosis.”^54,55 Furthermore, when apoptosis is massive, the capacity for rapid phagocytosis can be exceeded, and “secondary” necrosis can occur.⁵⁵

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