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In recent years, the number of anti-inflammatory and analgesic drugs in use has increased. Toxic effects are occasionally
reported, but only after a drug has been used for a significant period of time. Whether they are administered at a therapeutic
dose or at higher levels, anti-inflammatory drugs and analgesics can produce various clinical, biochemical and structural
changes. Frequently observed and extensively studied are the effects of nonsteroidal anti-inflammatory drugs (NSAIDs)
and other analgesics on the liver.
Clinicians sometimes forget that reactions to drugs and xenobiotics can cause a whole range of liver diseases; the final
diagnosis of liver disease should be based on clinical drug history, and laboratory and morphological findings. The
importance of etiological diagnosis cannot be over-emphasized: withdrawal, over time, of the toxic drug can be followed by
improvement or resolution of the liver damage.
Electron microscopy is an important step in detecting pharmacological and toxicological effects in human liver biopsies.
According to Phillips et al, electron microscopy
is "of equal or greater value than light microscopy" in the evaluation of
patients with known or suspected drug- or toxin-induced liver
damage[1]. Although light microscopy is certainly important in
the diagnosis of hepatotoxicity, a number of subcellular alterations can only be identified by electron microscopy. The
present review is focused on the contribution of electron microscopy in studies of the hepatotoxicity induced by a number
of frequently used analgesic and anti-inflammatory drugs.
firms the localization of changes according to this model; that is, cells of zone 1 are the first to receive blood and nutrients,
and usually the last to die and the first to regenerate. Alternatively, more distant cells may receive blood with less oxygen and
hepatotoxicity may be related to the gradient of blood supply and presence of the enzyme system responsible for the
conversion of the drug into a toxic metabolite. For example, in overdose, acetaminophen
becomes an intrinsic hepatotoxin that can cause perivenular necrosis. Thus, depending on the mechanism of toxicity and the drug involved, the acinar unit concept
may explain the variability of findings not only in different liver specimens, but also within a single section of the same
specimen.
Acute toxic cell injury, manifested by acute cell swelling and disintegration of
organelles[3,4], is now rarely the result of
medication toxicity and therefore is beyond the scope of this review. In less aggressive injury, drugs are metabolized in the
liver by various reactions, including oxidation, reduction, hydrolysis and conjugation, and thus fat-soluble compounds are
converted to hydrophilic ones, facilitating excretion into the urine. Hepatocytes are endowed with protective mechanisms
against toxicity: (1) the oxidizing enzymes of the smooth endoplasmic reticulum (SER); (2) the glutathione
oxidation_reduction system; (3) the peroxisomal
H2O2 system; and (4) various cytosolic
enzymes[5]. Only the organelles of hepatocytes that
are primarily affected by drug damage will be mentioned here.
The endoplasmic reticulum is consistently altered during toxicity. In the early stages of toxicity,
proliferation/reduplication of the SER can be observed. With ongoing toxicity, the SER comes to occupy most of the cell, both by proliferation and
dilatation of its cisternae. Hepatic clearance of many toxins requires enhanced synthesis of the enzyme UDP-glucuronyl
transferase by the SER. Pompella and Comporti, using confocal laser scanning fluorescence microscopy plus image video
analysis, show that the perinuclear SER is the first to be involved in oxidative stress and lipid
peroxidation[6]. The rough endoplasmic
reticulum (RER), the major site of protein synthesis, is also the primary site of damage by hepatotoxins. Electron
microscopic examination frequently reveals the detachment of ribosomes ("degranula-tion" of RER), which is considered to
limit protein synthesis.
Mitochondria are extremely sensitive to drugs, both at normal dosages and especially in overdose. Ultrastructural
changes have been classified as "reversible" and
"irreversible"[4]. Anoxia has been implicated in mitochondrial changes,
which explains the more severe findings in organelles located in acinar zone 3, away from the better oxygenated zone 1
(periportal). Mitochondrial components such as superoxide
dismutase[7] and NADP-dependent
dehydrogenase[8] have been localized by electron microscopy and can be used to analyze toxic injury. Mitochondria can be selectively injured by toxins
that interfere with oxidative phosphorylation or electron transport. Mitochondrial function is inhibited by certain drugs
through their effect on beta-oxidation energy production. In this way, nicotinamide adenine dinucleotide and flavin adenine
dinucleotide synthesis is inhibited, resulting in decreased ATP production. The elevation of the serum transaminase[alanine
transaminase (ALT) and aspartate transaminase (AST)] found in drug-induced liver diseases (DILD) has been linked to the
association of this enzyme with the outer parts of the inner mitochondrial
membrane[9].
Intramitochondrial dense granules (also named matrix granules or native granules) are osmiophilic and usually measure
20_50 nm in diameter. They are considered to have a role in mitochondrial calcium metabolism, and it is important to note that
their frequency can be altered by drugs. Manov
et al have shown that they disappear from cultured hepatoma-derived cells
after cells are exposed to high doses of
acetaminophen[10]. In their early description of the mechanism of hepatocyte injury
and cell death, Desmet and Vos describe the disappearance of matrix granules as an early, reversible phenomenon (stage
1a)[4].
Flocculent or "wooly" densities in mitochondria are completely different from matrical granules: they appear later in the
injury cascade, and are the most reliable early
manifestation of irreversible injury, cell death and ensuing
necrosis[11]. Ischemia/anoxia and a multitude of drugs and heavy metals, in addition to immune
cytolysis[12] can generate the wooly densities.
However, most frequently they are found in specimens undergoing delayed fixation or those that are retrieved post-mortem
or from comatose patients or experimental animals.
Close association of mitochondria and endoplasmic
reticulum[13] is seen in normal cells, but can be extensive under certain
conditions. Likewise, drugs and hormones that alter mitochondrial metabolism have a deleterious effect on the
RER[14]. (More specific mitochondrial changes, related to drugs, are listed in the following section and in Table 1).
There is little information concerning the effects of anti-inflammatory drugs and analgesics on peroxisomes. These
organelles contain catalases, oxidases and carrier
proteins[15]. Recently it has been shown that pretreatment with peroxisome
proliferators protect mice against acute acetaminophen toxicity, but not other
hepatotoxins[16]. Except for hyperplasia of
hepatocytes, and an increase in peroxisomes and endoplasmic reticulum, peroxisome proliferators do not produce specific
ultrastructural changes.
Golgi complexes of hepatocytes are frequently affected by toxins, thus producing widespread effects throughout the
cells. Poisoning of the Golgi complex of rat liver cells with tris (hydroxymethyl)-amino-methane causes vesiculation of Golgi
stacks on the concave side and swelling of lysosomes and secretory
vesicles[17]. Major toxic changes can be observed in the
nuclei and plasma membranes of hepatocytes. In liver biopsies, nuclei of individual cells can show evidence of apoptosis
(heterochromatin margination clumping and nuclear fragmentation). Apoptosis may be initiated through the stimulation of
death receptors located on the cell surface or through an intrinsic pathway involving the release of apoptotic signals from
mitochondria[18]. Both signals converge on a cascade of cysteine proteases known as caspases, which are central to the
initiation and execution of apoptosis. In cultured cells, nuclear changes are more conspicuous and are associated with other
changes (see later in this review). The
plasmamembranes of hepatocytes may show interruption and disintegration in the
necrotic foci. Covalent binding of the drug to intracellular proteins can cause a decrease in ATP levels, leading to actin
disruption. Disassembly of actin fibrils at the surface of the hepatocyte causes blebs and rupture of the
membrane[10,19]. Kupffer cells and sinusoidal lining cells may harbor prominent phagolysosomes, indicating uptake of remnants of dead cells.
There is no drug-specificity in this finding, which is prominent in chronic, rather than acute toxicities. Electron microscopy
can be used to identify stellate cells (Ito cells) in various stages of their transformation into fibroblasts, indicating potential
fibrosis. Lysosomes in liver cells play an important role in drug metabolism and elimination of drugs. Typical lysosomotropic
agents include gold-salts, methotrexate, and also acetaminophen and its metabolites. A number of experimental studies of
drug-induced lysosomal disorders of the liver in man and laboratory animals were reviewed by Schneider
et al. and show that many hepatotoxic drugs are in fact lysosomotropic
agents[20].
Mechanisms of drug-induced liver diseases
Drugs causing DILD can be classified in various ways on the basis of the mechanisms by which they act. The classical
divisions are: (1) drugs directly affecting the liver; and (2) drugs that mediate an immune response. The first group includes
drugs that cause "predictable intrinsic" reactions; these are immediate, dose-related and reproducible in animals (eg
acetaminophen). The second group, comprising drugs that cause "idiosyncratic" reactions, act through hypersensitivity
(immunoallergy) and metabolic-idiosyncratic reactions [eg phenytoin, isoniazid (INH)]. These latter reactions are not
predictable, are dose-independent and cannot be reproduced in experimental animals. Moreover,
they appear after a period of latency lasting weeks to
years[21]. Anti-inflammatory/analgesic drugs can be classified as follows:(1)intrinsic: aspirin,
acetaminophen, phenylbutazone; (2) immunoallergic idiosyncratic: ibuprofen, sulindac, phenylbutazone, piroxicam, diclofenac;
and (3) metabolic idiosyncratic: benoxaprofen, diclofenac, indomethacin, naproxen. It has been argued that these
classifications are not valid because some drugs may exhibit characteristics of both major groups: they may show initially immediate,
dose-related toxicity, only to produce, with ongoing use at lower dosages, signs of immune reaction (acetaminophen is
suspected to act in this way).
For drugs in which hepatotoxicity is considered to be due to idiosyncratic reactions, abnormal immune mechanisms have
been implicated. Covalent binding of a drug to the P-450 enzyme acts as an immunogen, activating cytolytic T cells and
cytokines and stimulating a multifaceted immune response. Recently it has been suggested that drug hepatotoxicity could
be classified as either hepatocellular or cholestatic
toxicity[22]. Drugs that affect transport proteins at the canalicular
membrane can interrupt bile flow. Loss of villous processes and interruption of transport pumps, such as multidrug
resistance-associated protein 3, prevent the excretion of bilirubin, causing cholestasis. Moreover, toxic metabolites excreted in bile may
cause injury to the bile duct epithelium.
Hepatotoxicity of specific drugs
(Gluco)corticosteroids The classical effect of prolonged corticosteroid therapy, both in children and adults, is
steatosis[1,23]. Lipid droplets can be visualized with an optical microscope provided an adequate histological technique is used.
Preliminary processing for electron microscopy, in which semi-thin sections of epoxy-embedded tissue are produced, is ideal
for visualizing fat droplets stained with toluidine blue 1% solution: the fat droplets appear yellow or green against the blue
coloration of the slides. In contrast, carbohydrate-containing compounds (ie, glycogen and mucopolysaccharides) exhibit
metachromasia with red or pink coloring. Electron microscopy unmistakably identifies the multiparticulate
a-glycogen molecules in hepatocytes thanks to their "flower" appearance. This is in contrast to the
b-glycogen monoparticulate particles present in other cell types.
A rarely recognized side-effect of glucocorticosteroid therapy is liver enlargement. This has been noticed in children
treated with large doses of prednisone for a variety of conditions [rheumatic fever, rheumatoid arthritis, bronchial asthma,
nephritic syndrome, systemic lupus erythematosus (SLE), idiopathic thrombocytopenic purpura, Stevens-Johnson syndrome,
aplastic anemia, hemolytic anemia, infantile spasms, and giant cavernous
hemangioma][24]. During our investigation of
prednisone-treated children, we found hepatomegaly in 7 of 122 patients treated with 2
mg/kg and 12 of 18 treated with 4 mg/kg prednisone. With the cessation of steroid therapy, liver size decreased within
7_14 d. One micrometer-thick sections stained with toluidine blue showed metachromasia of hepatocyte cytoplasm and a few
large lipid droplets in these cases. Electron microscopy revealed that the hepatocyte cytoplasm was filled with glycogen,
which displaced the mitochondria, RER and other organelles towards the plasma membrane or around the nucleus. The
appearance of the hepatocytes was similar to that observed in some glycogen storage diseases or untreated diabetes
mellitus.
Corticosteroids are also termed glucocorticosteroids because of their distinct effects on carbohydrate metabolism (ie
gluconeogenesis, promotion of liver glycogen deposition, and elevation of blood glucose concentrations). Several
mechanisms are considered to be involved in glucocorticoid-induced glycogen deposition: (1) glucocorticoids activate glycogen
synthetase a through an indirect mechanism involving synthesis of a phosphatase; (2) following steroid-induced hyperglycemia,
there is increased glycogen deposition through activation of phosphorylase a by glucose and subsequent activation of
synthetase a; and (3) glycogen synthetase is activated by insulin, which increases in glucocorticoid-induced
hyperglycemia[24,25].
Other noteworthy ultrastructural effects of corticosteroids are: (1) enlargement of mitochondria; (2) increase in frequency
of lysosomes; (3) decrease in frequency of peroxisomes; (4) increased volume of hepatocytic cytoplasm, but decreased area
of RER and SER following the accumulation of other cytosolic components (mainly fat droplets and
glycogen)[1,24].
Aspirin/acetylsalicylic acid overdose Since 1956, a number of reports have indicated the elevation of serum
transaminases (ALT, AST) in patients treated with
aspirin[26_28]. Elevations were noted especially in patients with a serum salicylate
concentration higher than 25_30 mg/100 mL for usually 10 or more days. Most of the subjects had rheumatoid arthritis,
rheumatic fever, or systemic lupus erythematosus. Light-microscopic observations made by Seaman
et al[29] and Iancu et
al[30], show preserved liver architecture, but also widened sinusoids and hepatic cytoplasmic vacuolization, indicating
degenerative changes. Ultrastructural changes were documented in a single human
case[30]. The patient, an 8-year-old boy with
rheumatic fever, had elevated transaminase
concentrations (AST 220 IU/mL and ALT 240 IU/mL), and a serum salicylate level
of 21 mg/mL. Electron microscopic observation of a liver biopsy specimen revealed hepatocytes with shrunken nuclei, and
dilated nuclear envelopes with clumping of chromatin (apoptosis). The rough endoplasmic reticulum was dilated and
degranulated. The SER had proliferated, and mitochondria were pleomorphic, had increased electron density in the matrix
and widened intracristal spaces (Figure 1). These findings led to the immediate discontinuation of aspirin therapy, followed
by return to normal of the transaminase levels.
Aspirin and Reye syndrome Reye syndrome (RS), which consists of acute liver failure with encephalopathy, has a
potentially fatal outcome (with approximately 40% of affected children dying). The syndrome was frequently diagnosed
during 1970_1980, mainly, but not only, in the USA. Initially, RS was considered to be linked to epidemics of varicella and
influenza B as well as aspirin ingestion. Indeed, a large group of affected children had increased aspirin levels in their serum.
After the use of aspirin diminished, the frequency of RS decreased dramatically. Presently, the rare cases of RS are related to
a wide variety of metabolic diseases presenting with a similar clinical and pathological picture. Among these, disorders of
oxidative phosphorylation, urea cycle defects, defects in fatty acid oxidation metabolism, systemic carnitine deficiency and
acyl-CoA dehydrogenase deficiency, should be
noted[31].
The classical RS, including that related to aspirin ingestion, typically has universal microvesicular hepatocyte steatosis
and major ultrastructural changes in the mitochondria. These organelles show marked dilatation, rarefied granular matrix and
assume an amoeboid shape. Crystolysis, disappearance of dense matrical bodies and occasional intramitochondrial whorls
were also observed. Peroxisomes occasionally appeared with increased frequency, whereas glycogen was depleted. The
mitochondrial alterations were less pronounced in the initial stages and were maximal at the height of the disease, when serum
ammonia and transaminases were elevated (Figure
2). In parallel, encephalopathy deepened. After this stage, which was
reached in 1_2 weeks, some patients recovered spontaneously or with aggressive therapy, and liver function, histological
changes and electron microscopic appearance returned to
normal[32_34].
Acetaminophen Acetaminophen is widely used as an analgesic and antipyretic with very few adverse effects at
therapeutic doses. Hepatotoxicity develops following suicide attempts or after accidental
overdose[35]. It has been reported that even
borderline high acetaminophen concentrations may induce liver toxicity in infants or in chronic
alcoholics[35,36]. The mechanism of liver cell injury produced by acetaminophen overdose remains controversial. Hepatotoxicity is attributed to its
transformation into a highly reactive metabolite,
N-acetyl-p-benzoquinone imine (NAPQI), by microsomal enzymes of the
P450 family[37]. NAPQI is detoxified by conjugation with glutathione (GSH). Once GSH is depleted, NAPQI covalently binds
to proteins, causing alterations in intracellular homeostasis that result in cell
necrosis[37_39]. Recently, it was demonstrated
that increases in acetaminophen-protein adducts in serum can be used as a predictor of the severity of
hepatotoxicity[39]. In addition to liver cell necrosis, acetaminophen can also induce
apopto-sis[40_45].
Acetaminophen: toxicity in patients There are few studies regarding the ultrastructural changes that occur during
acetaminophen overdose. Electron microscopic observations of acetaminophen-induced hepatic toxicity in humans was first
described by Dixon et al[46]. These researchers studied the lesions up to 28 d after acetaminophen overdose, and found
centrilobular necrosis, hydropic vacuolization, and macrophage infiltration followed by regeneration activity with rapid
restoration to normal. In a study of 12 acetaminophen overdoses and 10 other patients with fulminant hepatitis, McCaul
et al described differences in the ultrastructural pathology of these
groups[47]. Light- and electron microscopic changes were
more severe in the fatal non-viral, acetaminophen-induced cases. Acetaminophen overdose elicited prominent changes in
hepatocyte nuclei (corresponding to typical changes of apoptosis). Disarrangement of individual hepatocytes due to
breakdown in plasma membranes was a frequent finding, as was the presence of amorphous deposits in the endoplasmic
reticulum. Depletion of glycogen, mitochondrial swelling and detachment of ribosomes were less prominent in these cases.
Comparing his findings with those of Dixon et
al. in experimental animals[46], McCaul
et al attribute differenences in fine structure to the the timing of death in experimental animals versus
patients[47]. In our opinion, findings at such an advanced stage of necrosis or
necrapoptosis[48] are difficult to interpret because they occurred
when necrosis already involved the centrilobular and midzonal cells and, in some cases manifested as massive coagulative
necrosis. Biopsies obtained at earlier stages of acetaminophen toxicity are similar to the observations
obtained from the liver samples of experimental animals.
Acetaminophen: overdose in experimental
animals
Attempts to reproduce acetaminophen hepatotoxicity in humans have been made using experimental animals, which were
usually given large doses of the drug. Early studies showed initial (3_6 h) depletion of glycogen, loss of ribosomes,
cytoplasmic matrix swelling, and mitochondrial
abnormalities[49]. After that, as observed mainly in centrilobular cells, there was
rapid progression to frank coagulative necrosis. The extensive work of Ray
et al addressed the question of the role of
hepatocyte apoptosis and necrosis in the process of cellular
death[50]. Severe liver injury was produced in ICR mice by
administration of 350_500 mg/kg ip acetaminophen. Laboratory and morphological findings were similar to those of humans
with acute liver failure following acetaminophen overdose. Ultrastructural examination confirmed the presence of typical
apoptotic and necrotic features, similar to the changes found in our later studies with HepG2 and Hep3B cultured hepatoma
cells[10,42]. The observation that apoptosis can precede necrosis is of special interest in view of the recent finding that
N-acetylcysteine cannot arrest the apoptogenic
process[10].
More recently, Ruepp et al detected minor changes at the
electron microscopic level as
early as 15 min after administration of 500 mg/kg
acetaminophen[51]. The changes
started in the centrilobular zone and increased in
severity and distribution over time. Sixty min later, mitochondrial dilatation was visible as vacuolation by light microscopy. The
mitochondria were swollen and fused together. With lower doses (150 mg/kg) the changes
were much less severe. Similar findings, also located in the
centrilobular zones, were reported by Heinloth et
al[52], who exposed rats to 150 mg/kg acetaminophen. No ultrastructural
changes were found in controls or rats exposed to 50 mg/kg
acetaminophen.
Acetaminophen: in vitro electron-microscopic observations
Isolated rat hepatocytes The effects of
acetaminophen on the ultrastructure of isolated rat hepatocytes (IHC) were
studied by Fujimura et al[53]. These authors studied suspensions of IHC treated with 5 or 20 mmol/L acetaminophen. The
cells had surface blebs containing SER, dilatation of the Golgi apparatus, partial degranulation of the RER and enlargement
of the mitochondria. The altered mitochondria had matrix with low electron-density, and with loss of dense matrical granules.
The effect of NAPQI, the putative toxic metabolite of acetaminophen, was also investigated: with 500 µmol/L NAPQI, IHC
showed surface blebs containing various organelles. Disorderly distributions of cytoplasmic organelles, mild dilatation of
RER and SER, and cytoplasmic myeloid bodies were also observed.
Studies with HepG2 and Hep3B cultured cells
We studied the ultrastructure of HepG2 and Hep3B hepatoma-derived
cells exposed to acetaminophen for various time periods and at various
concentrations[10,42]. There were several types of
cells: (1) normal cells, similar to the controls illustrated in Figure 3A and 3B; (2) cells with discrete abnormalities such as
reduction in or absence of microvilli, as well as increased frequency of lipid droplets; (3) cells with apoptotic changes (Figure
3C and 3D); (4) typical necrotic cells, with extensive degeneration of the cytoplasm, vacuolization and disruption of the
plasma membrane; and (5) so-called "secondary necrotic" cells with morphological elements of both apoptosis and necrosis
(Figure 3E).
Variability in the frequency of mitochondrial dense granules in HepG2 cells exposed or not to acetaminophen was also
studied by Manov et al[10]. After acetaminophen treatment, the percentage of mitochondrial dense granule-positive
mitochondria decreased significantly. The disappearance of mitochondrial dense granules has been included among the
reversible changes in acute
hepatotoxicity[4], but their absence has also been observed in other
conditions[13].
Morphological changes associated with apoptosis such as cell shrinkage, chromatin condensation and margination, and
apoptotic bodies were also observed in the liver after high doses of
acetaminophen[40,54]. Quantitative analysis performed by
Ray et al on mouse liver indicated that 40% of cells died by apoptosis 24 h after acetaminophen
administration[50,55]. In
contrast, Gujral et al showed that necrosis, but not apoptosis, is the predominant mechanism of cell death after
acetaminophen overdose in
vivo[54]. Because apoptotic cells
in vivo are rapidly removed from the tissue by phago-cytes, this data
may be an underestimate. According to our in
vitro findings, both apoptosis and necrosis are observed
in HepG2 and Hep3B cells after acetaminophen
treatment[10,42]. To differentiate apoptotic from necrotic cells, we used ultrastructural morphometry
and cell staining with annexin V and propidium iodide. We found that after 24 h exposure to 10 mmol/L acetaminophen, HepG2
cells died predominantly by apoptosis, whereas necrosis was prominent after 48 h. In this late stage of toxicity the apoptotic
process may switch to secondary necrosis and apoptotic cells may become
indistinguishable from necrotic
ones[56]. Additionally, apoptosis in
vitro in the absence of phagocytes may normally
degenerate to secondary
necrosis[57].
In our in vitro investigation we found that
N-acetyl-cysteine, a known antioxidant used in acute acetaminophen poisoning,
does not prevent acetaminophen-induced apoptosis and
necrosis[10,42]. Exposure of Hep3B cells to acetaminophen and
N-
acetylcysteine for 48 h resulted in an increase in apoptotic cells in comparison with cells treated with acetaminophen
alone. In HepG2 cells 5 mmol/L of
N-acetylcysteine markedly increased the degree of necrosis and secondary necrosis in comparison with cells treated with
acetaminophen only. N-Acetylcysteine alone did not induce apoptotic or necrotic changes in HepG2 or Hep3B cells.
An interesting combination of hepatotoxic factors, namely acetaminophen and ethanol, was studied by
Neuman[44]. Electron microscopy of normal human primary hepatocytes treated with 40 mmol/L ethanol plus 10 mmol/L acetaminophen for
24 h demonstrated that the treated hepatocytes had a common specific attribute: their mitochondria were enlarged and
appeared swollen or elongated, with disrupted cristae, and lacked normal organization. The combination of 10 mmol/L
acetaminophen and 40 mmol/L ethanol was synergistic in producing mitochondrial damage after 24 h. Giant mitochondria, 3
µm in length, were observed within cells exposed to this treatment. The endoplasmic reticulum underwent enlargement and
vesiculation, and lipid accumulation was also seen.
The efflux pump P-glycoprotein (P-gp) has been shown to have an important role in intracellular drug concentration. To
evaluate the contribution of the P-gp transporter to the course of acetaminophen-induced toxicity, we compared toxicity to
cells caused by acetaminophen alone, and acetaminophen plus verapamil, a well known inhibitor of P-gp activity. If P-gp is
involved in acetaminophen transport, toxicity would be increased by inhibition of the P-gp pump activity. Among other
parameters, we are presently studying the ultrastructural changes caused by these agents. Preliminary observations show
that after exposure of HepG2 and Hep3B cells to verapamil (100 µmol/L for 24 h) numerous single-membrane-limited bodies
with variable content (autophagolysosomes) were seen, whereas other subcellular features remained apparently intact.
Treatment with acetaminophen plus verapamil increased the frequency of necrotic cells. In a "prenecrotic" stage, cells
displayed extensive SER dilatation/proliferation (Figure 3F) in parallel with other features of damage (eg autophagolysosomes,
reduction of surface microvilli, and increased
vacuolization). On the basis of this series of experiments we suspect that P-gp
is involved in acetaminophen transport and may play a significant role in acetaminophen-induced toxicity.
Nonsteroidal anti-inflammatory drugs The heterogeneity of this group notwithstanding, NSAIDs have common
therapeutic effects and similar side-effects. Individual drugs only very rarely have noxious effects, but their extremely widespread
use enhances the hazard. Despite their common toxic effects, individual features have been noticed among various NSAIDs
with respect to the liver pathology induced. For example the following pathologies are induced by the drugs indicated:
granulomatous hepatitis (phenylbutazone), hepatonecrotic lesions (phenylbutazone, sulindac, diclofenac, pirprofen,
piroxicam), and cholestatic hepatitis (sulindac, benoxprofen, ibuprofen, phenylbutazone, piroxicam).
Diclofenac The scarce side effects of diclofenac are significant because of its world-wide use by many patients.
Therapeutic use of diclofenac is associated with rare but sometimes fatal hepatotoxicity characterized by delayed onset of
symptoms and lack of a clear dose_response relationship. The toxicity has consequently been categorized as metabolic idiosyncrasy.
In fact, the acyl glucuronide of the drug has been demonstrated to be reactive and capable of covalent modification of cellular
proteins, binding covalently to liver proteins in rats depending on the activity of multidrug resistance protein 2, a hepatic
canalicular transporter[58]. From a toxicodynamic point of view, both oxidative stress (caused by putative diclofenac cation
radicals or nitroxide and quinone imine-related redox cycling) and mitochondrial injury (protonophoretic activity and opening
of the permeability transition pore) alone or in combination, have been implicated in diclofenac toxicity. In some cases,
immune-mediated liver injury is involved, as inferred from inadvertent rechallenge data and from a number of experiments
demonstrating T cell sensitization. Caspases 8 and 9 are apparently active caspases in diclofenac-induced apoptosis. In
addition, an early dose-dependent increase of Bcl-X
L expression parallel to the generation of reactive oxygen species in the
mitochondria was found. In conclusion, the mitochondrial pathway is very likely the only pathway involved in
diclofenac-induced apoptosis, which is related to CYP-mediated metabolism of diclofenac, with the highest apoptotic effect produced by
the metabolite 5OH-diclofenac. To date, cumulative damage to mitochondrial targets seems a plausible putative mechanism
to explain the delayed onset of liver failure, perhaps even superimposed on an underlying silent mitochondrial
abnormality[58]. Although
Gomez-Lechon et al have demonstrated that diclofenac induces apoptosis by alteration of mitochondrial
function and generation of reactive oxygen species
(ROS)[59], liver injury could not be reproduced in current animal models.
Nevertheless, it is noteworthy that ultrastructural damage was found in the liver of rainbow trout exposed to various
concentrations of diclofenac, thus illustrating differences in reactivity between mammals and other
vertebrates[60].
Nimesulide Nimesulide is a selective cyclo-oxygenase-2 (COX-2) inhibitor that has been recently linked with rare but
serious and unpredictable adverse reactions in the liver, such as fulminant hepatic failure. It is thought that the
hepatotoxicity induced by nimesulide is of the idiosyncratic type. In addition, the weakly acidic sulfonanilide drug undergoes bioreductive
metabolism of the nitroarene group to form reactive intermediates that have been implicated in oxidative stress, covalent
binding, and mitochondrial injury[61]. Tian
et al investigated the effect of nimesulide on the
proliferation and apoptosis of SMMC-7721 human hepatoma
cells[62]. Under the electron microscope, these cells exhibited characteristics of apoptosis,
including plasma membrane blebbing, cytoplasmic condensation, pyknotic nuclei, condensed chromatin and apoptotic
bodies. Compared with control groups, groups treated with 300 µmol/L and 400 µmol/L nimesulide had many more cells with
apoptotic characteristics. This observation is of interest because of the putative antitumor effect of NSAIDs.
Indomethacin The incidence of liver injury associated with indomethacin appears to be low, although exact figures are
not available. The reactions usually develop within the first few months of treatment, sometimes in association with
manifestations of hypersensitivity, and are fatal in rare instances. The histological findings range from an acute viral-like hepatitis to
confluent or multilobular necrosis; chronic active hepatitis with cholestasis and fatty changes are also occasionally observed.
Sorensen and Acosta reported that cells exposed to high concentrations of indomethacin were severely damaged, as
evidenced by marked cellular necrosis, nuclear pleomorphism, margination of chromatin, swollen mitochondria, reductions in
the number of microvilli, smooth endoplasmic reticulum proliferation, and cytoplasmic
vacuolation[63].
Celecoxib Celecoxib, a specific COX-2 enzyme inhibitor, has been widely used to alleviate pain and inflammation in
osteoarthritis and rheumatoid arthritis. It has minimal gastrointestinal, platelet, and renal side effects, but
has been associated with acute hepatocellular and cholestatic
injury[64]. Results of an in
vitro study indicate that celecoxib may
inhibit proliferation and induce apoptosis in human cholan-giocarcinoma cells through its effect on COX-dependent
mechanisms and the PGE2 pathway. Celecoxib as a chemo-preventive and chemotherapeutic agent may be primarily effective for
COX-2-expressing cholangiocarcinoma, but may also be effective for other tumors as
well[65].
Ibuprofen Patients with hepatitis C may show increased serum transaminase concentrations when treated with ibuprofen.
One morphological feature that deserves to be mentioned here is the "vanishing bile duct syndrome", which has been
reported in children with chronic rejection after
transplantation[21,66], and also in adults, and has been associated with more
than 30 drugs, including ibuprofen[67]. More severe reactions, such as subfulminant hepatitis requiring liver transplantation
following ibuprofen overdose, although very rare, have also been
described[68].
Naproxen As with other NSAIDs, borderline abnormalities in one or more liver tests may occur in up to 15% of patients
treated with naproxen. The ALT test is probably the most sensitive indicator of liver dysfunction. Meaningful (3 times the
normal upper limit) elevations of ALT or AST occurred in controlled clinical trials in less than 1% of patients. Severe hepatic
reactions, including jaundice and cases of fatal hepatitis, have been reported with naproxen as with other
NSAIDs[69]. Although such reactions are rare, if abnormal liver tests persist or worsen, and if clinical signs and symptoms consistent with
liver disease develop, or if systemic manifestations occur (eg eosinophilia or rash), naproxen treatment should be
discontinued[70].
Sulindac Sulindac has been associated with various toxic side-effects, including neurological, hematological, renal and
liver damage. From a histopathological perspective, cholestatic and/or hepatocellular damage are the morphologic
expressions of toxicity. Tarazi et al analyzed 91 cases of sulindac-associated hepatic injury, among which there were four fatal
outcomes[71]. The morphological features of sulindac hepatotoxicity were reported by Wood and
co-workers[72], who described a cholestatic hepatitis with marked anisonucleosis, cytoplasmic invaginations into the nucleus and binuclearity of
hepatocytes.
Piroxicam Isolated severe
cholestatic[73] and
fatal[74] cases have been reported in various age groups in response to
piroxicam treatment. Liver morphology in these cases indicated acute hepatitis with cholestasis and confluent necrosis.
Microvesicular steatosis has also been reported.
Disease-modifying antirheumatic drugs
The disease-modifying antirheumatic drug (DMARD) group comprises both chemical and biological
(immunosupp-ressive) compounds. In recent years, biological compounds have been used with increased frequency, mainly in rheumatoid
arthritis. Infliximab, anakinra, etanercept and leflunomide are the major representatives of this group. For leflunomide,
hepatotoxicity, defined as a 3-fold increase in ALT above the normal upper limit, has been found in up to 10% of patients, but
cases of acute liver failure have been rare. The pathological findings included a variety of abnormalities, including centrilobular
necrosis with portal or periportal inflamma-tion, steatosis, focal piecemeal necrosis, and periportal fibrosis. Too few
ultrastructural observations have been reported to enable a meaningful description. However, among DMARDs, gold
compounds and methotrexate (MTX) deserve further discussion because of their longer use and better-known
hepatotoxicity[75].
Gold hepatotoxicity Among DMARDs, gold continues to have a place, although it is now seldom used. Although the
more frequent complications of crysotherapy are gold nephritis and dermatitis, occasional cases of severe, even lethal
hepatotoxicity are still
reported[76]. When transaminase concentrations increase, liver biopsy becomes compul-sory. In gold
nephropathy, electron microscopy reveals typical lysosomes, which are termed
aurosomes[13] (Figure 4; TC Iancu and O
Ben-Itzhak, unpublished micrograph), but gold deposits can be found in various other organs, including the liver, where they
induce fibrosis and cirrhosis. Histopatholo-gically, inflammation and severe loss of parenchyma can be seen, while
macrophages contain dark granules that are shown by electron microscopy to be typical
aurosomes[77].
Methotrexate Methotrexate is presently used as the initial DMARD, especially for patients whose rheumatoid arthritis is
more active. It is frequently effective and has low toxicity. During recent years, various regimens of MTX therapy have been
used, and its side-effects have become better
known[78]. The macrovesicular steatosis, fibrosis and cirrhosis that were noted
earlier are now rare because MTX is not given in daily but weekly doses. However, hepatitis with bridging
fibrosis[79] and methotrexate-induced
cirrho-sis[80] were still reported until 1990. Phillips
et al described several liver ultrastructural changes
in MTX-treated patients (Table 1)[1].
Earlier studies (in 1988) showed that patients with rheumatoid arthritis treated with MTX had an increased frequency of
abnormal, pleomorphic lysosomes and increased collagen
in the space of Disse[81]. A year later, Kremer
and Kaye showed that these features were present also in
untreated patients[82]. Their observations were reinforced in 1995, when they reported
that there was no significant increase in collagen deposition after prolonged MTX
therapy[83]. Ros
et al carried out a light and electron microscopic analysis of 42 liver biopsies from patients with rheumatoid arthritis treated
for 4 years with MTX (weekly doses of 7.5_15
mg)[84]. The availability of pre-MTX biopsies enabled a valid comparison with
post-MTX findings. Electron microscopy appeared to be more sensitive for identifying increases in collagen fibers in the
space of Disse. By using light microscopy, the authors concluded that 14% of rheumatoid arthritis patients had mild
perisinusoidal fibrosis before MTX therapy, whereas electron microscopy revealed increased collagen presence in the space
of Disse in 50%. After 4 years of MTX therapy, no significant increase in fibrosis was found. The authors concluded that
MTX therapy is safe, regardless of the mild fibrotic changes found in the initial biopsies. Recently, Maurice
et al also stressed the minimal toxicity of low-dosage, long-term MTX
therapy[85]. These authors monitored patients on MTX with
normal serum assays of the amino terminal peptide of type III procollagen, and found no hepatic fibrosis on biopsy.
Maurice et al concluded that liver biopsy could be avoided in 45% of cases.
Conclusions
The study of ultrastructural changes in liver cells following exposure to xenobiotics or excessive doses of medication is
helpful for:
1) Detection of diagnostic features, particularly in complex syndromes (specific changes are scarce; various agents
mostly produce similar features of damage).
2) Assessment of the degree of damage, from mild, reversible changes to severe, irreversible (apoptosis, necrapo-ptosis,
necrosis) changes, which are usually related to duration of exposure and concentration.
3) Identification of target-specificity: there are marked differences in the subcellular changes produced by the same agent
in different cell types, as seen in cultures of primary cells (eg hepatocytes), tumoral cells (eg HepG2, Hep3B) and biopsy
samples (from humans or experimental animals).
The information provided by ultrastructural study has contributed to the unraveling of the mechanisms of hepatotoxicity.
Electron microscopy continues to be an essential tool for the study of drug-induced liver diseases.
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