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REGULATORY TOXICOLOGY AND PHARMACOLOGY
ARTICLE NO.
27, 119–130 (1998)
RT981201
Serum Transaminase Elevations as Indicators of Hepatic Injury
Following the Administration of Drugs
David E. Amacher
Drug Safety Evaluation, Pfizer Central Research, Groton, Connecticut 06340
Received December 20, 1997
During the preclinical, early clinical, late-stage clinical, and postmarketing phases of the pharmaceutical
discovery and development process, one important aspect of drug safety assessment involves monitoring for
possible drug-induced hepatic injury. Hepatic injuries
vary in nature from direct, intrinsic effects that are
observed in most recipients and more than one species
to rare idiosyncratic responses seen only in a few clinical subjects. Histological types of injuries vary from
hepatocellular to hepatobiliary with multiple cellular
effects characteristic of each type. Of the various clinical laboratory markers for hepatic injury, serum
transaminases, especially alanine aminotransferase
(ALT), are the most universally important indicators
for studies ranging from early preclinical animal testing to postmarketing patient monitoring. This review
examines the characteristics of hepatic toxicity that
result in serum ALT changes, the differences in the
etiology of hepatic responses which govern when liver
injury is most likely to be detected during the four
phases of the drug discovery and development process,
and those modulating factors which affect the utility
of ALT as a dependable marker of hepatic injury in
clinical populations. The paper concludes with a summary of some ancillary methods for early preclinical
screening such as in vitro metabolism and toxicity
assays, gene and protein expression analysis, and
some strategies for enhancing the probability for the
early detection of idiosyncratic hepatotoxic responses
which are infrequent but significant factors in the
safety assessment process. q 1998 Academic Press
Key Words: review; serum transaminases; alanine
aminotransferase; liver injury.
1. DRUG HEPATOTOXICITY AND THE
PHARMACEUTICAL DEVELOPMENT PROCESS
Because of its central role in drug metabolism, the
liver is particularly susceptible to injury following systemic exposure to xenobiotics. Accordingly, liver toxicity as suggested by slightly elevated serum enzymes of
hepatic origin is one of the more frequently encoun-
tered adverse effects in early clinical trials of therapeutic agents, although the actual incidence of hepatic injury is extremely low. During the pharmaceutical discovery, development, and marketing processes, drugassociated hepatotoxicity can be encountered at any of
four stages.
The first stage involves the detection of hepatotoxic
phenomena in preclinical safety assessment studies
using rats, dogs, monkeys, or other mammals at dose
levels in excess of the intended therapeutic dose. A
significant incidence of drug-associated hepatocellular
or hepatobiliary injury detected by clinical chemistry
procedures and confirmed by histological methods in
one or more species generally precludes further development unless there is clear evidence that a similar
effect is not anticipated in humans. An exception could
be the demonstration that animal toxicity is due to a
specific metabolite that would not be a major metabolite in humans. Most intrinsically hepatotoxic (type A)
compounds are detected in preclinical animal trials
and are not advanced to clinical trials. The proportion
of compounds causing some degree of hepatic effects
such as hepatomegaly, serum enzyme elevations, hepatobiliary effects, or necrosis at high doses in animals
can reach as high as 50% of drugs tested in preclinical
studies.
The next level where evidence of hepatotoxicity may
be evaluated is in early clinical development. At therapeutic doses, untoward findings could include, for example, the reporting of serum transaminase elevations
that are 2 times the upper limit of normal, jaundice,
or 1.5 times the normal alkaline phosphatase (Alk P;
EC 3.1.3.1) activity in a small treated population of
healthy volunteers during early clinical trials (Batt and
Ferrari, 1995). While these clinical chemistry changes
are usually transient and not accompanied by clinical
evidence of hepatic toxicity, drugs eliciting these
changes are typically not continued in development unless there is a compelling reason to do so.
The third stage typically involves the reporting of a
few patients within a controlled clinical population who
are found to have transaminase levels two to three
times the upper limit of normal but who are otherwise
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All rights of reproduction in any form reserved.
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asymptomatic. A fairly large number of drugs fall
within this category. These are not typically discontinued for this phenomenon alone. For example, the
U.S. Physicians’ Desk Reference indicates that liver enzyme elevations may be found in up to 15% of patients
taking nonsteroidal anti-inflammatory drugs (NSAIDs).
One NSAID in particular, sulindac, actually accounts
for most of the risk (Fry and Seeff, 1995; Garcia Rodriguez et al., 1994). Another example is the 12% incidence of asymptomatic hepatic enzyme abnormalities
reported in ketoconazole recipients (Mosca et al., 1985).
The disease under treatment may also affect the liver
adversely leading to elevated hepatic enzymes. Most of
these reactions are host-dependent, idiosyncratic responses which are not easily predicted in preclinical
testing or observed in early clinical trials because of
the small number of subjects.
The final stage involves similar findings in a large
postmarketing population. These cases involve verylow-frequency, idiosyncratic (type B) events that are
not detectable in controlled clinical populations of 500–
1000, for example, due to mathematical probability.
These result from a novel aspect of the patient, not the
drug, and are more uncertain in etiology because they
are typically reported in an uncontrolled nonclinical
setting. Thus, drug-induced hepatic injury ranges from
frequent, dose-related effects in multiple species to rare
but profound hepatic effects only detectable in susceptible individuals at therapeutic doses. Serum transaminase activities are the single diagnostic indicator for
all of these effects and are the subject of this review.
results. In humans, the incidence of fatty liver disease
noted at autopsy for accident victims ranges from 40
to 80% (Hodgson et al., 1989) suggesting a high incidence from unknown causes in the general adult population. Necrotic cell death is accompanied by a disruption of cell membrane integrity and the loss of liverspecific enzymes which forms the basis of the serum
chemistry analyses used systematically for its detection. Clinical manifestations of hepatic necrosis and
degeneration range from jaundice to fulminant hepatic
failure. Cholestatic injury occurs when there is a disruption or impairment of bile flow. Canalicular cholestasis can be caused by several mechanisms including
leaky paracellular junctions, a diminished contractility
of canaliculus, diminished transcytosis, impaired
transporters, and toxin concentration in the paracanicular area (Moslen, 1996). Toxins or their metabolites
can also damage the intrahepatic bile ducts. Chlorpromazine, oral contraceptives, and androgens used to
increase muscle mass can produce a cholestatic response. Hepatobiliary lesions lead to elevated serum
levels of bile constituents or enzymes localized in the
hepatobiliary tree such as Alk P or gamma glutamyltranspeptidase (GGT; EC 2.3.2.2). In humans, most hepatic drug reactions appear within 3 months of starting
therapy (Davis, 1989).
2. HISTOLOGICAL TYPES OF TOXIC HEPATIC INJURY
Three categories of noninvasive laboratory tests are
used to identify the type and extent of drug hepatotoxicity based on the presence or absence of specific markers in the blood of exposed individuals. The first category of clinical assays is that used to assess hepatocellular damage leading to liver cell necrosis. Evidence of
this type of injury is based on the detection the hepatic
transaminases, alanine aminotransferase (ALT), and
aspartate aminotransferase (AST). These are not normal components of blood and serve no known function
outside the organ of origin. A third specific marker of
hepatocellular damage, serum F protein, has been recently described (Foster et al., 1989), although it is not
yet as widely used as the transaminases. Based on liver
biopsy specimens, chronic ALT elevations in asymptomatic patients have also been associated with fatty
liver (Hulteranz et al., 1986). In fact, nonalcoholic
steatosis has been cited as a very common cause of
chronic ALT elevations in the general population
(Craxi and Almasio, 1996). Thus, transaminase elevations are indicative of hepatocellular death and fatty
degeneration in particular, but can also be used in conjunction with other serum enzymes for distinguishing
non-hepatocellular injury as described below.
The second group of tests includes markers for hepatobiliary (chlolestatic) effects. This type of injury in-
In general, the type and degree of acute toxic liver
injury following drug administration depend upon the
dose and duration of exposure. In addition, many chemicals preferentially affect certain cell populations but
not others in the same host. Major histologic lesions of
the liver due to chemical toxicity include fatty change,
hepatocellular death, and hepatobiliary lesions. Granulomatous and mixed histological changes may also be
seen (Pirmohamed et al., 1992) or venoclusive effects,
especially with some antineoplastic drugs (McDonald
and Tirumali, 1984). Steatosis or fatty change refers
to an increase in hepatic lipid content greater than 5%
by weight (Moslen, 1996). Lipid accumulates as intracytoplasmic vacuoles in either a zonal or diffuse pattern. Microvesicular and macrovesicular steatoses, differing in the lipid vacuole size and the mechanism for
their formation, are distinguishing characteristics of
specific toxicants. Macrovesicular steatosis is characterized by a single large fat droplet which displaces
the nucleus to the cell periphery, but in microvesicular
steatosis there are a number of small lipid droplets
that do not displace the nucleus (Stricker, 1992a).
Steatosis is reversible unless it is persistent enough to
cause secondary changes or so severe that cell death
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3. BIOCHEMICAL TESTS USED FOR THE DETECTION
OF HEPATIC INJURY
3.1. Categories of Laboratory Tests
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cludes biliary obstruction or hepatic infiltrative processes resulting in the retention of bile acids in liver
and leading to drug-induced jaundice if severe. Serum
markers include Alk P from the cell canalicular membrane, 5* nucleotidase (5*NT), and GGT. Neither Alk P
nor GGT, which is also inducible by alcohol and certain
drugs (see below), is liver-specific. However, marked
serum Alk P elevations, especially when accompanied
by 5*NT or GGT elevations, suggest mechanical bile
duct obstruction, primary sclerosing cholangitis, primary biliary cirrhosis, or drug-induced hepatitis
(Herlong, 1994). Thus, serum enzyme profiles as opposed to individual enzyme changes are used for diagnostic purposes, especially for monitoring hepatobiliary
effects.
The final category of diagnostic procedures is based
upon altered liver function. These are methods that
monitor serum albumin, cholesterol, prothrombin time,
or serum bilirubin as general indicators of the synthetic
and general metabolic capacity of the liver (Fregia and
Jensen, 1994) as opposed to marking some specific toxic
injury. The serum bilirubin assay will indicate liver
injury; however, elevated serum enzyme assays as described above usually reflect hepatotoxicity earlier
(Herlong, 1994). Although drug-associated hepatic dysfunction is uncommon in general, severely altered liver
function, especially those processes leading to coagulation disorders or bilirubin encephalopathy, are indicative of severe hepatic injury.
3.2. Aminotransferases
Aminotransferases are a group of enzymes that catalyze the reversible transfer of the amino group from
an a-amino acid to an oxo acid. ALT and AST shunt
their amino acid and oxo acid substrates into several
intermediate pathways. Cytosolic ALT is associated
with the utilization of pyruvate in glycolysis, mitochondrial ALT is involved in the conversion of alanine to
pyruvate for gluconeogenesis, and AST plays an important role in the transport of reducing equivalents
across the mitochondrial membrane (Sakagishi, 1995;
Rej, 1989). Hepatocellular damage with the subsequent disruption of the plasma membrane allows leakage of intracellular enzymes such as ALT or AST into
the bloodstream. With some hepatotoxicants, increased hepatic synthesis of aminotransferases has
also been suggested as a source of increased serum
enzyme levels in hepatocellular injury (Pappas, 1986).
The range of normal is determined by either cutoff
values of {2 SD or 97.5 percentile cutoffs in a population without known disease. Due to half-lives of approximately 17 h for AST and 47 h for ALT, the presence of these enzymes in serum is considered an indicator of recent hepatocyte injury (Scheig, 1996). Some
drugs actually decrease the activities of serum ALT
and AST. Examples include oxodipine which causes
hepatic damage and yet decreases ALT activities in
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dogs and rats (Waner and Nyska, 1991), cefazolin
which significantly depresses ALT and AST activities
in rat liver (Dhrami et al., 1979), and isoniazid which
significantly inhibits hepatic and serum AST in rodents (Yamada et al., 1984a,b). In addition to inhibitory effects by antivitamin B6 compounds, transaminase levels are effected by other nutritional events.
For example, healthy human volunteers ingesting a
choline-deficient diet for 4 weeks showed significantly
elevated levels of ALT compared their cohorts (Zeisel
et al., 1991). A discussion of the characteristics of each
of these important enzymes follows.
3.2.1. ALT. ALT (L-alanine:2-oxoglutalate aminotransferase, EC 2.6.1.2) is a pyridoxal enzyme which
catalyzes the reversible interconversion of L-alanine /
a-ketoglutarate to pyruvate / L-glutamate with peridoxal phosphate as a coenzyme. The presence of low
levels of ALT in the peripheral circulation represents
normal cell turnover or release from nonvascular
sources. Drug-related increases in aminotransferase
activity are typically transitory with values returning
to within normal reference limits within a few weeks
(Rej, 1989).
ALT is widely distributed. Human isozymes are
found in the cytosol and mitochondria of liver, kidney,
and skeletal and cardiac muscles (Sakagishi, 1995). Mitochondrial ALT comprises only a small portion of the
tissue activity and has not been demonstrated in normal human serum. The largest pool of ALT is in the
cytosol of hepatic parenchymal cells (Sherman, 1991).
Considerable differences in both the organ distribution
and intracellular compartmentalization of ALT have
been found among species (Hoffmann et al., 1989).
Some nonhuman primates, for example, show little or
no ALT organ specificity (Clampitt and Hunt, 1978).
However, overall, serum ALT is one of the most universal markers for hepatic injury across species.
In the clinical laboratory, the measurement of ALT
is a routine part of serum chemistry panels used to
assess hepatic injury. ALT values in healthy blood donors, however, can be influenced by age, sex, dietary
change, geographical location, ethnicity, obesity, longterm acetaminophen use, alcohol use, and marital status (Sherman, 1991), a complicating factor when monitoring human populations for transaminase elevations
following drug exposure. Even the difference in mean
ALT values between males and females in a donor population can be significant (Mijovic et al., 1987). ALT
activities are elevated for a few days following major
abdominal or thoracic surgery (Stricker et al., 1992a)
and can also be elevated by the disease being treated.
The incidence of hepatic damage due to cotrimaoxazole,
for example, is around 20% higher in AIDS patients
(Westphal et al., 1994) compared to other diseases. Cognizant of these modulating factors in clinical populations, ALT is the single most important indicator of
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hepatocellular injury for preclinical animal studies,
clinical trials, and postmarketing monitoring.
3.2.2. AST. AST (EC 2.6.1.1) is found in both the
cytosol and mitochondria of hepatocytes (Herlong,
1994), but high tissue levels are also found in heart,
skeletal muscle, kidney, brain, and pancreas (Rej,
1989). Accordingly, muscle trauma and surgery by itself can lead to AST serum elevations (Clermont and
Chalmers, 1967). An estimated 60–70% of AST activity
in human hepatocytes is localized within mitochondria
(Schmidt and Schmidt, 1990). Hence, when found in
blood, AST is considered to be a sensitive indicator of
mitochondrial damage, especially in the hepatic centrilobular regions which are particularly sensitive to toxic
and hypoxic liver injury (Schmidt and Schmidt, 1990).
It should be noted, however, that depending upon the
assay used a number of drugs can reportedly produce
spurious elevations in AST (Davis, 1989). Serum AST
is affected to a greater degree by alcohol consumption
than ALT (Lewis, 1984). Within these limitations, AST
in conjunction with ALT is a very important marker of
hepatic injury.
4. NON-DRUG-RELATED TRANSAMINASE ELEVATIONS
The term transaminitis has been applied to mild elevations of serum aminotransaminases in the absence
of other clinical laboratory abnormalities in asymptomatic individuals (Hodgson et al., 1989; Rej, 1989). When
observed after drug exposure, these small elevations
are often considered as being uninterpretable. A second
difficulty in monitoring transaminase levels in patient
populations is the underlying incidence of mildly abnormal results in a classical panel of liver tests used
in routine monitoring of asymptomatic, unaffected individuals. These may be associated with population
characteristics previously discussed and are not usually encountered in preclinical animal toxicology studies. Liver injury tests are abnormal in up to 8% of a
healthy population (Hodgson et al., 1989). Liver enzyme elevations noted during hospital admission have
been estimated at 5.7–7.6% (Grohmann et al., 1984;
Van Dijke et al., 1986). In one study, 25–30% of asymptomatic, normal workers had serum chemistries on a
five-test liver panel in excess of the established normal
range in the absence of risk factors for liver disease
(Wright et al., 1988). Of course, specific estimates of
the incidence of abnormal ALT values alone are lower
in donor populations. In one study, the frequency of
elevated ALT in a donor population was reported to
be 4.6% above 40 IU/liter in 1986 and appeared to be
increasing with time (Mijovic et al., 1987). In the absence of any intentional drug exposure, extremely high
transaminase levels (ú8- to 10-fold normal) in a patient
population may indicate acute viral hepatitis, drug- or
toxin-mediated liver necrosis, or other damage; persistent mild elevations (2- to 8-fold) are characteristic of
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chronic hepatitis, steatosis, metabolic diseases (Fregia
et al., 1994), alcoholic liver disease, and, much less frequently, drug-induced liver disease and nonalcoholic
steatosis (Renner and Dallenbach, 1992). In addition,
some disease states, especially malignancies, produce
hepatic effects culminating in elevated transaminases.
5. GUIDELINES FOR SERUM TRANSAMINASE
ELEVATION AS AN INDICATOR OF LIVER INJURY
By international consensus, liver injury, as might be
detected by liver tests, has been defined as an increase
greater than two times the upper limit of normal range
(ULN) in ALT or conjugated bilirubin or a combined
increase in AST, Alk P, and total bilirubin provided
that one of them is greater than two times the ULN
(Benichou, 1990). This injury is further defined as hepatocellular when (1) there is an increase of greater
than two times the ULN for ALT alone or (2) there is
a ratio of serum ALT activity/serum Alk P activity
greater than five or cholestatic when there is an increase of greater than two times the ULN of Alk P
alone or a ratio equal or less than two. Acute liver
injury is distinguished from chronic liver injury when
the increases have lasted less or more than 3 months,
respectively. Severe liver injury and fulminant liver
injury are defined by (1) the presence of jaundice, prothrombin õ50% (or equivalent), and hepatic encephalopathy and (2) the rapid development of hepatic encephalopathy and severe coagulation disorders, respectively.
6. SERUM ENZYME ELEVATIONS DUE TO HEPATIC
ENZYME INDUCTION
Enzyme induction is one reported iatrogenic effect
leading to elevated hepatic serum enzyme levels in patient populations that are not directly indicative of hepatic injury. This has been well documented, especially
for anticonvulsants, but also for some other drugs. In
one such study, 56 children of 63 receiving phenobarbital and/or diphenylhydantoin had elevated GGT; 6 had
elevated ALT and AST for more than 20 weeks in the
absence of specific histopathology (Aiges et al., 1980).
Further evidence of hepatic enzyme induction by anticonvulsants in asymptomatic patients was cited by
Wall et al. (1992) in a study of 206 adults and children.
Of these, serum GGT was elevated in 74.6%, Alk P in
29.7%, and ALT in 25.2%. Of 242 patients administered
antiepileptic drugs, 40 exhibited high levels of serum
GGT and nearly all cases indicated hepatic microsomal
enzyme induction as measured by antipyrine half-life,
leading Hirayanagi et al. (1991) to conclude that, in
these patients, elevated serum GGT did not necessarily
indicate hepatocellular damage. Elevated circulating
levels of GGT are commonly observed in patients taking cytochrome P450 enzyme-inducing drugs such as
rifamicin (Davis, 1989). Similar studies with co-antiep-
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ileptic drug therapy (Haidukewych and John, 1986) indicated ALT elevations up to three times and AST elevations up to two times the upper limit of normal in
more than one-quarter of the patient population. These
were not considered clinically significant but instead
were attributed to enzyme induction. Liver biopsies in
similar patients undergoing long-term anticonvulsant
therapy showed no signs of chronic liver damage (Jacobsen et al., 1976). Both ethanol and glucocorticoids
have been shown to induce GGT activity in rat liver
(Nishimura et al., 1981; Billon et al., 1980). In studies
with both man and rat, the primary cause for increased
GGT associated with prolonged alcohol exposure has
been attributed to hepatic enzyme induction rather
than liver cell injury (Teschke et al., 1983), although
it is clear that ethanol is hepatotoxic in general. In
developing rats, cortisol enhances hepatic activities of
ALT (Herzfeld and Raper, 1979).
7. TYPES OF DRUG HEPATOTOXICTY
7.1. Intrinsic Hepatotoxicity
One of two major categories of chemicals that can
produce dose-dependent, hepatic injury, either directly
or indirectly via a metabolite, includes the intrinsic or
‘‘direct’’ (type A) hepatotoxicants. These responses often can be anticipated from the known pharmacology
of the drug, are generally detectable in animal models,
and occur more frequently or with greater severity
when exposure is increased, i.e., with increasing dose
levels or duration of dosing. Salicylates (Lewis, 1984)
and acetaminophen (Fry and Seeff, 1995) are examples
of analgesics that produce intrinsic liver toxicity. At
high blood levels, aspirin can produce hepatocellular
injury, confirmed by liver biopsy studies (focal necrosis), with 10- to 40-fold elevations for serum transaminases noted (Zimmerman, 1978). Chemotherapeutic
agents often possess predictable, dose-dependent hepatotoxicity (King and Perry, 1995), even though they do
not intentionally target slowly dividing hepatocytes.
Tetracycline, methotrexate, mercaptopurine (Finlayson, 1973), and possibly suldinac (Boelsterli et al.,
1995) are other examples of direct hepatotoxins. Intrinsic hepatotoxins produce injury in a large percentage
of exposed individuals after a short fixed latent period
either by direct, nonselective physicochemical distortion or disruption of hepatocytes or by indirect interference with specific metabolic processes leading to structural damage (Lewis, 1984). This type of toxicity can
be alleviated by dose reduction in patient populations.
7.2. Idiosyncratic Hepatotoxicity
Although a number of drugs, as for example some
antineoplastic agents (McDonald and Tirumali, 1984),
exhibit intrinsic toxicity in man or animals, most hepatotoxic drug reactions in humans are considered to be
idiosyncratic (King and Perry, 1995), i.e., due to un-
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usual susceptibility of an individual. Occurring at therapeutic doses after a variable latent period, these responses are characterized by an incidence of hepatic
injury that is very low in frequency within a population, dose-independent, and not reproducible in experimental animals (Zimmerman, 1978). Due to their low
incidence, idiosyncratic responses are generally not detected until late in the drug development process after
a large number of patients have been treated. Although
rare, serious adverse liver responses may include fulminant hepatitis and cholestasis. These account for
many drug-induced deaths worldwide. Drugs withdrawn from the market due to idiosyncratic drug reactions, including or due solely to hepatotoxicity, include
benoxaprofen, ibufenac, temafloxacin, tenilic acid (Park
et al., 1992), nomifensine, and perhexilene (Breckenridge, 1996).
Etiologically, these reactions are of two major types:
(1) aberrant metabolism-based responses leading to the
accumulation of toxic metabolites in susceptible individuals and (2) hypersensitivity or immune-based toxicity. Other mechanisms may include abnormal receptor sensitivity, latent biochemical abnormalities, or
multifactorial causes (Park et al., 1992). Almost all hepatotoxic reactions to antibacterials (George and Crawford, 1996) or NSAIDs (Boelsterli et al., 1995), especially of the indole, pyrazole, and propionic acid classes
(Lewis, 1984), are idiosyncratic. Mechanisms may be
primarily metabolite-dependent (isoniazid, diclofenac),
hypersensitivity-mediated (beta-lactams, sulindac), or
both (sulfonamides, erythromycin derivatives) (Westphal et al., 1994; Miyamoto et al., 1997; Boelsterli et
al., 1995). Some notable histamine (H2)-receptor antagonists and a number of antidepressants are associated
with hepatic idiosyncratic reactions, presumably mediated via chemically reactive metabolites (Black, 1987;
Pirmohamed et al., 1992).
7.2.1. Metabolism-based toxicity. Interindividual
variations of drug effects are most frequently explained
by genetic variation or polymorphism of drug metabolism. Based on immunogenicity and catalytic activities,
approximately 20 human cytochrome P450 isozymes
have been identified (Larrey and Pageaux, 1997) comprising at least 10 distinct P450 gene families (Watkins, 1990). Of these, cytochromes CYP1A2, CYP2C9,
CYP2C19, CYP2E1, and, in particular, CYP2D6 and
CYP3A are especially important for the hepatic metabolism of drugs (Brockmoller and Roots, 1994). While
bioactivation of a drug to a toxic metabolite may represent õ1% of the overall metabolism of that drug (Pirmohamed et al., 1996), specific reactive metabolites
may directly lead to hepatic injury or may act via the
immune system. Genetic deficiencies in hepatic cytochrome CYP2D6, CYP2C19, N-acetyltransferase 2
(NAT2), and glutathione synthetase have been cited
to explain individual variations in drug hepatotoxicity
(Larrey and Pageaux, 1997; Meyer, 1996). Seven muta-
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tions of the NAT2 gene are known to define numerous
alleles associated with decreased function, and two mutant alleles of CYP2C19 have been described in poor
metabolizers (Meyer and Zanger, 1997; Goldstein and
de Morias, 1994). Oxidative polymorphism for cytochrome CYP2D6, which metabolizes drugs typically
having a positively charged nitrogen atom (Pirmohamed et al., 1996), includes poor, intermediate, and
ultrarapid metabolizers (Meyer and Zanger, 1997)
leading to a wide range of interindividual variability
in CYP2D6-based response in a healthy population
(Spina et al., 1994). For example, patients receiving the
antianginal agent perhexiline maleate who are deficient in CYP2D6 can develop hepatotoxicity due to perhexiline accumulation (Morgan et al., 1984). Although
it is not subject to genetic polymorphism (Ereshefsky,
1996), both genetic and nongenetic factors lead to
marked (5- to 20-fold) individual variability in metabolic clearance via CYP3A (Wilkinson, 1996) which
comprises 25% of the total hepatic cytochrome system.
This leads to wide variations in individual exposure
levels to drugs such as cyclosporine (Watkins, 1990).
Polymorphism has also been described for CYP2C9 and
CYP2E1 (Meyer and Zanger, 1997). The latter is important for the metabolism of general anesthetics such
as halothane which causes hepatitis in 1 in 35,000 patients on first exposure and in 1 in 3700 patients upon
multiple exposures (Pirmohamed et al., 1996).
7.2.2. Immune system-based toxicity. Immunebased injuries represent the other major mechanism
for idiosyncratic responses in humans. Autoimmunity
triggered by xenobiotics involves the modification of
host tissues or immune cells by the chemical so that
self-antigens are erroneously targeted; drug hypersensitivity or allergy refers to a situation where the immune system responds in an exaggerated or inappropriate manner (Burns et al., 1996) by one of four mechanisms (Combs and Gell, 1975). Most autoimmune
diseases are associated with specific alleles of the major
histocompatibility complex (MHC) class II genes (Fronek et al., 1991). The high level of polymorphism that
is characteristic of the MHC genes may lead to subpopulations of individuals who are highly immune to responses toward drug-related antigens (Park et al.,
1992). Four major categories of drugs known to cause
autoimmune responses include hydrazines, derivatives
of aromatic amines, sulfhydryl-containing compounds,
and compounds with a phenol ring (Adams and Hess,
1991). Specific examples of drugs known or suspected
of causing hepatitis via an autoimmune mechanism
include methyldopa, oxypehnisatin, isoniazid, nitrofurantoin, clometacin, fenofibrate, papaverine, and tienilic acid (Bigazzi, 1988). Although other nongenetic
factors may be involved in the etiology of altered immune responses, the role of drug metabolism polymorphism leading to bioactivation is an important one. Extrahepatic metabolism of some drugs associated with
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a generalized type of idiosyncratic drug reaction mediated through the immune system may also involve the
liver (Uetrecht, 1988).
7.3. Mixed or Species-Relevant Toxicity
Some drugs appear to cause idiosyncratic hepatic injury in humans and yet can also produce hepatotoxicity, perhaps by a different mechanism, in animal models at high doses. Thus, the same drug can be an intrinsic hepatotoxicant in one or more animal models and
yet cause an idiosyncratic liver response in some humans. Some of the angiotensin-converting enzyme inhibitors used for the treatment of hypertension and
congestive heart disease fall into this category. Captopril, for example, has been associated with the rare
incidence of hepatotoxicity in humans (Rahmat et al.,
1985; Tabibian et al., 1987). In mice, acute high doses
of captopril cause moderate increases in ALT, decreases in hepatic GSH, and histological evidence of
hepatic necrosis with 24 h (Helliwell et al., 1985). Enalapril maleate which has been reported to cause a rare
but potentially serious hepatotoxicity in humans, was
demonstrated by Jurima-Romet and Huang (1992) to
produce centrilobular necrosis and significant moderate increases in ALT and AST 24 h after acute exposure
in Fisher 344 rats.
8. THERAPEUTIC CLASS AND DRUG-INDUCED
HEPATIC INJURY
The frequencies of drug-induced hepatitis at therapeutic doses vary among drug classes with some classes
more consistently demonstrating low levels of hepatic
injury than others. These can vary from 1/100 for a few
compounds, to 1/10,000 for some intermediate compounds, to 1/100,000 for others (Larrey and Pageaux,
1997). For one antitumor antibiotic, elevations of aminotransferases can occur in up to 100% of patients
(King and Perry, 1995). In one large study (Sameshima
et al., 1984) based on 8156 case histories of drug-induced hepatic injury over a 70-year period in Japan,
the greatest number of cases was due to antibiotics
(34%), followed by CNS drugs (15%), chemotherapeutic
drugs (14%), cardiovascular drugs (11%), carcinostatic
drugs (7%), hormones and hormone antagonists (6%),
diagnostic aids (4%), and other drugs (9%). A smaller
study based on 46 patients representing 20% of patients admitted for acute or chronic hepatitis during
a 3-year period cited anti-inflammatories, antibiotics,
antihypertensives, and diuretic drugs as being most
frequently implicated (Grippon et al., 1985). In the U.S.
Physicians Desk Reference, transaminase elevations,
albeit usually minor and infrequent, are not uncommon
for drugs classified as antineoplastic compounds, neurotrophic drugs, and antibiotics/antibacterials. Table 1
summarizes available information for some of these
drug categories.
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SERUM TRANSAMINASES AND HEPATIC INJURY
TABLE 1
The Relative Incidence of Liver Injury and Elevated Transaminase Levels for Various Drug Classes
Class
Hepatic effects
Nonsteroidal
anti-inflammatory
drugs
Incidence of acute liver injury is about 3.7
per 100,000 NSAID users. Transaminase
levels abnormal in 5–15% of patients
taking a particular drug.
Hepatotoxic manifestations include
venoclusive disease, hepatocellular
necrosis, and fatty change. Incidence of
1.3% in 980 cases of hepatic injury in one
study.
Estimated frequency of hepatotoxic
reactions is between 1 and 10 per 100,000
drug prescriptions. Antibiotics accounted
for one-third of all suspected druginduced hepatic injury in one study. Mild
transient transminase elevations reported
in up to 30–50% of recipients for some
antibiotics.
In one study, cardiovascular agents were
implicated in 27.3% of 980 cases of liver
injury. Diuretics accounted for 13.9% in
another study. Mild ALT/AST elevations
are common at the onset of treatment.
Anticancer drugs
Antibacterials
Cardiovascular
9. DRUGS FREQUENTLY ASSOCIATED
WITH HEPATOTOXICITY
Some marketed drugs have been associated with a
high, predictable incidence of suspected hepatotoxicity
in human populations, as, for example, they may cause
asymptomatic, transient elevations in serum transaminase levels in ¢10% of the patient population. It should
be noted that the actual frequency of isolated, asymptomatic elevations of transaminases in patient populations may actually be understated because liver function tests are not routinely ordered during routine clinical practice (Jick, 1995). Some examples are shown in
Table 2. Likewise, drug interaction, disease complications, viral infections, or combinations of drugs may be
responsible, in part, for some of these effects.
10. DISCUSSION
Drug-induced liver toxicity can be detected at various
stages of drug development depending upon the mechanism responsible for hepatic injury. Intrinsic hepatotoxicants are effectively identified in the preclinical
testing stage using animal models at doses much
higher than the intended human dose while, at the
other extreme, rare but sometimes fatal idiosyncratic
responses can only be discovered after the exposure of
thousands of patients to therapeutic levels of the drug.
Elevated serum transaminases, especially ALT, are the
single most important laboratory indicators of hepatic
effects from early preclinical testing to the postmarket
monitoring of patients responding to a variety of histologic effects. Baseline serum transaminase levels in hu-
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Mechanism of injury
References
Mostly idiosyncratic responses.
Garcia Rodriguez et al. (1994),
Boelsterli et al. (1995)
Diverse injuries, both intrinsic
and idiosyncratic.
King and Pery (1995),
McDonald and Tirumali
(1984), Jean-Paston and
Jouaglard (1984)
Injuries include necrosis,
cholestasis, cholangitis, and
other. Mostly idiosyncratic
except intrinsic for
tetracyclines and some
macrolides.
George and Crawford (1996),
Stricker et al. (1992b),
Stricker (1986)
Autoimmune or hypersensitivity
reactions with antiarrhythmic
agents; heptocellular pattern
with antihypertensive agents.
Jean-Pastor and Jouglard
(1984), Stricker et al.
(1992b), Stricker (1986)
man populations are affected by a variety of both genetic and environmental factors. In addition, some serum enzymes of hepatic origin used for assessment of
liver injury or disease can be increased by hepatic enzyme induction instead of or in addition to membrane
leakage caused by hepatic damage. Within these limitations, guidelines for the detection of hepatic injury
via these biochemical markers have been established.
The hepatic metabolism of drugs to reactive toxicants
or protein-binding moieties is the single most important, but by no means the only mechanism leading
to hepatic injury. Unlike preclinical laboratory animals
which are relatively homogenous in terms of their constitutive hepatic cytochrome P450 isozymes, human
populations are remarkably diverse with cytochrome
P450 enzyme polymorphisms and other individual differences in drug-metabolizing enzyme systems being
prevalent. Subtle individual differences in the hepatic
detoxification processes or the formation of minor but
highly reactive metabolites in susceptible individuals
can lead to rare but unpredictable consequences as an
increasing proportion of the population is exposed to a
drug at therapeutic doses. Frequently, these idiosyncratic responses are caused by an aberrant immune
response to the drug or a drug-altered self-antigen. For
both direct, intrinsic toxic responses in humans or laboratory animals and isolated idiosyncratic responses in
a clinical setting, early evidence of hepatic injury is
obtained via biochemical methods including the monitoring of serum transaminases.
While knowledge of the typical human metabolism
of experimental drugs will not predict idiosyncratic hepatic toxicity, it can be useful in determining similari-
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DAVID E. AMACHER
TABLE 2
Drugs Frequently Associated with Elevated Transaminase Levels
Description of
transminase response
Drug name
Amiodarone
(antiarrhythmic)
Approximately 25% of users
develop transminase
elevations ú2 times normal
during treatment.
Mild liver enzyme elevations
in 10–42% of recipients;
serum transaminases
typically elevated first week
of treatment.
Elevated transaminases (ú3
times upper limit of normal)
in 25% of patients.
Chlorpromazine
(anticonvulsant)
Cognex (tacrine–HCl)
cholinesterase
inhibitor (cognition
activator)
Ibufenac
(nonsteroidal antiinflammatory)
Iproniazid
(antidepressant)
Elevated transaminase levels
in ú30%; hepatocellular
jaundice in 5% of
individuals.
Abnormal transaminases in up
to 20% of recipients.
Isoniazid
(antibacterial)
Serum transaminases elevated
in 10–20% of patients.
a-Methyldopa
(antihypertensive)
Transient increases in
transaminases, Alk P,
bilirubin in 2.8% of patients.
Peak ALT levels 3–19 times
baseline values in some
patients after 3–8 weeks of
treatment.
ALT and/or ALT elevated ú3
times upper limit of normal
in 15% of patients.
Transient increases in
transaminases, Alk P, and
bilirubin in 2.8% of patients.
Peak ALT levels 3–19 times
baseline values in some
patients after 3–8 weeks of
treatment.
ALT and/or ALT elevated ú3
times upper limit of normal
in 15% of patients.
Elevated in 56% of patients.
Naltrexone–HCl
(Revia) (opioid
antagonist)
Precose (acarbose)
a-glucosidase
inhibitor (diabetes)
a-Methyldopa
(antihypertensive)
Naltrexone–HCl
(Revia) (opioid
antagonist)
Precose (acarbose)
a-glucosidase
inhibitor (diabetes)
Proleukin
(aldesleukin;
interleukin-2
product)
(anticancer)
Riluzole (Rilutek)
(amyotrophic
lateral sclerosis
treatment)
Tienilic acid
(uricosuric diuretic)
AST and ALT elevated.
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Frequent, minor, dose-related
elevations of ALT, AST, and
LDH reported.
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Mechanisms
Reference
Phospholipidosis.
Metabolic idiosyncratic.
Lewis et al. (1989),
Sticker et al. (1992)
Cholestatic or mixed.
Reactive metabolite
responsible,
imunoallergy.
Davis (1989), Pessayre
(1992), De Mol et al.
(1984), Stricker et al.
(1992b)
Consistent with
hepatocellular injury.
Mechanism of
hepatotoxicity is
unclear.
Watkins et al. (1994)
Hepatocellular necrosis.
Removed from clinical
use in 1967.
Intrinsic toxicity.
Boelsterli et al. (1995),
Fry and Seeff (1995)
Hepatocellular necrosis.
Withdrawn from most
markets.
Liver injury reported in
0.5% of adults; higher
in children
Reactive metabolite
Sticker et al. (1992b)
Reactive metabolite
responsible for
metabolic
idiosyncratic
response.
Reactive metabolite
responsible,
hypersensitivity.
Direct hepatotoxin.
Davis (1989), Timbrell
et al. (1980), Stricker
(1986), Stricker et al.
(1992b), PDR
Davis (1989), Finlayson
(1973), Dybing et al.
(1976)
PDR
Not reported.
PDR
Reactive metabolite
responsible,
hypersensitivity.
Direct hepatotoxin.
Davis (1989), Finlayson
(1973), Dybing et al.
(1976)
PDR
Not reported.
PDR
Not reported.
PDR
Dose-related (intrinsic).
Bryson et al. (1996)
Reactive metabolite
responsible,
immunoallergy.
Dansette et al. (1991),
Stricker (1986)
Reactive metabolite.
Koch-Weser and Browne
(1980), Pessayre (1992),
Porubek et al. (1984),
PDR
Acute hepatitis in about
6% of patients within
the first few weeks.
No cases of hepatic
failure reported.
Hepatic abnormalities
improved or resolved
upon discontinuation.
Acute hepatitis in about
6% of patients within
the first few weeks.
No cases of hepatic
failure reported.
Hepatic abnormalities
improved or resolved
upon discontinuation.
Elevated bilirubin,
alkaline phosphatase,
jaundice.
Elevated ALT in 10% and AST
in 14% of patients at 100
mg/kg.
Valproic acid
(anticonvulsant)
AID
Hepatic histological
responses
1 in 10,000 patients
develop hepatitis of
the immunoallergic
type. Withdrawn from
the market.
Microvesicular steatosis
06-19-98 12:50:03
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SERUM TRANSAMINASES AND HEPATIC INJURY
ties between species that may lead to intrinsic toxicity
or to explain tolerance or sensitivity to the hepatic toxicity of a compound in one species versus another. Current in vitro laboratory procedures utilize isolated microsomal preparations or specific recombinant human
cytochrome products in conjunction with chromatographic procedures for the detection and identification
of drug metabolites. In vitro methods have been used
to predict the in vivo drug metabolism of verapamil,
loxidine, diazepam, lidocaine, phenacetin, and some
other compounds in the human liver (Iwatsubo et al.,
1997). In a similar manner, in vitro comparisons of
drug toxicity in metabolizing rat, dog, monkey, and,
where possible, human hepatocyte cultures can be used
for the preclinical assessment of drug hepatic toxicity
using the same clinical chemistry end points, namely
the transaminases, that are used in vivo. Primary hepatocytes cultured on an extracellular collagen matrix
in time form bile canaliculi (LeCluyse et al., 1994) providing the opportunity to study the effects of drugs
on the hepatobiliary system in vitro. Examples of the
successful use of hepatocyte cultures from humans and
other species to study hepatic effects include the metabolism of adinazolam, the cytotoxicity of trospectomycin, and the human-specific hepatotoxicity of panadiplon (Ulrich et al., 1995).
Recent developments that may have application in
preclinical testing for potential liver toxicity include
procedures that have evolved in conjunction with combinatorial chemistry and pharmacological screening
methods (see Lam, 1997). Screening methods of particular interest include genomic and proteonomic assays
that are sensitive and plenary by nature, although they
may be of limited value in revealing specific toxicities.
These methods depend heavily upon automation, the
availability of specific probes or libraries for pattern
recognition, and advanced computer systems. The ultimate advantage of these techniques would be the detection of early cellular events, as, for example, markers
of oxidative damage, at therapeutic dose levels that
may predispose the organism to frank toxicity with sustained exposure. Examples of gene expression in liver
after toxic injury include heat shock and oxidative
stress-inducible genes (Schiaffonati and Tiberio, 1997).
Both induction and suppression of gene expression can
occur with some gene families (Runge-Morris, 1997).
Reactive intermediates of xenobiotics often bind covalently to proteins as conjugates or adducts leading to
either direct toxicity or immune-mediated toxicity
(Pumford et al., 1997). In addition to the modification
of cellular proteins, xenobiotics may modify protein expression (Richardson et al., 1994). Two-dimensional gel
electrophoresis techniques have been described that
allow the resolution and quantitation of hundreds of
liver proteins that may be altered by the toxic effects
of xenobiotics (Anderson et al., 1996). If a clear association between either altered gene expression or specific
changes in protein structure and abundance can be
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established with specific liver injury, these techniques
may be useful in detecting hepatotoxic potential preclinically, even at therapeutic doses.
Idiosyncratic responses remain difficult to assess at
a preclinical or early clinical level. Using separate cultures from several donors, it may be possible to expose
human hepatocytes to higher concentrations of a drug
than would be found in clinical practice in order to
forcibly detect idiosyncratic responses due to minor reactive metabolites that do not involve the immune system. Because most compounds that have been tested
do cause observable changes in liver gene expression
(Anderson et al., 1996) and idiosyncratic hepatotoxins
such as diclofenac, halothane, tienilic acid, and valproic
among others are known to form protein adducts (Pumford and Halmes, 1997), protein analysis by two-dimensional gel electrophoresis may provide a useful preclinical screening too. Future developments in assays may
include determinations of drug effects on transgenic
animals expressing human MHC class I and II and
associated genes (Campbell and Milner, 1993), utilization of the sensitive reporter antigen/popliteal lymph
node assay (Albers et al., 1997), the comparative testing
of a compound at high doses or concentrations in
transgenic animals or cell lines expressing various constitutive human cytochrome P450s, or the testing of
preclinical candidates in rodent strains genetically diverse in terms of the hepatic cytochrome P-450 system.
11. CONCLUSIONS
• The incidence of drug-induced hepatic injury varies among the different drug classes as well as within
a drug class. Intrinsic responses are generally detected
early in the discovery and development process, while
host-dependent, idiosyncratic responses are usually
not detectable during the early phases of development
due to their rarity.
• Of the various clinical laboratory markers for hepatic injury, serum transaminases, especially ALT, are
the most universally important indicators for studies
ranging from early preclinical animal testing to postmarketing patient monitoring. Serum ALT is elevated
in response to several categories of hepatic lesions in
multiple species. However, slight transaminase elevations following drug exposure in humans must be interpreted with care due to the many extrinsic factors
known to elevate transaminases.
• The hepatic metabolism of drugs to reactive toxicants or protein-binding moieties is a frequently encountered mechanism leading to hepatic injury. Subtle
individual differences in the hepatic detoxification processes or the formation of minor but highly reactive
metabolites in susceptible individuals can lead to rare
but unpredictable consequences as an increasing proportion of the population is exposed to a drug at therapeutic doses. Improvements in the metabolic profiling
of drugs using in vitro microsomal systems expressing
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DAVID E. AMACHER
multiple human cytochromes during the preclinical
testing phase may enhance our ability to anticipate
both intrinsic and idiosyncratic toxic responses.
• Although the monitoring of serum transaminases
will continue to be an important clinical laboratory procedure for the assessment of preclinical hepatotoxicity,
new developments involving comparative toxicity
assays in human and animal hepatocyte cultures, techniques to detect altered gene expression or changes in
protein structure and abundance, or improved methods
for the detection of immunogenicity will augment the
safety assessment process.
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