Drug-induced liver injury (DILI)
is a major health problem that challenges not only health care professionals
but also the pharmaceutical industry and drug regulatory agencies. According to
the United States Acute Liver Failure Study Group, 1 DILI accounts for more
than 50% of acute liver failure, including hepatotoxicity caused by overdose of
acetaminophen (APAP, 39%) and idiosyncratic liver injury triggered by other
drugs (13%). Because of the significant patient morbidity and mortality
associated with DILI, 2 the U.S. Food and Drug Administration (FDA) has removed
several drugs from the market, including bromfenac, ebrotidine, and
troglitazone. Other hepatotoxic drugs, such as risperi-done, trovafloxacin, and
nefazodone, have been assigned “ black
box ” warnings. DILI is the most common cause for the withdrawal of drugs from
the pharmaceutical market.
Although the exact mechanism of
DILI remains largely unknown, it appears to involve 2 pathways — direct
hepato-toxicity and adverse immune reactions. In most instances, DILI is
initiated by the bioactivation of drugs to chemically reactive metabolites,
which have the ability to interact with cellular macromolecules such as
proteins, lipids, and nucleic acids, leading to protein dysfunction, lipid
peroxidation, DNA damage, and oxidative stress. Additionally, these reactive
metabolites may induce disruption of ionic gradients and intracellular calcium
stores, resulting in mitochondrial dysfunction and loss of energy production.
This impairment of cellular function can culminate in cell death and possible
liver failure.
Hepatic cellular dysfunction and
death also have the ability to initiate immunological reactions, including both
innate and adaptive immune responses. Hepatocyte stress and/or damage could
result in the release of signals that stimulate activation of other cells,
particularly those of the innate immune system, including Kupffer cells (KC),
natural killer (NK) cells, and NKT cells. These cells contribute to the
progression of liver injury by producing proinflammatory mediators and secreting
chemokines to
further recruit inflammatory cells to the liver. It has been demonstrated that
various inflammatory cytokines, such as tumor necrosis factor (TNF)- ,
interferon (IFN)- , and interleukin (IL)-1, produced during DILI are involved
in pro moting tissue damage.
However,
innate immune cells are also the main source of IL-10, IL-6, and certain
postglandins, all of which have been shown to play a hepatoprotective role.
Thus, it is the delicate balance of inflammatory and hepatoprotective mediators
produced after activation of the innate immune system that determines an
individual ’ s susceptibility and adaptation to DILI.
In addition to the innate
immune responses, clinical features of certain DILI cases strongly suggest that
the adaptive immune system is activated and involved in the pathogenesis of
liver injury. With regard to the involvement of the adaptive immune system in
DILI, our current understanding is based on the hapten hypothesis and the p-i (
p harmacological interaction of drugs with i mmune receptors) concept. Evidence
to support these hypotheses is gained by the detection of drug-specific
antibodies and T cells in some patients with DILI.
Figure.8.1:-Pathogenesis of drug-induced liver diseases. Upstream
events in the hepatocytes affect viability of individual cells but sensitize to
downstream processes leading to clinically overt organ damage. The latter
involves a balance of effects of cytokines, chemokines, and inflammatory
mediators, mainly produced by nonparenchymal cells and the effects on repair
processes a such as regeneration.
Figure.8.2:- Role
of cytokine balance in determining susceptibility to toxins. Drugs or metabolites
may directly injure hepatocytes to a minor extent, but may markedly sensitize
to the lethal effects of TNFa and IFNg. The latter are modulated by cytokines
that promote or inhibit their production or actions. Abbreviations: TNFa, tumor
necrosis factor a; IFNg, interferon g.
DILI
DILI can affect both parenchymal and
nonparenchymal cells of the liver, leading to a wide variety of pathological
con ditions, including acute and chronic hepatocellular hepatitis,
fibrosis/cirrhosis, cholestasis, steatosis, as well as sinusoidal and hepatic
artery/vein damage. The predominant forms of DILI include acute hepatitis,
cholestasis, and a mixed pattern. Acute hepatitis is defined as a marked
increase in aminotransferases coinciding with hepatocellular necrosis.
Cholestasis is characterized by jaundice with a concurrent elevation in
alkaline phosphatase, conjugated bilirubin, and -glutamyl transpeptidase.
Mixed-pattern DILI includes clinical manifestations of both hepatocellular and
cholestatic injury.
The clinical diagnosis and
prediction of DILI remain a major challenge due to various confounding factors.
These factors include preexisting liver disease, multiple drug usage by
patients, and most important, lack of reliable screening methods and diagnostic
standards. As a general rule, alanine transaminase (ALT) levels greater than 3
times the upper limits of normal (ULN) have been identified as a marker for
liver injury. Hyman Zimmerman noted that elevated ALT accompanied by jaundice
was associated with a mortality between 5% and 50%. This observation has since been referred to as
“ Hy ’ s rule ” and is currently employed by the FDA in the evaluation of
hepatotoxicity for newly developed drugs. However, it must be noted that 3 ×
ULN of ALT levels are not necessarily predictive of overt severe liver toxicity
or acute liver failure.
MECHANISMS
OF DILI
Drug-Induced Direct Hepatotoxicity
Direct hepatotoxicity is often
caused by the direct action of a drug, or more often a reactive metabolite of a
drug, against hepatocytes. One classically studied drug used to examine the
mechanisms of hepatotoxicity is APAP. APAP is a popular over-the-counter
analgesic that is safe at therapeutic doses but at overdose can produce centrilobular
hepatic necrosis, which may lead to acute liver failure. APAP is metabolized to
a minor electrophilic metabolite, N-acetyl-p-benzoquinoneimine (NAPQI), which
during APAP overdose depletes glutathione and initiates covalent binding to
cellular proteins. These events lead to the disruption of calcium homeostasis,
mitochondrial dysfunction, and oxi-dative stress and may eventually culminate
in cellular damage and death. Fortunately, drug candidates that induce
significant direct hepatotoxicity at therapeutic doses are more likely to be
detected during preclinical toxicity screening and thus rarely reach the
pharmaceutical market.
In most instances of DILI, it appears
that hepatocyte damage triggers the activation of other cells, which can
initiate an inflammatory reaction and/or an adaptive immune re -sponse. These
secondary events may overwhelm the capacity of the liver for adaptive repair
and regeneration, thereby contributing to the pathogenesis of liver injury.
Drug-Induced
Immune-Mediated Liver Injury
Innate and Adaptive Immune System of the
Liver
The innate immune system provides a
first line of defense against microbial infection, but it is not sufficient in
eliminating infectious organisms. The lymphocytes (T and B cells) of the
adaptive immune system provide a more versatile means of defense and possess “
memory, ” which is the ability to respond more vigorously to repeated exposure
to the same microbe. Moreover, cells of the innate immune system play an
integral role in the initiation of adaptive immunity by presenting antigens and
are crucial in determining the subsequent T-cell- or antibody-mediated immune response.
Because of the liver ’ s continuous exposure to pathogens, toxins, tumor cells,
and harmless dietary antigens, it possesses a range of local immune mechanisms
to cope with these challenges. The liver contains large numbers of both innate
and adaptive immune cells, including the largest populations of tissue
macrophages (KC), NK cells, and NKT cells. The liver also possesses a unique
combination of intrahepatic lymphocytes, which include not only the
conventional CD4 + and CD8 + T cells but also high percentages of T cells and
CD4 − CD8 − T cells. Collectively, the innate and adaptive immune cells
contribute to the unique immune responses of the liver, including removal of
pathogenic microorganisms, clearance of particles and soluble molecules from circulation,
deletion of activated T cells, and induction of tolerance to food antigens
derived from the gastrointestinal tract.
KC play an essential role in the
phagocytosis and removal of pathogens entering the liver via portal-venous
blood. Upon activation, KC produce various cytokines and other mediators,
including prostanoids, nitric oxide, and reactive oxygen intermediates. These
KC products play prominent roles in promoting and regulating hepatic
inflammation, as well as modulating the phenotype of other cells in the liver,
such as NK and NKT cells. Furthermore, KC represent a major population of
antigen-presenting cells (APCs) within the liver. It has been demonstrated that
KC can activate T cells in vitro, but they do so less efficiently than peritoneal-exudate
macrophages. Studies of organ transplantation using animal models have further
shown that inhibition of KC abrogated the prolonged survival of allografts
induced by portal vein infusion of allogeneic donor cells. Collectively, this
evidence suggests that KC play an important role in the delicate balance
between the induction of immunity and tolerance within the liver.
Unique to the liver are the
remarkably high frequencies of NK and NKT cells, which account for ~50% of
intrahepatic leukocytes. These cells act as a first line of defense against
certain pathogens and invading tumor cells prior to the adaptive immune
response of B and T lymphocytes. One characterized function of hepatic NK and
NKT cells is their cytotoxic capacity against other cells. It has been
demonstrated that freshly isolated liver NK cells spontaneously induce the
cyto-toxicity of various cell lines, whereas NKT cells are cytotoxic in the
presence of IL-2. This cytotoxicity is further enhanced by IL-12 and IL-18,
which are produced by activated KC. Another function ascribed to NK and NKT
cells is their ability to produce high levels of T helper (Th) 1 and Th2 cyto-kines
upon stimulation. NK cells have been shown to represent a major source of IFN-
in many types of liver disease. NKT cells produce either IFN- or IL-4, or in
some cases both cytokines, depending on the differentiation state of the cells and
the stimuli. It has also been demonstrated that IL-4 produced by NKT cells may
be associated with the initiation and regulation of Th2 responses.
The liver ’ s adaptive immune
responses are unique in that the liver is known to favor induction of
immunological tolerance rather than immunity. This is supported by numerous
studies demonstrating that (1) dietary antigens derived from the
gastrointestinal tract are tolerized in the liver; (2) allogeneic liver organ
transplants are accepted across major histocompatibility complex (MHC) barriers
(3) preexposure to donor cells through
the portal vein of recipient animals increased their acceptance of solid tissue
allografts and (4) preexposure of soluble antigens via the portal vein leads to
systemic immune tolerance. Several mechanisms have been suggested to account
for this tolerance, including apoptosis of activated T cells, immune deviation,
and active suppression.
The liver has been dubbed the “
elephant ’ s graveyard ” for activated T cells. These cells accumulate in the
liver before undergoing apoptosis. Studies using T-cell receptor transgenic
models have demonstrated that following antigen exposure, the activated T cells
undergo apoptosis after a transient accumulation within the liver. Immune
deviation may account for liver-induced tolerance, as it has been shown that
Th2 cytokine production is preferentially maintained when adoptively
transferred Th1 and Th2 cells are recovered from the liver. It has also been
reported that liver sinusoidal endothelial cells (LSEC) are capable of
selectively suppressing IFN- -producing Th1 cells while concurrently promoting
the outgrowth of IL-4-expressing Th2 cells. Active suppression of T-cell
activation resulting in liver-induced tolerance is also likely to occur within
the liver because of its unique anatomy and composition of “ tolerogenic ”
APCs. Within the liver, blood flow slows down through the narrow sinusoids (7-12
µm) and is temporarily obstructed by KC, which reside in the sinusoidal lumen.
Because of this reduction in blood flow, circulating T cells can interact with
LSEC and KC. Consequently, naïve T cells, which would normally encounter APCs
in lymphoid tissues, could be primed directly by LSEC and/or KC within the
liver. Current evidence suggests that LSEC and KC as well as hepatic dendritic
cells are important in the induction of tolerance, rather than the activation
of T-cell responses. It has been further demonstrated that although LSEC are
capable of presenting antigen to T cells, LSEC-activated CD4 + or CD8 + T cells
fail to differentiate into Th1 cells or cytotoxic effector cells, respectively.
In addition, studies have shown that KC and hepatic dendritic cells are not
effective APCs when compared with their counterparts in lymphoid tissues.
In summary, it is the unique
composition of innate and adaptive immune cells in the liver and the
characteristic response of the liver to endogenous and exogenous antigens that
may account for the mechanism of immune-mediated DILI, as well as the low
occurrence and unpredictable nature of these reactions.
Role of Innate Immunity in DILI
Drug-induced stress and/or damage
of hepatocytes may trigger activation and inflammatory responses of the innate
immune system within the liver. Evidence to support this idea has been mainly
obtained from studies of liver injury induced by overdose of APAP, which is one
of the few drugs that provide an experimental animal model of DILI. There is
growing evidence that the initial NAPQI-induced hepatocyte damage may lead to
activation of innate immune cells within the liver, thereby stimulating hepatic
infiltration of inflammatory cells. Activated cells of the innate immune system
produce a range of inflammatory mediators, including cytokines, chemokines, and
reactive oxygen and nitrogen species that contribute to the progression of
liver injury. Some of these mediators, such as IFN- , Fas, or Fas ligand, are
directly involved in causing liver damage; mutant mice lacking these factors
are resistant to APAP hepatotoxicity. On the other hand, the innate immune
cells also represent a major source of hepatoprotective factors, as it has been
demonstrated that transgenic mice deficient in IL-10, IL-6, or COX-2 are more
susceptible to APAP-induced liver injury.
The innate immune cells reported to
participate in APAP hepatotoxicity include NK and NKT cells, macrophages, and
neutrophils. A recent study demonstrated that depletion of NK and NKT cells
protected mice from APAP-induced liver injury. This protective mechanism seems
to involve eliminating the production of IFN- and various other pro-inflammatory
chemokines as well as decreasing neutrophil accumulation within the liver. APAP
hepatotoxicity has also been attributed in part to the activation of KC
secondary to hepatocyte damage. KC activation results in the release of a wide
range of proinflammatory mediators, such as TNF- , which may directly induce
tissue damage, and IL-12 and IL-18, which are important activators of NK and
NKT cells. However, other studies suggest that KC may play a protective role in
addition to their protoxicant effect, as KC are the predominant source of IL-10
and IL-6, which are important in counteracting inflammatory responses and/or stimulating
liver regeneration. Although many studies have shown neutrophil accumulation in
the liver of APAP-treated animals, the pathogenic role of these cells remains
controversial. One study has shown that in vivo injection of rabbit
antineutrophil antiserum protected rats against APAP toxicity, while another
study suggested that neutrophils are recruited into the liver to remove
cellular debris and do not directly cause tissue damage.
Our understanding of APAP-induced
liver injury underscores the role of the innate immune system as an important
regulator in the progression and severity of tissue damage. However, the
temporal activation of different cell populations and their production of
various mediators, as well as the interplay among these cells, warrant further
investigation. A proposed mechanism of DILI involving parenchymal cell damage
and subsequent activation of the innate immune system is presented in Figure 1
.
Role of Adaptive Immune Response in DILI
The clinical features of some cases
of DILI strongly suggest an involvement of the adaptive immune system. These
clinical characteristics include (1) concurrence of rash, fever, and
eosinophilia; (2) delay of the initial reaction (1-8 weeks) or requirement of
repeated exposure to the culprit drug; (3) rapid recurrence of toxicity on
reexposure to the drug; and (4) presence of antibodies specific for native or drug-modified
hepatic proteins. (5) Drugs suspected to induce these types of reactions
include halothane, tienilic acid, dihydralazine, diclofenac, phenytoin, and
carbamazepine.
Our current understanding of
drug-induced adaptive immune responses is largely based on the hapten
hypothesis and the p-i concept. The hapten hypothesis proposes that drugs, or
more often reactive metabolites of the drugs, act as haptens and covalently
bind to endogenous proteins to form immunogenic drug-protein adducts. These
immunogenic adducts elicit either antibody or cytotoxic T-cell responses.
The hapten hypothesis is supported by
the detection of antibodies that recognize drug-modified hepatic proteins in the
sera of DILI patients. For example, antibodies that recognize
trifluoroacetate-altered hepatic proteins have been detected in the sera of
patients with halothane-induced hepatitis. Such drug-specific antibodies or
autoantibodies that recognize native liver proteins have also been found in
patients with liver injury caused by other drugs, such as tienilic acid,
dihydralazine, and diclofenac. The p-i concept suggests that certain drugs can
bind to T-cell receptors, mimicking a ligand and its receptor interaction, and
cause T-cell activation in an MHC-dependent fashion. In patients who developed
drug-induced systemic reactions of the liver and other organs, drug-specific T
cells have been detected, and in some cases, T-cell clones were generated.
Despite the detection of
drug-specific antibodies and T cells, it has been difficult to directly prove the
pathogenic role of the adaptive immune system in DILI, in part because of the
lack of animal models. An important reason for the difficulty in developing
animal models is that the default response of the liver to antigens is
immunological tolerance. This tolerogenic response could also explain the low
occurrence of this type of DILI in humans. As described in the above section,
the anatomy, cellular composition, and microenvironment of the liver favor
tolerance rather than pathogenic immunity. Therefore, DILI mediated by adaptive
immune reactions against a drug can occur only when the tolerance mechanism is
deficient or abrogated in susceptible individuals. As such, animal models of
this type of DILI can be developed only after the barrier of such tolerance is
overcome.
Figure.8.3:- General interrelationship between toxicity (including
immune response; “hapten hypothesis”) anddisposition of a drug with reactive
metabolites (left ). Scheme focusing on major processes relevant for the
dispositionof reactive acyl glucuronides (right ), for which an antigen
character of adducts was shown and antibodies have beendetected, also in vivo,
in some cases. Acyl glucuronides are generating increasing interest as
potential mediators ofhypersensitivity reactions and cellular toxicity.
Figure.8.4:- Illustration of the proposed mechanism of
DILI, which involves drug metabolism, hepatocyte damage, activation of innate
immune cells, and production of tissue-damaging and tissue-protective
mediators. CYP indicates cytochrome P450; IFN, interferon; IL, interleukin; NK,
natural killer cell; NKT, natural killer T cell; TNF, tumor necrosis factor.
Figure.8.5:- A 3-step mechanistic
working model of hepatotoxicity. First, initial injury is exerted through
direct cell stress, direct mitochondrial inhibition and/or specific immune
reactions. Second, initial injury can lead to mitochondrial permeability
transition (MPT). Direct cell stress causes MPT via the intrinsic pathway. The
intrinsic pathway involves activation of intracellular stressor cascades and
pro-apoptotic proteins including Bax. Alternatively MPT can be initiated
through the death receptor-mediated extrinsic pathway that is activated by
immune reactions and/or after sensitization to TNF and FasL binding to death
receptors. Cytokines modulate the sensitivity of its activation. Third, MPT
leads to necrosis or apoptosis depending on the availability of ATP. In
hepatocytes activation of initiator caspase 8 through the extrinsic pathway is
not sufficient to directly activate apoptosis, but amplification through
pro-apoptotic factors including Bid and ceramides lead to MPT, which will then
lead to the apoptotic pathway that is activated in the presence of sufficient
remaining ATP production. Necrosis occurs if there is no ATP available, which
is required for energy-consuming apoptotic pathways. Several highlighted
amplification mechanisms (A) may play an important role at different levels for
the idiosyncratic occurrence of hepatotoxicity.
Figure.8.6:- Risk factors for
hepatotoxicity. First, risk factors can be classified into environmental vs.
genetic factors. From a mechanistic point of view risk factors can further
affect all different levels of events leading to the final outcome of
drug-induced liver injury, which is mostly dichotomous, i.e. full recovery vs.
acute liver failure. Note that risk factors affecting events downstream of
initial injury are rather unspecific regarding different hepatotoxins. The
figure presents a selection of well-described risk factors, but one must assume
that other factors also play a role, of which many currently remain unknown.
Figure.8.7:- Six Mechanisms of
Liver Injury. Injury to liver cells occurs in patterns specific to the
intracellular organelles affected. The normal hepatocyte shown in the center of
the figure may be affected in at least six ways, labeled A through F.
Disruption of intracellular calcium homeostasis leads to the disassembly of
actin fibrils at the surface of the hepatocyte, resulting in blebbing of the
cell membrane, rupture, and cell lysis. In cholestatic diseases, disruption of
actin filaments (B) may occur next to the canaliculus, the specialized portion
of the cell responsible for bile excretion. Loss of villous processes and the
interruption of transport pumps such as multidrug-resistance–associated protein
3 (MRP3) prevent the excretion of bilirubin and other organic compounds. Many
hepatocellular reactions involve the heme-containing cytochrome P-450 system
(C), generating high-energy reactions that can lead to the covalent binding of
drug to enzyme, thus creating new, nonfunctioning adducts. These enzyme–drug
adducts migrate to the cell surface (D) in vesicles to serve as target
immunogens for cytolytic attack by T cells, stimulating a multifaceted immune
response involving both cytolytic T cells and cytokines. Activation of
apoptotic pathways by tumor necrosis factor a (TNF-a) receptor or Fas may
trigger the cascade of intercellular caspases (E), which results in programmed
cell death with loss of nuclear chromatin. Certain drugs inhibit mitochondrial
function by a dual effect on both b-oxidation (affecting energy production by
inhibition of the synthesis of nicotinamide adenine dinucleotide and flavin
adenine dinucleotide, resulting in decreased ATP production) and the
respiratory-chain enzymes (F). Free fatty acids cannot be metabolized, and the
lack of aerobic respiration results in the accumulation of lactate and reactive
oxygen species. The presence of reactive oxygen species may further disrupt
mitochondrial DNA. This pattern of injury is characteristic of a variety of
agents, including nucleoside reverse-transcriptase inhibitors, which bind directly
to mitochondrial DNA, as well as valproic acid, tetracycline, and aspirin.
Toxic metabolites excreted in bile may damage bile-duct epithelium (not shown).
DD denotes death domain.
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