62, 212–220 (2001)
Copyright © 2001 by the Society of Toxicology
TOXICOLOGICAL SCIENCES
Vascular and Hepatocellular Peroxynitrite Formation during
Acetaminophen Toxicity: Role of Mitochondrial Oxidant Stress
Tamara R. Knight, Angela Kurtz, Mary Lynn Bajt, Jack A. Hinson, and Hartmut Jaeschke 1
Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas
Received February 14, 2001; accepted May 7, 2001
Peroxynitrite may be involved in acetaminophen-induced liver
damage. However, it is unclear if peroxynitrite is generated in
hepatocytes or in the vasculature. To address this question, we
treated C3Heb/FeJ mice with 300 mg/kg acetaminophen and assessed nitrotyrosine protein adducts as indicator for peroxynitrite
formation. Vascular nitrotyrosine staining was evident before liver
injury between 0.5 and 2 h after acetaminophen treatment. However, liver injury developed parallel to hepatocellular nitrotyrosine
staining between 2 and 6 h after acetaminophen. The mitochondrial content of glutathione disulfide, as indicator of reactive
oxygen formation determined 6 h after acetaminophen, increased
from 2.8 ⴞ 0.6% in controls to 23.5 ⴞ 5.1%. A high dose of
allopurinol (100 mg/kg) strongly attenuated acetaminophen protein-adduct formation and prevented the mitochondrial oxidant
stress and liver injury after acetaminophen. Lower doses of allopurinol, which are equally effective in inhibiting xanthine oxidase,
were not protective and had no effect on nitrotyrosine staining and
acetaminophen protein adduct formation. In vitro experiments
showed that allopurinol is not a direct scavenger of peroxynitrite.
We conclude that there is vascular peroxynitrite formation during
the first 2 h after acetaminophen treatment. On the other hand,
reactive metabolites of acetaminophen bind to intracellular proteins and cause mitochondrial dysfunction and superoxide formation. Mitochondrial superoxide reacts with nitric oxide to form
peroxynitrite, which is responsible for intracellular protein nitration. The pathophysiological relevance of vascular peroxynitrite
for hepatocellular peroxynitrite formation and liver injury remains
to be established.
Key Words: peroxynitrite; nitrotyrosine; acetaminophen; allopurinol; liver failure; mitochondria; oxidant stress.
Acetaminophen is a commonly used analgesic and antipyretic, which is considered very safe in the therapeutic range. In
overdose, it produces centrilobular hepatic necrosis in humans
and animals. It is well established that the formation of the
reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI)
during the metabolism of acetaminophen by cytochrome P-450
1
To whom correspondence should be addressed at the Department of
Pharmacology and Toxicology, University of Arkansas for Medical Sciences,
4301 W. Markham St., Mailslot 638, Little Rock, AR 72205–7199. Fax: (501)
686-8970. E-mail: [email protected].
is an important step in the development of the hepatotoxicity
(Dahlin et al., 1984). NAPQI is detoxified by glutathione,
resulting in the depletion of this sulfhydryl reagent (Mitchell et
al., 1973). If NAPQI formation exceeds the capacity of cellular
glutathione, NAPQI covalently binds to intracellular proteins
(Jollow et al., 1973). In recent years, a substantial number of
proteins have been identified as targets for NAPQI (Pumford et
al., 1997; Cohen and Khairallah, 1997; Qui et al., 1998).
However, none of these proteins appeared to be inactivated to
the extent that it could explain massive cell necrosis. Therefore, the molecular mechanism of acetaminophen-induced hepatocellular necrosis is not completely understood.
More recently, the concept emerged that nitric oxide and
peroxynitrite may be critical mediators for acetaminophen hepatotoxicity (Gardner et al., 1998, 1999; Hinson et al., 1998;
Michael et al., 1999). Peroxynitrite is a strong oxidant generated by the spontaneous reaction of nitric oxide and superoxide
(Koppenol, 1998). Peroxynitrite or secondary metabolites can
cause tyrosine nitration as well as induce oxidative damage to
proteins, DNA and lipids (Squadrito and Pryor, 1998). During
acetaminophen toxicity, the inducible nitric oxide synthase
(iNOS) is upregulated in the liver (Gardner et al., 1998) and
there is evidence for increased formation of NO (Gardner et al.,
1998, Hinson et al., 1998). Reduced liver injury after acetaminophen was observed in animals treated with the iNOS
inhibitor aminoguanidine (Gardner et al., 1998) and in iNOS
knock-out mice (Gardner et al., 1999). However, the location
of peroxynitrite formation remains unclear. Inhibitors of
Kupffer cell activity, which reduce the capacity of Kupffer
cells to generate reactive oxygen (Liu et al., 1995), attenuated
hepatocellular nitrotyrosine staining (Michael et al., 1999) and
acetaminophen-induced liver injury (Goldin et al., 1996;
Laskin et al., 1995; Michael et al., 1999). This suggests that the
resident macrophages of the liver could be a source of superoxide and/or nitric oxide after acetaminophen treatment. However, intracellular sources of reactive oxygen such as mitochondria cannot be excluded (Jaeschke, 1990). Thus, the
objective of this investigation was to differentiate formation of
peroxynitrite in the vascular space and in hepatocytes during
the development of acetaminophen-induced liver injury and to
address whether mitochondria-derived superoxide could be
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ACETAMINOPHEN-INDUCED PEROXYNITRITE FORMATION
responsible for hepatocellular peroxynitrite generation and injury.
MATERIALS AND METHODS
Animals. Male C3Heb/FeJ mice (Jackson Laboratory, Bar Harbor,
Maine), with an average weight of 18 to 20 g, were housed in an environmentally controlled room with 12-h light/dark cycle and allowed free access to
food and water. The animals were fasted overnight before the experiments.
Animals received an intraperitoneal injection of 300 mg/kg or 150 mg/kg
acetaminophen (Sigma Chemical Co., St. Louis, MO) dissolved in warm saline
solution (15 mg/ml). Some of the animals were treated twice with allopurinol
(10 or 100 mg/kg) (Sigma) suspended in water. The first dose of allopurinol
was administered by gavage 18 h before, and the second dose 1 h before
acetaminophen. Control animals received only water (16 ml/kg). Some animals
were treated ip with 250 mg/kg phorone (Aldrich Chemical Co., Inc. Milwaukee, WI) dissolved in corn oil to deplete hepatic glutathione levels (Jaeschke
et al., 1987).
Experimental protocols. At selected times after acetaminophen treatment,
the animals were killed by cervical dislocation. Blood was drawn from the
vena cava into heparinized syringes and centrifuged. The plasma was used for
determination of alanine aminotransferase (ALT) activities. Immediately after
collecting the blood, the livers were excised and rinsed in saline. A small
section from each liver was placed in 10% phosphate-buffered formalin to be
used in immunohistochemical analysis. A portion of the remaining liver was
homogenized for isolation of mitochondria or frozen in liquid nitrogen and
stored at – 80°C for later analysis of glutathione.
Isolation of mitochondria. A portion of the liver was homogenized in
ice-cold isolation buffer (pH 7.4) containing 220 mM mannitol, 70 mM
sucrose, 2.5 mM Hepes, 10 mM EDTA, 1 mM EGTA, and 0.1% bovine serum
albumin.
The liver homogenate was centrifuged at 600 ⫻ g for 8 min at 4°C to
remove nuclei and cellular debris. The supernatant was removed and centrifuged at 10,000 ⫻ g for 10 min at 4°C to pellet the mitochondria; the pellet was
washed once with 2 ml of isolation buffer. The mitochondrial pellet was
resuspended in 3% sulfosalicylic acid containing 0.1 mM EDTA, vigorously
vortexed, and centrifuged to sediment the precipitated protein. A part of the
supernatant was diluted in 100 mM potassium phosphate buffer (pH 6.5) for
the determination of total glutathione (GSH ⫹ GSSG) and another part was
added to 10 mM N-ethylmaleimide (NEM) in potassium phosphate buffer for
the determination of glutathione disulfide (GSSG).
Methods. Plasma ALT activities were determined with the test kit DG
159- UV (Sigma Chem. Co., St. Louis, MO) and expressed as IU/liter. Protein
concentrations were assayed using the bicinchoninic acid kit (Pierce, Rockford, IL). Plasma levels of nitrite/nitrate were determined with the Colorimetric
Non-Enzymatic Nitric Oxide Assay Kit (Oxford Biochemical Research, Inc.,
Oxford, MI). Total soluble GSH and GSSG were measured in the liver
homogenate and mitochondrial homogenate with a modified method of Tietze,
as described in detail (Jaeschke and Mitchell, 1990). Briefly, the frozen tissue
or isolated mitochondria were homogenized at 0°C in 3% sulfosalicylic acid
containing 0.1 mM EDTA. An aliquot of the homogenate was added to 10 mM
NEM in phosphate buffer (KPP) and another aliquot was added to 0.01 N HCl.
The NEM-KPP sample was centrifuged and the supernatant was passed
through a C 18 cartridge to remove free NEM and NEM-GSH adducts (Sep-pak;
Waters Associates, Waltham, MA). The HCl sample was centrifuged and the
supernatant was diluted with KPP. All samples were assayed using dithionitrobenzoic acid (DTNB). All data are expressed in GSH-equivalents.
Immunohistochemistry. Protein adducts of acetaminophen and nitrotyrosine were assessed by immunohistochemistry as described in detail previously (Michael et al., 1999) using an anti-nitrotyrosine antibody (Hinson et al.,
2000) or an anti-acetaminophen antiserum (Matthews et al., 1996). Some of
the stained sections were evaluated independently by two of the authors. The
extent of the staining and the intensity of the staining was graded separately for
213
vascular staining and parenchymal cell staining. The maximal staining in each
compartment was set as 100% (2-h time point for the vasculature; 6 h for
parenchymal cells) and the percentage estimated for earlier time points.
Western blotting. The expression of iNOS protein in the liver was determined by Western blotting as previously described for other proteins (Bajt et
al., 2000; Lawson et al., 1999). Briefly, liver tissue was homogenized in 25
mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (pH 7.5)
containing 5 mM EDTA, 2 mM DTT (dithiothreitol), 0.1% CHAPS (3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonate), 1 ␮g/ml pepstatin,
leupeptin, and aprotinin. Homogenates were centrifuged at 14,000 ⫻ g at 4°C
for 20 min. Cytosolic extracts (10 ␮g per lane) were resolved by 4 –20%
SDS–polyacrylamide gel electrophoresis under reducing conditions. Separated
proteins were transferred to polyvinylidine difluoride membranes (PVDF,
Immobilin-P, Millipore, Bedford, MA). The membranes were first blocked
with 5% milk in TBS (20 mM Tris, 15 M NaCl, 0.1% Tween 20, and 0.1%
BSA) overnight at 4°C, followed by incubation with primary antibody for 2 h
at room temperature. A rabbit polyclonal iNOS antibody (Upstate Biotechnology, Lake Placid, NY) was used as a primary antibody. The membranes were
washed and then incubated with the secondary antibody anti-rabbit IgG conjugated with horseradish peroxidase (Santa Cruz Biotechnology). Proteins
were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech. Inc., Piscataway, NJ), according to the manufacturer’s instructions.
Densitometric analysis of the gels was performed with a GS170 Calibrated
Imaging Densitometer (Biorad, Hercules, CA) using Quantity One 4.0.3 software (Biorad).
Nitration of BSA in vitro. The nitration of proteins by peroxynitrite
(Upstate Biotechnology, Lake Placid, NY) was determined spectrophotometrically at 438 nm as the intensely yellow phenolate of nitrotyrosine (Hinson et
al., 2000). Briefly, BSA (Sigma Chem. Co., St. Louis, MO) was added to 60
mM carbonate buffer (pH 9.6) with a final concentration of 2 mg/ml. Varying
concentrations (50 ␮M, 200 ␮M, 500 ␮M, 1 mM) of allopurinol or N-acetylcysteine were added to the BSA-carbonate buffer. Peroxynitrite was then
added to each solution (final concentration of 1.40 mM) to nitrate BSA. A
spectrum was recorded and the amount of nitrotyrosine in the peroxynitritetreated BSA was determined at the absorbance maximum of the phenolate ion
at 438 nm.
Statistics. All results were expressed as mean ⫾ SE. Comparisons between multiple groups were performed with one-way ANOVA followed by
Bonferroni t-test. If the data were not normally distributed, we used the
Kruskal-Wallis Test (nonparametric ANOVA) followed by Dunn’s Multiple
Comparisons Test; p ⬍ 0.05 was considered significant.
RESULTS
Administration of a moderately toxic dose of 300 mg/kg
acetaminophen caused liver injury in C3Heb/FeJ mice. Based
on the release of ALT, cell damage was first evident at 2 h (Fig.
1). The injury progressed to severe damage at 6 h. Immunohistochemical staining for nitrotyrosine (NT) protein adducts in
liver sections of controls indicated no adduct formation (Fig.
2A). However, at 1 h after acetaminophen administration, an
exclusive staining in the vasculature around central veins was
detectable (Fig. 2B). There was no staining of hepatocytes at
that time. In contrast, at 4 h after acetaminophen treatment,
individual hepatocytes in the pericentral area were intensely
stained for NT protein adducts (Fig. 2C). However, a significant number of hepatocytes were unstained. At 6 h, a confluent
staining of all hepatocytes in the central area was observed
(Fig. 2D). The staining in the vascular compartment and in
hepatocytes was then evaluated in all samples of each time
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KNIGHT ET AL.
FIG. 1. Time course of plasma ALT activities in C3Heb/FeJ mice after
administration of acetaminophen (300 mg/kg, ip). Data represent means ⫾ SE
of n ⫽ 5 animals per group; *p ⬍ 0.05 (compared to t ⫽ 0).
point. The extent and intensity of NT staining in each compartment was visually assessed by 2 investigators. For each
time point, the percent of the maximal staining in the vasculature (2 h) and in hepatocytes (6 h) was estimated. Vascular
staining for NT was negligible at 30 min after acetaminophen
injection but then increased steeply up to 2 h (Fig. 3). In
contrast, hepatocellular staining was not detectable up to 1 h
(Figs. 2B and 3). After that time, the number of NT-positive
hepatocytes increased progressively up to 6 h with the confluent staining of all hepatocytes in the centrilobular area (Fig. 3).
A comparison of the liver injury data in Figure 1 with the
staining pattern showed that the vascular staining preceded
liver injury. However, hepatocellular staining increased parallel to the ALT release. To evaluate nitric oxide formation,
plasma levels of nitrite/nitrate were measured. No significant
change of basal nitrite/nitrate concentrations (43 ⫾ 2 ␮M, n ⫽
5) was observed up to 6 h after acetaminophen treatment (data
not shown). Consistent with these results, the baseline expression of iNOS protein was not increased in livers of acetaminophen-treated animals (Fig. 4). In contrast, a decrease of hepatic iNOS protein levels was evident at 6 h after
acetaminophen.
The xanthine oxidase inhibitor allopurinol has been shown
to prevent a mitochondrial oxidant stress and protect against
acetaminophen-induced liver injury (Jaeschke, 1990). Therefore, animals were pretreated with low doses (2⫻ 10 mg/kg) or
high doses (2⫻ 100 mg/kg) of allopurinol. In previous studies
it was shown that both doses completely inhibited xanthine
oxidase activities in the liver. However, only the high dose of
allopurinol protected against acetaminophen-induced liver injury (Jaeschke, 1990). Based on plasma ALT values, these
findings were confirmed (Fig. 5). A dose of 100 mg/kg allopurinol completely prevented acetaminophen-induced liver in-
jury at 6 h. The lower dose of allopurinol was ineffective (Fig.
5). NT staining showed again the confluent staining of hepatocytes around the centrilobular area in livers of animals
treated with acetaminophen alone (Fig. 6B) or with acetaminophen and the low dose of allopurinol (Fig. 6C). In contrast,
treatment with the high dose of allopurinol prevented hepatocellular staining (Fig. 6D). However, the vascular staining was
only attenuated (Fig. 6D). Evaluation of NT staining at 1 h
after acetaminophen confirmed that the high dose of allopurinol did not eliminate vascular staining (data not shown).
To address the mechanism of the protective effect of allopurinol against acetaminophen hepatotoxicity, acetaminophen
protein adducts were visualized by immunohistochemistry.
Consistent with previous findings (Matthews et al., 1996; Michael et al., 1999), extensive centrilobular staining for acetaminophen protein adducts was observed 6 h after acetaminophen treatment (Fig. 6E). The low dose of allopurinol did not
affect this staining pattern (data not shown); however, the high
dose of allopurinol substantially reduced (Fig. 6F) or in some
cases completely eliminated staining for acetaminophen protein adducts.
Since increased hepatocellular superoxide formation is a
prerequisite for intracellular peroxynitrite generation, liver
GSSG levels were measured as indicator for an increased
oxidant stress 6 h after acetaminophen treatment. Compared to
control animals, the liver content of total glutathione (GSH ⫹
GSSG) was reduced by 37% in acetaminophen-treated animals, but the levels of GSSG and the GSSG-to-GSH ratio were
significantly increased (Table 1). This significant intracellular
oxidant stress induced by acetaminophen was prevented by the
high dose of allopurinol (Table 1). Since previous data suggest
that most of the GSSG is located in mitochondria at 24 h
(Jaeschke, 1990), mitochondria were isolated for determination
of GSH and GSSG at 6 h. The total glutathione content of
hepatic mitochondria was similar in control and acetaminophen-treated animals (Table 1). However, GSSG levels were
increased 6-fold and the GSSG-to-GSH ratio was increased
8-fold above baseline values after acetaminophen (Table 1),
indicating a mitochondrial oxidant stress. Treatment with the
high dose of allopurinol had no significant effect on the total
glutathione content but prevented the increase of mitochondrial
GSSG levels (Table 1). Thus, allopurinol eliminated the mitochondrial oxidant stress induced by acetaminophen. To determine the time when the oxidant stress was first evident, mitochondria were isolated at different time points after
acetaminophen treatment. Similar to the total glutathione content in the liver (Jaeschke, 1990), the mitochondrial glutathione
content decreased rapidly to levels 20 –25% of controls at 0.5
to 2 h after acetaminophen (Fig. 7). After 2 h, mitochondrial
glutathione levels began to recover. The mitochondrial GSSG
content decreased to 49% at 15 min but then increased to
70 – 80% of baseline between 0.5 and 2 h (Fig. 7). At 4 h,
mitochondrial GSSG levels were significantly higher than control values. Therefore, the GSSG-to-GSH ratio of 0.050 ⫾
ACETAMINOPHEN-INDUCED PEROXYNITRITE FORMATION
215
FIG. 2. Immunohistochemical staining of liver sections for nitrotyrosine (NT) protein adducts in controls and 1, 4, and 6 h after 300 mg/kg of acetaminophen.
Control (A): The liver was histologically normal with no evidence of NT staining or hepatocyte injury (400 ⫻). One-hour acetaminophen (B): NT staining present
in vascular lining cells but not in hepatocytes of centrilobular areas (400 ⫻). Four-hour acetaminophen (C): Intensely NT stained hepatocytes were scattered
among unstained cells in centrilobular areas (400 ⫻). Six-hour acetaminophen (D): Confluent hepatocellular staining for NT in centrilobular areas (400 ⫻); CV,
central vein.
0.009 in controls did not change significantly at 15 min but
increased to 0.156 ⫾ 0.0124 (p ⬍ 0.05) at 30 min. This
increased ratio was maintained for the duration of the experiment. To address the concern that the increased GSSG-to-GSH
ratio may not reflect an enhanced oxidant stress but merely be
due to the decline in GSH, animals were treated with 250
mg/kg phorone, a known GSH-depleting agent. Phorone treatment reduced hepatic glutathione levels by 70% at 90 min. The
mitochondrial GSH content deceased by 85% to 0.41 ⫾ 0.09
nmol/mg protein and the GSSG levels dropped below the
detection limit of the assay (0.01 nmol/mg protein). Thus, the
GSSG-to-GSH ratio observed in controls was not increased
after phorone treatment. Similar to treatment with phorone, a
nontoxic dose of acetaminophen (150 mg/kg) did not increase
the mitochondrial GSSG-to-GSH ratio (data not shown).
To determine if allopurinol was protective by acting as a
direct scavenger of peroxynitrite, we performed an in vitro
experiment using a BSA-carbonate buffer containing various
concentrations of allopurinol or N-acetyl cysteine. Peroxynitrite was then added to each solution and the nitration of BSA
was determined by reading the absorbance maximum of phe-
nolate at a wavelength of 438 nm. As shown in Figure 8, 1 mM
allopurinol had no effect on the nitration of BSA by peroxynitrite. Similar results were obtained with lower concentrations
of allopurinol (data not shown). In contrast, 1 mM N-acetyl
cysteine completely prevented BSA nitration (Fig. 8). The
effect of N-acetyl cysteine was dose-dependent (data not
shown).
DISCUSSION
The major objective of this investigation was to localize
peroxynitrite formation during acetaminophen-induced liver
injury. Our data demonstrated a clear initial nitrotyrosine staining of vascular lining cells of the centrilobular areas. This
suggests that peroxynitrite was formed in sinusoids during the
early phase after acetaminophen overdose. Peroxynitrite could
be formed in the vascular lumen by superoxide derived from
Kupffer cells and nitric oxide from Kupffer and endothelial
cells. Since basal levels of NO are present in the vasculature,
increased peroxynitrite formation may depend to a large degree
on superoxide formation by Kupffer cells. In fact, use of
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KNIGHT ET AL.
FIG. 3. Time course of nitrotyrosine (NT) staining in vascular lining cells
and hepatocytes after treatment with acetaminophen (300 mg/kg, ip). Staining
intensity and extent of staining in the vasculature and hepatocytes was estimated as percent of maximal staining in the respective compartment, i.e., at 2 h
in the vasculature and at 6 h in hepatocytes. Five high-power fields (400 ⫻) in
each of 3– 4 slides per time point were evaluated.
gadolinium chloride, which inhibits Kupffer cell-derived superoxide formation (Liu et al., 1995), attenuated acetaminophen-induced liver injury (Laskin et al., 1995; Michael et al.,
1999). Isolation of Kupffer cells from acetaminophen-treated
animals demonstrated the activation and priming of these cells
for superoxide formation (Laskin et al., 1986). Gadolinium
chloride treatment also prevented the later hepatocellular NT
staining (Michael et al., 1999), which suggests an important
role of Kupffer cell-derived vascular oxidant stress in acetaminophen hepatotoxicity. Other vascular sources of reactive
FIG. 4. Western-blot analysis of the inducible nitric oxide synthase
(iNOS; molecular weight 125 kDa) in livers from controls and acetaminophen
(AAP)-treated animals. Liver samples were obtained at different time points
after 300 mg/kg of AAP. Each lane represents a sample from a single animal.
The bar graph shows the densitometric analysis of each lane. The dashed line
indicates the control level.
FIG. 5. Effect of allopurinol (AP) on acetaminophen-induced liver injury.
Plasma ALT activities were measured 6 h after a single dose of 300 mg/kg
acetaminophen (AAP). Vehicle-treated controls are compared with animals
that received 2⫻ 10 mg/kg AP or 2⫻100 mg/kg AP 18 h and 1 h before AAP
injection. Data represent means ⫾ SE of n ⫽ 5 animals per group; *p ⬍ 0.05
(compared to controls); #p ⬍ 0.05 (compared to AAP/H 2O).
oxygen formation, e.g., neutrophils, can be excluded, because
these cells accumulate at later time points (Lawson et al.,
2000). Further support for the relevance of sinusoidal peroxynitrite generation was also provided by the observation that
the overexpression of vascular glutathione peroxidase reduced
liver injury and prolonged survival after acetaminophen overdose (Mirochnitchenko et al., 1999). Glutathione peroxidase
can not only metabolize peroxides but is also an effective
reductase for peroxynitrite (Sies et al., 1997). Thus, vascular
peroxynitrite formation occurs early in the pathophysiology of
acetaminophen-induced liver injury and may be relevant for
later hepatocellular events.
In addition to the initial vascular NT staining, our data
showed a progressive intracellular staining in hepatocytes.
Based on the magnitude of NT staining in hepatocytes, it
appears unlikely that vascular peroxynitrite could be responsible for the intracellular NT adduct formation in hepatocytes.
Thus, peroxynitrite must have been generated in hepatocytes.
Previous data indicated the development of an intracellular
oxidant stress, located mainly in mitochondria, during acetaminophen hepatotoxicity (Jaeschke et al., 1990). Mitochondrial dysfunction occurs rapidly, i.e., within 1–2 h after acetaminophen administration (Donnelly et al., 1994; Esterline et
al., 1989; Meyers et al., 1988; Ramsay et al., 1989). All studies
showed an inhibition of state-3 (ADP-stimulated) respiration of
mitochondria. Consistent with these findings, hepatic ATP
levels declined rapidly after acetaminophen treatment in vivo
(Jaeschke, 1990; Tirmenstein and Nelson, 1990). The enhanced state-4 (resting) respiration, with succinate as a substrate (Donnelly et al., 1994; Meyers et al., 1988), suggests
uncoupling of the mitochondrial respiratory chain with electron
ACETAMINOPHEN-INDUCED PEROXYNITRITE FORMATION
217
FIG. 6. Immunohistochemical analyses of liver sections for nitrotyrosine (NT) protein adducts (A–D) and acetaminophen protein adducts (E, F) 6 h after
acetaminophen administration. Control (A): The liver was histologically normal with no evidence of NT staining or hepatocyte injury (400 ⫻). Six-hour
acetaminophen (B): Confluent hepatocellular staining for NT in centrilobular areas (200 ⫻). Six-hour acetaminophen/10 mg/kg allopurinol (C): Similar confluent
hepatocellular staining in centrilobular areas as after acetaminophen alone (200 ⫻). Six-hour acetaminophen/100 mg/kg allopurinol (D): NT staining present in
vascular lining cells but not in hepatocytes of centrilobular areas (400 ⫻). Six-hour acetaminophen (E): Staining for acetaminophen protein adducts showed a
confluent hepatocellular staining in centrilobular areas (200 ⫻). Six-hour acetaminophen/100 mg/kg allopurinol (F): Reduced acetaminophen protein-adduct
formation in livers of animals treated with the high dose of allopurinol (200 ⫻); CV, central vein.
leakage, which may result in superoxide formation. These
findings are consistent with the dramatic increase in mitochondrial GSSG content (Jaeschke, 1990). The baseline GSSG
levels measured in mitochondria were higher than the level in
the intact liver (Table 1). This may reflect some potential thiol
oxidation during the isolation process. However, the significantly higher GSSG levels at 4 and 6 h after acetaminophen,
and the higher GSSG-to-GSH ratio compared to identically
isolated control mitochondria, suggests a higher oxidant stress
at 0.5 to 6 h after drug treatment. In addition, glutathione
depletion with phorone did not increase the GSSG-to-GSH
ratio in mitochondria. These data further support the conclusion that the increase of the GSSG-to-GSH ratio at 0.5 to 2 h
reflects an oxidant stress. These findings are consistent with
earlier observations showing a rapid mitochondrial dysfunction
1–2 h after acetaminophen treatment (Esterline et al., 1989;
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TABLE 1
Effect of Allopurinol on Hepatic and Mitochondrial Glutathione
Levels after Acetaminophen Overdose
Hepatic levels
Saline ⫹ H 2O
AAP ⫹ H 2O
AAP ⫹ AP
Mitochondrial levels
Saline ⫹ H 2O
AAP ⫹ H 2O
AAP ⫹ AP
GSH ⫹ GSSG
GSSG
GSSG-to-GSH ratio
4.7 ⫾ 0.4
2.9 ⫾ 0.3*
5.0 ⫾ 0.1**
44.2 ⫾ 7.8
60.9 ⫾ 7.3*
39.8 ⫾ 3.3**
0.011 ⫾ 0.001
0.021 ⫾ 0.002*
0.008 ⫾ 0.001**
6.6 ⫾ 1.0
5.5 ⫾ 1.6
4.7 ⫾ 0.8
0.18 ⫾ 0.03
1.13 ⫾ 0.27*
0.18 ⫾ 0.04**
0.028 ⫾ 0.006
0.235 ⫾ 0.051*
0.040 ⫾ 0.008**
Note. Hepatic GSH ⫹ GSSG in ␮mol/g liver; hepatic GSSG in nmol/g liver;
mitochondrial GSH ⫹ GSSG and GSSG in nmol/mg protein. Hepatic and
mitochondrial content of total glutathione (GSH ⫹ GSSG) and glutathione
disulfide (GSSG) were determined 6 h after ip administration of 300 mg/kg
acetaminophen (AAP) or saline (20 ml/kg). Animals were treated po with
water (2⫻ 16 ml/kg) or allopurinol (2⫻ 100 mg AP/kg) 18 h and 1 h before
acetaminophen injection. All results are given in GSH equivalents. Data
represent means ⫾ SE of n ⫽ 5 animals per group.
*p ⬍ 0.05 (compared to saline).
**p ⬍ 0.05 (compared to AAP/H 2O).
Donnelly et al., 1994; Meyers et al., 1988; Ramsay et al.,
1989).
Treatment with a high dose of allopurinol attenuated the
acetaminophen-induced mitochondrial oxidant stress (Table 1),
prevented the decline of hepatic ATP levels (Jaeschke, 1990),
and abolished cell injury (Fig. 5). Furthermore, allopurinol
prevented NT staining in hepatocytes but had only a moderate
effect on vascular NT adduct formation (Fig. 6). These results
suggest that the mitochondrial superoxide formation may promote peroxynitrite generation and protein nitration. Interestingly, no significant increase in iNOS protein levels or plasma
nitrite/nitrate concentrations were observed, suggesting no relevant increase over baseline in NO formation during the first
6 h after acetaminophen administration. Thus, the enhanced
formation of superoxide may have primarily determined the
increase in peroxynitrite generation. There are reports showing
a moderate increase in plasma nitrite/nitrate levels (Hinson et
al., 1998) and an induction of iNOS in the liver (Gardner et al.,
1998) 6 – 8 h after acetaminophen. In addition, ex vivo stimulation with endotoxin and interferon-␥ of hepatic macrophages
and hepatocytes from acetaminophen-treated rats showed enhanced formation of nitric oxide compared to control cells
(Gardner et al., 1998, 1999). Our data do not contradict these
findings. We can not rule out a moderate increase in nitric
oxide formation, which may not result in higher plasma nitrite/
nitrate levels. It appears that under our experimental conditions, basal or modestly increased levels of nitric oxide are
sufficient to react with superoxide and generate peroxynitrite.
Acetaminophen overdose induces conversion of xanthine
dehydrogenase to the oxidase (Jaeschke, 1990), which can be
a significant source of superoxide under certain pathophysio-
FIG. 7. Time-dependent changes of the mitochondrial content of glutathione
(GSH ⫹ GSSG) and glutathione disulfide (GSSG) after a single dose of acetaminophen (300 mg/kg). All results are given in GSH equivalents. Data represent
means ⫾ SE of n ⫽ 4 animals per group; *p ⬍ 0.05 (compared to t ⫽ 0).
logical conditions (Jaeschke and Mitchell, 1989). Allopurinol
is a potent inhibitor of xanthine oxidase; doses as low as 5–10
mg/kg completely inhibit this enzyme activity in the liver in
vivo (Jaeschke, 1990). However, only the high dose (100
mg/kg) of allopurinol prevented the intracellular oxidant stress
and NT staining in hepatocytes. This indicates that xanthine
oxidase is not a relevant contributor to the intracellular reactive
oxygen formation after acetaminophen treatment and, therefore, is unlikely to be involved in peroxynitrite formation.
Other intracellular sources such as the microsomal P450 sys-
FIG. 8. Effect of allopurinol (AP) on protein nitration by peroxynitrite
(PN). Nitration of bovine serum albumin (BSA) was determined spectrophotometrically as the intensely yellow phenolate of nitrotyrosine at 438 nm. PN
was added to BSA-carbonate buffer (final concentration of 2 mg/ml; pH 9.6)
containing 1 mM of AP or N-acetyl cysteine (NAC). A spectrum was recorded
and the amount of nitrotyrosine in the peroxynitrite-treated BSA was calculated by reading the absorbance maximum of the phenolate ion at 438 nm.
ACETAMINOPHEN-INDUCED PEROXYNITRITE FORMATION
tem can be excluded because no oxidant stress could be detected during the initial metabolism of acetaminophen in
mouse livers (Smith and Jaeschke, 1989). Thus, based on the
cellular localization of GSSG, it can be concluded that mitochondria are the main source of superoxide.
Although it was previously shown that allopurinol can prevent acetaminophen-induced liver injury, the molecular mechanism of this hepatoprotection remained unclear (Jaeschke,
1990). Protein nitration by peroxynitrite in vitro was not inhibited by allopurinol. These results suggest that allopurinol
did not directly react with peroxynitrite. Furthermore, allopurinol treatment had no effect on hepatic glutathione depletion
or biliary acetaminophen-glutathione conjugate excretion
(Jaeschke, 1990). This suggests that allopurinol does not affect
the kinetics or extent of NAPQI formation. Consistent with
these findings, allopurinol did not enhance the pentobarbital
sleeping time in mice (Jaeschke, 1990). This indicated that
allopurinol does not inhibit the isoenzymes of P450 responsible
for pentobarbital and, at least in part, acetaminophen metabolism. Thus, there is no evidence to suggest that allopurinol
inhibited the metabolic activation of acetaminophen. However,
our current data indicate that allopurinol reduced or eliminated
acetaminophen protein adduct formation. The molecular mechanism of how allopurinol interferes with the binding of NAPQI
to cellular proteins is unclear at present and requires further
investigation. Covalent binding of acetaminophen to mitochondrial proteins was first reported by Jollow et al. (1973). Subsequent studies showed the highest cellular levels of 3-(cystein-S-yl) acetaminophen protein adducts in the plasma
membrane and mitochondrial fractions (Pumford et al., 1990).
Since then, a number of mitochondrial proteins have been
identified as targets for covalent binding (Cohen and Khairallah, 1997; Pumford et al., 1997; Qiu et al., 1998). The time
course of acetaminophen protein adduct formation (Pumford et
al., 1990) correlated well with the onset of mitochondrial
dysfunction (Donnelly et al., 1994; Meyers et al., 1988). The
fact that allopurinol strongly reduced covalent binding (Fig. 6)
and eliminated the decline in hepatic ATP levels (Jaeschke,
1990; Tirmenstein and Nelson, 1990) as well as the mitochondrial oxidant stress (Table 1), suggests that covalent binding of
NAPQI to these proteins is responsible for mitochondrial dysfunction. Thus, the reduced NT staining and hepatoprotective
effect of allopurinol after acetaminophen overdose is likely
related to the reduced mitochondrial superoxide formation due
to the lack of NAPQI protein binding. However, our data do
not allow us to distinguish whether mitochondrial dysfunction
per se is sufficient to cause cell death or if the subsequent
peroxynitrite formation is a critical event in the pathophysiology. More specific intervention studies are necessary to clarify
this issue.
In summary, our investigation demonstrated an initial vascular nitrotyrosine staining after acetaminophen treatment,
which indicates that peroxynitrite formation took place in the
sinusoids. The subsequent intracellular peroxynitrite genera-
219
FIG. 9. Current hypothesis of the mechanism of acetaminophen hepatotoxicity; EC, endothelial cells; NAPQI, N-acetyl-p-benzoquinone imine.
tion in hepatocytes increased in parallel with parenchymal cell
injury. Mitochondria are the most likely source of superoxide
after acetaminophen overdose. Allopurinol, which attenuated
acetaminophen protein-adduct formation and mitochondrial
dysfunction and oxidant stress, eliminated hepatocellular nitrotyrosine staining and injury. We conclude that after acetaminophen treatment, the reactive metabolite NAPQI binds to intracellular proteins and causes mitochondrial dysfunction and
superoxide formation (Fig. 9). Mitochondrial superoxide reacts
with nitric oxide to form peroxynitrite, which is responsible for
intracellular protein nitration. The pathophysiological relevance of vascular peroxynitrite for hepatocellular peroxynitrite
formation and liver injury remains to be established.
ACKNOWLEDGMENT
This work was supported in part by NIH grants ES-06091 and GM-58884.
REFERENCES
Bajt, M. L., Lawson, J. A., Vonderfecht, S. L., Gujral, J. S., and Jaeschke, H.
(2000). Protection against Fas receptor-mediated apoptosis in hepatocytes
and nonparenchymal cells by a caspase-8 inhibitor in vivo: Evidence for
postmitochondrial processing of caspase-8. Toxicol. Sci. 58, 109 –117.
Cohen, S. D., and Khairallah, E. A. (1997). Selective protein arylation and
acetaminophen-induced hepatotoxicity. Drug Metab. Rev. 29, 59 –77.
Dahlin, D. C., Miwa, G. T., Lu, A. Y. H., and Nelson, S. D. (1984). N-acetylp-benzoquinone imine: A cytochrome P-450-dependent oxidation product of
acetaminophen. Proc. Natl. Acad. Sci. U.S.A. 81, 1327–1331.
Donnelly, P. J., Walker, R. M., and Racz, W. J. (1994). Inhibition of mitochondrial respiration in vivo is an early event in acetaminophen-induced
hepatotoxicity. Arch. Toxicol. 68, 110 –118.
Esterline, R. L., Ray, S. D., and Ji, S. (1989). Reversible and irreversible
inhibition of hepatic mitochondrial respiration by acetaminophen and its
toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI). Biochem. Pharmacol. 38, 2387–2390.
Gardner, C. R., Heck, D. E., Chiu, H., Laskin, J. D., Durham, S. K., and
Laskin, D. L. (1999). Decreased hepatotoxicity of acetaminophen in mice
lacking inducible nitric oxide synthase. In Cells of the Hepatic Sinusoid (E.
220
KNIGHT ET AL.
Wisse, D.L. Knook, R. de Zanger, and R. Fraser, Eds.), Vol. 7, pp. 104 –105.
Kupffer Cell Foundation, Leiden.
Gardner, C. R., Heck, D. E., Yang, C. S., Thomas, P. E., Zhang, X. J.,
DeGeorge, G. L., Laskin, J. D., and Laskin, D. L. (1998). Role of nitric
oxide in acetaminophen-induced hepatotoxicity in the rat. Hepatology 26,
748 –754.
Goldin, R. D., Ratnayaka, I. D., Breach, C. S., Brown, I. N., and Wickramasinghe, S. N. (1996). Role of macrophages in acetaminophen (paracetamol)induced hepatotoxicity. J. Pathol. 179, 432– 435.
Hinson, J. A., Michael, S. L., Ault, S. G., and Pumford, N. R. (2000). Western
blot analysis for nitrotyrosine protein adducts in livers of saline-treated and
acetaminophen-treated mice. Toxicol. Sci. 53, 467– 473.
Hinson, J. A., Pike, S. L., Pumford, N. R., and Mayeux, P. R. (1998).
Nitrotyrosine-protein adducts in hepatic centrilobular areas following toxic
doses of acetaminophen in mice. Chem. Res. Toxicol. 11, 604 – 607.
Jaeschke, H. (1990). Glutathione disulfide formation and oxidant stress during
acetaminophen-induced hepatotoxicity in mice in vivo: The protective effect
of allopurinol. J. Pharmacol. Exp. Ther. 255, 935–941.
Jaeschke, H., Kleinwaechter, C., and Wendel, A. (1987). The role of acrolein
in allyl alcohol-induced lipid peroxidation and liver cell damage in mice.
Biochem. Pharmacol. 36, 51–57.
Jaeschke, H., and Mitchell, J. R. (1989). Mitochondria and xanthine oxidase
both generate reactive oxygen species after hypoxic damage in isolated
perfused rat liver. Biochem. Biophys. Res. Commun. 160, 140 –147.
Jaeschke, H., and Mitchell, J. R. (1990). Use of isolated perfused organs in
hypoxia and ischemia/reflow oxidant stress. Methods Enzymol. 186, 752–
759.
Jollow, D. J., Mitchell, J. R., Potter, W. Z., Davis, D. C., Gillette, J. R., and
Brodie, B. B. (1973). Acetaminophen-induced hepatic necrosis: II. Role of
covalent binding in vivo. J. Pharmacol. Exp. Ther. 187, 195–202.
Koppenol, W. H. (1998). The basic chemistry of nitrogen monoxide and
peroxynitrite. Free Radic. Biol. Med. 25, 385–391.
Laskin, D. L., Gardner, C. R., Price, V. F., and Jollow, D. J. (1995). Modulation of macrophage functioning abrogates the acute hepatotoxicity of
acetaminophen. Hepatology 21, 1045–1050.
Laskin, D. L., and Pilaro, M. (1986). Potential role of activated macrophages
in acetaminophen hepatotoxicity: I. Isolation and characterization of activated macrophages from rat liver. Toxicol. Appl. Pharmacol. 86, 204 –215.
Lawson, J. A., Farhood, A., Hopper, R. D., Bajt, M. L., and Jaeschke, H.
(2000). The hepatic inflammatory response after acetaminophen overdose:
Role of neutrophils. Toxicol. Sci. 54, 509 –516.
Lawson, J. A., Fisher, M. A., Simmons, C. A., Farhood, A., and Jaeschke, H.
(1999). Inhibition of Fas receptor (CD95)-induced hepatic caspase activation and apoptosis by acetaminophen in mice. Toxicol. Appl. Pharmacol.
156, 179 –186.
Liu, P., McGuire, G. M., Fisher, M. A., Farhood, A., Smith, C. W., and
Jaeschke, H. (1995). Activation of Kupffer cells and neutrophils for reactive
oxygen formation is responsible for endotoxin-enhanced liver injury after
hepatic ischemia. Shock 3, 56 – 62.
Matthews, A. M., Roberts, D. W., Hinson, J. A., and Pumford, N. R. (1996).
Acetaminophen-induced hepatotoxicity. Analysis of total covalent binding
vs. specific binding to cysteine. Drug Metab. Dispos. 24, 1192–1196.
Meyers, L. L., Beierschmitt, W. P., Khairallah, E. A., and Cohen, S. D. (1988).
Acetaminophen-induced inhibition of mitochondrial respiration in mice.
Toxicol. Appl. Pharmacol. 93, 378 –387.
Michael, S. L., Pumford, N. R., Mayeux, P. R., Niesman, M. R., and Hinson,
J. A. (1999). Pretreatment of mice with macrophage inactivators decreases
acetaminophen hepatotoxicity and the formation of reactive oxygen and
nitrogen species. Hepatology 30, 186 –195.
Mirochnitchenko, O., Weisbrot-Lefkowitz, M., Reuhl, K., Chen, L., Yang, C.,
and Inouye, M. (1999). Acetaminophen toxicity: Opposite effects of two
forms of glutathione peroxidase. J. Biol. Chem. 274, 10349 –10355.
Mitchell, J. R., Jollow, D. J., Potter, W. Z., Gillette, J. R., and Brodie, B. B.
(1973). Acetaminophen-induced hepatic necrosis: IV. Protective role of
glutathione. J. Pharmacol. Exp. Ther. 187, 211–217.
Pumford, N. R., Halmes, N. C., and Hinson, J. A. (1997). Covalent binding of
xenobiotics to specific proteins in the liver. Drug Metab. Rev. 29, 39 –57.
Pumford, N. R., Roberts, D. W., Benson, R. W., and Hinson, J. A. (1990).
Immunochemical quantitation of 3-(cystein-S-yl)acetaminophen protein adducts in subcellular liver fractions following a hepatotoxic dose of acetaminophen. Biochem. Pharmacol. 40, 573–579.
Qiu, Y., Benet, L. Z., and Burlingame, A. L. (1998). Identification of the
hepatic protein targets of reactive metabolites of acetaminophen in vivo in
mice using two-dimensional gel electrophoresis and mass spectrometry.
J. Biol. Chem. 273, 17940 –17953.
Ramsay, R. R., Rashed, M. S., and Nelson, S. D. (1989). In vitro effects of
acetaminophen metabolites and analogs on the respiration of mouse liver
mitochondria. Arch. Biochem. Biophys. 273, 449 – 457.
Sies, H., Sharov, V. S., Klotz, L. O., and Briviba, K. (1997). Glutathione
peroxidase protects against peroxynitrite-mediated oxidations. J. Biol.
Chem. 272, 27812–27817.
Smith, C. V., and Jaeschke, H. (1989). Effect of acetaminophen on the hepatic
content and biliary efflux of glutathione disulfide in mice. Chem. Biol.
Interactions 70, 241–248.
Squadrito, G. L., and Pryor, W. A. (1998). Oxidative chemistry of nitric oxide:
The role of superoxide, peroxynitrite and carbon dioxide. Free Radic. Biol.
Med. 25, 392– 403.
Tirmenstein, M. A., and Nelson, S. D. (1990). Acetaminophen-induced oxidation of protein thiols. Contribution of impaired thiol-metabolizing enzymes and the breakdown of adenine nucleotides. J. Biol. Chem. 265,
3059 –3065.