Liver Disease Caused by Drugs

Genetic and Environmental Determinants of Cytochrome P450 Enzymes

Genetic Factors

Genetic determinants predispose to drug-induced liver disease, as they do for other types of drug reaction, such as penicillin allergy. The contention that atopic patients may be at increased risk of some types of drug hepatitis is unproven, however. 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. Documented examples of drugs associated with a familial predisposition to adverse hepatic drug reactions are few and include valproic acid and phenytoin. Inherited mitochondrial diseases are a risk factor for valproic acid–induced hepatotoxicity. Some forms of drug-induced liver disease, particularly drug-induced hepatitis and granulomatous reactions, are associated with the reactive metabolite syndrome (see later). Initial studies showed only no or weak associations between specific HLA haplotypes and some types of drug-induced liver disease. Genome-wide association studies (GWAS) have revealed stronger associations between specific HLA haplotypes and several drugs, including flucloxacillin and amoxicillin-clavulanic acid ( Table 88.3 ).

Age

Most hepatic drug reactions are more common in adults than in children. Exceptions include valproic acid hepatotoxicity, which is most common in children under 3 years of age but rare in adults, and Reye syndrome, in which salicylates play a key role. 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. ^{, , ,} The increased frequency of adverse drug reactions in older subjects is largely the result of increased exposure, polypharmacy, and altered drug disposition. In addition, the 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. ^, Drug-induced chronic hepatitis caused by nitrofurantoin, diclofenac, or minocycline has an even more pronounced female preponderance. ^, Conversely, equal sex frequency or even male preponderance is common for some cholestatic drug reactions (e.g., amoxicillin-clavulanic acid). Azathioprine-induced liver disease occurs more frequently in male renal transplant recipients than in female recipients.

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. ^{, ,} The mechanisms include enhanced CYP-mediated metabolism of the second drug to a toxic intermediate (see later). Examples 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). This mechanism may account for interactions between OCS and other drugs to produce cholestasis. Drugs or their metabolites may also interact through 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. ^,

Previous Drug Reactions

A history of an adverse drug reaction generally increases the risk of reactions to the same drug as well as other agents. 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 89 ), erythromycins, phenothiazines and tricyclic antidepressants, isoniazid and pyrazinamide, 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 DILI. A Spanish study examined the risk of DILI in persons with a history of DILI (with a different drug). Recurrent DILI was infrequent (1.2%) and was attributable most commonly to drugs that were structurally similar or had similar targets, whereas others exhibited features consistent with AIH, thereby raising the possibility that immune-mediated processes may be mechanistically involved or that the correct diagnosis was actually AIH.

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 and is an independent risk factor for NASH and hepatic fibrosis in persons taking methotrexate or tamoxifen. Fasting also predisposes to acetaminophen hepatotoxicity, and a role for undernutrition has been proposed in isoniazid hepatotoxicity.

Preexisting Liver Disease

In general, liver diseases such as alcohol-associated cirrhosis and cholestatic disorders do not predispose to adverse hepatic reactions. Exceptions include toxicity due to some anticancer drugs, niacin, pemoline, and hycanthone. Preexisting liver disease is a critical determinant of methotrexate-induced hepatic fibrosis. Patients with chronic HBV or HCV infection or HIV/AIDS appear to be at heightened risk of liver injury during antituberculous or ART therapy, after exposure to ibuprofen and possibly other NSAIDs, after myeloablative therapy in preparation for bone marrow transplantation (resulting in sinusoidal obstruction syndrome), and possibly after taking antiandrogens, such as flutamide and cyproterone acetate. A particularly strong association has been reported between HCV infection and the risk of liver injury during ART; the risk may be increased 2- to 10-fold. ^,

Other Diseases

RA increases the risk of salicylate hepatotoxicity, and a curious, unexplained observation is that sulfasalazine hepatitis is more common in patients with RA than in those with IBD. Diabetes mellitus, obesity, and chronic kidney disease predispose to methotrexate-induced hepatic fibrosis, whereas HIV/AIDS confers a heightened risk of sulfonamide hypersensitivity. A retrospective cohort study found that the age- and sex-standardized incidence of drug-induced ALF 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 6 months of exposure. Renal transplantation is a risk factor for azathioprine-associated vascular injury, whereas kidney disease predisposes to tetracycline-induced fatty liver. Finally, sinusoidal obstruction syndrome induced by anticancer drugs is more common after bone marrow transplantation and in persons with HCV infection. ^{, , , ,}

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. Notwithstanding these comments, there is increasing evidence that “direct hepatotoxins,” such as acetaminophen, activate innate immune mechanisms in the liver in response to stress with release of danger-activated molecular patterns; the latter (as well as bacterial products such as endotoxin, a pathogen-associated molecular pattern) trigger Toll-like receptors to activate pro-inflammatory and cell death pathways (see Chapter 72 ).

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 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 polymorphonuclear leukocytes (neutrophils) 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-δ-glutamyl-L-cysteine-glycine) is the most important antioxidant in the mammalian liver.

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. ^{, , ,} Glutathione synthesis, via expression of the rate-limiting enzyme glutamate cysteine ligase is also a response to mitochondrial injury by such agents as acetaminophen. Glutathione in its reduced form (GSH) 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. GSH is also a cofactor for conjugation reactions catalyzed by the glutathione S -transferases involved with phase 3 transport of drug metabolites into bile. 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 mitochondrial integrity and 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 via a transport system that is altered by chronic ethanol exposure and in some forms of lipotoxicity (e.g., with cholesterol) and is therefore another potential target for drug toxicity.

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 appears to be a component of the biochemical stress that characterizes toxic liver injury. In one experiment, healthy volunteers were administered a variety of low-molecular-weight heparins, known to cause transient serum ALT elevations. In addition to aminotransferase increases in >90% of cases, markers of subcellular injury (cytosolic, mitochondrial), apoptosis (M30 fragmentation product of cytokeratin [CK] 8/19), microRNA (miRNA)-122, DNA, and high-mobility group box-1 (HMGB1) increased; HMGB1 is a DAMP released in necrosis. The authors concluded that heparins as a class caused self-limited and mild necrosis with secondary activation of an innate immune response. Secondary reactions, including post-translational modification of proteins via adenosine diphosphate ribosylation or protease activation, cleavage of DNA by activation of endogenous endonucleases, and disruption of lipid membranes by activated phospholipases may also play a role in DILI. Some of these catabolic reactions could be initiated by a rise in the cytosolic ionic calcium concentration (Ca ²⁺ ) _i , as a result of increased Ca ²⁺ entry or release from internal stores in the endoplasmic reticulum and mitochondria. ^, The potential role of endoplasmic reticulum stress in DILI is less well defined. ^,

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 [Ca ²⁺ ] _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. Mitochondrial injury, particularly that signaled via activation of the c-Jun N-terminal kinase (JNK), appears to be critically involved with acetaminophen and most likely several other hepatotoxins. ^, In turn, the balance between these factors can trigger the onset of cell death or facilitate protection of the cell, as discussed later.

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. 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 its 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.

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 at least some necrosis and a hepatic inflammatory response. Whether 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. ^,

The operation of hepatocellular apoptosis can be determined by detection of the caspase-3-cleaved fragmentation product (M30) of cytokeratins 8 and 18 that is specific to hepatocytes. 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 TNF 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 T NF- r elated a poptosis- i nducing l igand (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 → caspase activation → apoptosis.

Mitochondria play a pivotal role in pathways that provoke or oppose apoptosis. ^{, , ,} In the external pathway, activation of the death domain of pro-apoptotic receptors recruits adapter molecules, Fas-associated death domain and TNF receptor-associated death domain, which bind and activate procaspase 8 to form the death-inducing signaling complex. In turn, caspase 8 cleaves Bid, a pro-apoptotic member of the B cell lymphoma/leukemia (Bcl-2) family, to tBid. Then, tBid causes translocation of Bax to the mitochondria, where it aggregates with Bak to promote permeability of the mitochondria. 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) allows formation of the “apoptosome,” which activates caspase 9 and eventually caspase 3 to execute cell death ( Fig. 88.1 ). 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-xL, antagonize apoptosis and serve as survival factors by regulating the integrity of mitochondria; the protective mechanism is not yet fully understood but involves myeloid cell leukemia sequence 1 (Mcl-1). Stress-activated protein kinases, particularly JNK, are also pro-apoptotic, targeting Mcl-1 degradation and phosphorylating and inactivating the mitochondrial protective protein Bcl-xL.

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. 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 “necrapoptosis.” ^, Furthermore, when apoptosis is massive, the capacity for rapid phagocytosis can be exceeded, and “secondary” necrosis can occur.

Intracellular processes and activation of pro-apoptotic death receptors are not mutually exclusive pathways of cell death in toxic liver injury. In fact, drug toxicity could predispose the injured hepatocyte to apoptosis mediated by TNF-R or Fas-operated pathways by several mechanisms, including blockade of nuclear factorkappa B (NF-ĸB), which usually is a hepatoprotective transcription factor in hepatocytes, and inhibition of purine and protein synthesis. Furthermore, activation of Kupffer cells (e.g., by endotoxin) and recruitment of activated inflammatory cells can increase production of TNF.

Caspase inhibition is an important protective mechanism against cell death. Such anti-apoptotic pathways include chemical blockade of the cysteine thiol group by nitric oxide (NO) or ROS and cellular depletion of glutathione. Protein inhibitors include IAP family members, heat shock proteins, and FLICE (caspase-8)-inhibitory proteins (FLIP). FLIP inhibit caspase-8 activation as a decoy for Fas-associated death domain binding. Bcl-2 and Bcl-X _L inhibit mitochondrial permeability, whereas phosphatidylinositol 3-kinase/Akt phosphorylates caspase 9 and activates NF-ĸB.

Role of Hepatic Nonparenchymal Cells and the Innate Immune Response

In addition to migratory cells, activation of nonparenchymal liver cell types is likely to play an important role in drug and toxin-induced liver injury. Kupffer cells function as resident macrophages and antigen-presenting cells, whereas dendritic cells and natural killer (NK) T cells are also resident in the liver and play a role in antigen processing and innate immunity. Some of the toxic effects of activated Kupffer cells, as well as of recruited leukocytes, may be mediated by release of cytokines, such as TNFα, interleukin (IL)-1β and Fas-L, which under some circumstances can induce cell death in hepatocytes by apoptosis or necrosis. In addition, activated Kupffer cells release ROS, nitroradicals, leukotrienes, and proteases. It has been suggested, however, that the sterile inflammatory response may aid in clearing cell debris and pave the way for tissue repair.

Endothelial cells of the hepatic sinusoids or terminal hepatic veins are vulnerable to injury by some hepatotoxins because of their low glutathione content. Such hepatotoxins include the pyrrolizidine alkaloids, which are an important cause of the sinusoidal obstruction syndrome (hepatic veno-occlusive disease). Other types of drug-induced vascular injury may be caused primarily by involvement of the sinusoidal endothelial cells.

Hepatic stellate cells are the principal liver cell type involved in matrix deposition in hepatic fibrosis. Stellate cells are activated in methotrexate-induced hepatic fibrosis. The possibility that vitamin A, ROS, or drug metabolites can transform stellate cells into collagen-synthesizing myofibroblasts is of considerable interest.