Mechanisms of Acetaminophen-Induced Hepatotoxicity: Role of Oxidative Stress and Mitochondrial Permeability Transition in Freshly Isolated Mouse Hepatocytes

Angela B. Reid, Richard C. Kurten, Sandra S. McCullough, Robert W. Brock, and
Jack A. Hinson
Departments of Pharmacology and Toxicology (A.B.R., S.S.M., R.W.B., J.A.H.) and Physiology and Biophysics (R.C.K.), College
of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas
Received August 10, 2004; accepted October 1, 2004

ABSTRACT
Freshly isolated mouse hepatocytes were used to determine
the role of mitochondrial permeability transition (MPT) in acetaminophen
(APAP) toxicity. Incubation of APAP (1 mM) with
hepatocytes resulted in cell death as indicated by increased
alanine aminotransferase in the media and propidium iodide
fluorescence. To separate metabolic events from later events in
toxicity, hepatocytes were preincubated with APAP for 2 h
followed by centrifugation of the cells and resuspension of the
pellet to remove the drug and reincubating the cells in media
alone. At 2 h, toxicity was not significantly different between
control and APAP-incubated cells; however, preincubation with
APAP followed by reincubation with media alone resulted in a
marked increase in toxicity at 3 to 5 h that was not different
from incubation with APAP for the entire time. Inclusion of
cyclosporine A, trifluoperazine, dithiothreitol (DTT), or N-acetylcysteine
(NAC) in the reincubation phase prevented hepatocyte
toxicity. Dichlorofluorescein fluorescence increased during the
reincubation phase, indicating increased oxidative stress. Tetramethylrhodamine
methyl ester perchlorate fluorescence decreased
during the reincubation phase indicating a loss of
mitochondrial membrane potential. Inclusion of cyclosporine A,
DTT, or NAC decreased oxidative stress and loss of mitochondrial
membrane potential. Confocal microscopy studies with
the dye calcein acetoxymethyl ester indicated that MPT had
also occurred. These data are consistent with a hypothesis
where APAP-induced cell death occurs by two phases, a metabolic
phase and an oxidative phase. The metabolic phase
occurs with GSH depletion and APAP-protein binding. The
oxidative phase occurs with increased oxidative stress, loss of
mitochondrial membrane potential, MPT, and toxicity.
Acetaminophen is a commonly used analgesic/antipyretic
that produces necrosis of the centrilobular cells of the liver
when taken in overdose (Bessems and Vermeulen, 2001;
James et al., 2003a). The initial step in toxicity is cytochrome
P-450 metabolism to the reactive metabolite N-acetyl-p-benzoquinone
imine (NAPQI). At therapeutic doses, NAPQI is
efficiently detoxified by glutathione (GSH). In overdose, conjugation
of the reactive metabolite with GSH leads to GSH
depletion, and NAPQI covalently binds to cysteine residues
on proteins to form acetaminophen adducts. Covalent binding
of NAPQI to proteins is an excellent correlate of acetaminophen
toxicity (Cohen et al., 1997; James et al., 2003a).
These adducts occur only in the hepatic centrilobular cells
that develop necrosis (Roberts et al., 1991), and toxicity has
not been reported to occur in the absence of their formation or
by GSH depletion alone (Mitchell et al., 1974). Necrosis is
believed to be the principal mechanism for toxicity. Since
acetaminophen toxicity does not occur with either an activation
of caspases or a substantial increase in apoptotic hepatocytes,
it has been concluded that its toxicity is not mediated
by an apoptotic mechanism (Lawson et al., 1999; Gujral et
al., 2002). Even though much is known about the importance
of acetaminophen metabolism leading to toxicity, the cellular
events producing necrosis are unknown.
We have previously shown that acetaminophen toxicity in
mice is accompanied by increased NO synthesis and by the
formation of nitrotyrosine-protein adducts (Hinson et al.,
This work was supported by National Institutes of Health Grants R01-GM-
58887 and T32-ES-10952. The use of the facilities in the University of Arkansas
for Medical Science’s Digital and Confocal Microscopy Laboratory was
supported by National Institutes of Health Grants 1 P20 RR 16460, PAR-98-
092, and 1-R24 CA82899.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.104.075945.

ABBREVIATIONS: NAPQI, N-acetyl-p-benzoquinoneimine; GSH, glutathione; MPT, mitochondrial permeability transition; APAP, acetaminophen;
NAC, N-acetylcysteine; AM, acetoxymethyl ester; TMRM, tetramethylrhodamine methyl ester perchlorate; DCF, 2 ,7 -dichlorodihydrofluorescein;
JC-1, 5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolcarbocyanine iodide; DTT, dithiothreitol; ALT, alanine aminotransferase; ROS, reactive
oxygen species.
0022-3565/05/3122-509–516$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 312, No. 2
Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 75945/1188007
JPET 312:509–516, 2005 Printed in U.S.A.
509
1998).

These adducts colocalize with acetaminophen-protein
adducts in the centrilobular areas of the liver. Nitrotyrosineprotein
adducts are believed to be formed by nitration of
tyrosine by peroxynitrite, a highly reactive species generated
from superoxide and NO. Peroxynitrite is normally detoxified
by GSH, but GSH is depleted by the reactive metabolite of
acetaminophen. It has been shown that the NO is primarily
from inducible nitric oxide synthase (Michael et al., 2001),
and multiple cell types (Kupffer cells, hepatocytes, and endothelial
cells) may participate in NO synthesis in the liver
following exposure to toxic doses of acetaminophen. The
source of the superoxide has not yet been defined. We originally
postulated that the superoxide was formed from
NADPH oxidase on Kupffer cells because we and others
found that acetaminophen toxicity was decreased following
administration of Kupffer cell inactivators (Blazka et al.,
1995; Laskin et al., 1995; Michael et al., 1999). However, we
recently showed that NADPH oxidase-deficient mice were
equally susceptible to acetaminophen toxicity and had similar
levels of nitrated tyrosine in the hepatic centrilobular
cells (James et al., 2003b). It was postulated that the superoxide
generation resulted from mitochondrial permeability
transition (MPT) (Jaeschke et al., 2003; James et al.,
2003a,b).
MPT is an abrupt increase in the permeability of the inner
mitochondrial membrane to ions and small molecular weight
solutes. Oxidants such as peroxides and peroxynitrite, Ca2 ,
and Pi promote MPT, whereas Mg2 , ADP, low pH, and high
membrane potential oppose the onset of MPT. Associated
with the permeability change is inner mitochondrial membrane
depolarization, uncoupling of oxidative phosphorylation,
release of intramitochondrial ions and metabolic intermediates,
and mitochondrial swelling. These changes result
in decreased ATP synthesis. MPT occurs with release of
superoxide from the mitochondria and is a lethal event for
the cell (Lemasters, 1999; Zorov et al., 2000). In this study,
we have investigated the role of MPT in acetaminopheninduced
hepatotoxicity in freshly isolated mouse hepatocytes
and the relationship of MPT to increased oxidative stress.

Materials and Methods
Reagents. Acetaminophen (APAP; 4-acetamidophenol), Hepes,
heparin sodium salt grade 1-A from porcine intestinal mucosa, penicillin
G sodium salt, Waymouth MB 752/1 media with glutamine
and without sodium bicarbonate, propidium iodide solution of 1
mg/ml in water, N-acetylcysteine (NAC), trifluoperazine, Percoll,
and Trypan Blue 0.4% solution were obtained from Sigma-Aldrich
(St. Louis, MO). Collagenase A from Clostridium histolyticum was
acquired from Roche Diagnostics (Indianapolis, IN). Cyclosporine A
was obtained from Bedford Laboratories (Bedford, OH). Calcein AM
of high-purity grade as well as tetramethylrhodamine methyl ester
perchlorate (TMRM), 2 ,7 -dichlorodihydrofluorescein (DCF) diacetate,
and 5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolcarbocyanine
iodide (JC-1) were purchased from Molecular Probes (Eugene,
OR). Dithiothreitol (DTT)-Cleland’s reagent of electrophoresis
purity (DTT) was acquired from Bio-Rad (Hercules, CA). Alanine
aminotransferase (ALT) (SGPT) colorimetrics was obtained from
TECO Diagnostics (Anaheim, CA). All chemicals were of the highest
grade commercially available.
Animals. Six-week-old male B6C3F1 mice were obtained from
Harlan (Indianapolis, IN). All animal experimentation and animal
protocols were approved by the University of Arkansas for Medical
Sciences Animal Care and Use Committee. Animal experiments
were carried out in accordance with the Guide for the Care and Use
of Laboratory Animals as adopted by the U.S. National Institutes of
Health. Mice were acclimatized 1 week prior to the experiments and
fed ad libitum until the time of sacrifice.
Hepatocyte Isolation and Incubations. Freshly isolated hepatocytes
were prepared from male B6C3F1 mice by collagenase perfusion
following a modification of the method of Grewal and Racz
(Grewal and Racz, 1993; Rafeiro et al., 1994). Briefly, for each individual
experiment, hepatocytes were isolated from a single mouse as
previously described followed by centrifugation at 140g for 8 min in
a 90% Percoll gradient to purify the cells followed by a wash in media
and a 3-min centrifugation at 140g to wash the Percoll from cells.
Preparations yielding 40 million cells and cell viability 90% as
determined by Trypan blue exclusion were used for the experiments.
The hepatocytes were incubated at a concentration of 1,000,000
cells/ml in Waymouth’s media (supplemented with 25 mM Hepes, 10
IU heparin/ml, and 500 IU penicillin G/ml) in 125-ml Erlenmeyer
flasks at 37°C under an atmosphere of 95% O2-5% CO2. APAP (1
mM) was added to experimental hepatocytes, but no APAP was
added to control flasks. At 2 h following drug addition, the hepatocytes
were centrifuged for 2 min at 140g. Supernatants were discarded,
and fresh media were added to the cells. This procedure was
repeated to remove the APAP not covalently bound and has been
reported to remove greater than 99% of the acetaminophen (Tee et
al., 1986). Following the final wash, some cells were resuspended in
fresh media containing 1 mM NAC, 10 M cyclosporine A, 10 M
trifluoperazine, or 0.5 mM DTT. The toxicity data were obtained
from three to four separate incubations that were performed on
separate mice on different days.
Toxicity Assays. Toxicity was quantified by the presence of ALT
in the media, which occurred as a result of lysis of or leakage from
hepatocytes. ALT in the media was determined with a colorimetric
endpoint method using a commercial kit (TECO Diagnostics).
Briefly, the hepatocytes were separated from the media by centrifugation.
ALT substrate (50 l) was added to each sample of media (10
l) followed by incubation in a heating bath at 37°C for 30 min. ALT
Color Reagent (50 l) was subsequently added to each sample and
reincubated for 10 min. ALT Color Developer (200 l) was added to
each sample, and the mixture was placed in heating bath for 5 min.
The absorbance of samples was determined spectrophotometrically
in a Bio-Rad 550 plate reader at a wavelength of 490 nm. The
relative absorbance was used to calculate the ALT values as described
in the kit. Cell death was also determined by addition of
propidium iodide to the cell suspension. Upon lysis of hepatocytes,
propidium iodide enters the cell and binds DNA resulting in an
increase in fluorescence. The increased fluorescence can be quantified
as a relative index of cell death. Using a modified method of
Nieminen et al. (1995), aliquots of hepatocytes (1 ml) were placed in
12-well plates, and the cells were incubated with 30 M propidium
iodide solution at room temperature for 30 min. Fluorescence in each
well was measured using a Millipore CytoFluor 2350 fluorescence
scanner (Millipore Corporation, Billerica, MA) using 560-nm excitation
and 645-nm emission filters.
Fluorescence Assays. The relative mitochondrial membrane potential
was determined by a mitochondrial membrane specific cationic
dye JC-1 (Reers et al., 1995). JC-1 enters the mitochondria
based on high negative membrane potential. JC-1 emits fluorescence
as a monomer at 535 nm or an aggregate at 590 nm. The monomer
indicates a low membrane potential, whereas the aggregate indicates
a high membrane potential. Briefly, hepatocytes of 2-ml aliquots
were centrifuged at 140g for 2 min and supernatants discarded.
Cells were resuspended with 6.5 M JC-1 in 3 ml of JC-1
buffer (containing 137 mM NaCl, 3.6 mM KCl, 0.5 mM MgCl2, 1.8
mM CaCl2, and 10 mM Hepes) and incubated for 25 min at 37°C in
atmosphere of 95% O2-5% CO2. Following incubation, cells were
centrifuged and washed to remove excess dye, resuspended in 2 ml of
JC-1 buffer, and excited at 490 nm in a Hitachi F-2500 Fluorescence
Spectrophotometer. Membrane potential was calculated using Hita-
510 Reid et al.
chi Fluorescence Spectrophotometer FL Solutions software to calculate
the area under the curve of the monomer and aggregate peak,
and a 590:535 nm ratio of the peaks was determined using DataQWindaq
software.
The production of ROS was determined in isolated hepatocytes
using the dye DCF diacetate. In the hepatocytes, endogenous esterases
hydrolyze the acetate ester, trapping free DCF inside the cells.
ROS convert the nonfluorescent dichlorofluorescein to the highly
impermeable fluorescent dichlorofluorescein. Briefly, hepatocytes of
2-ml aliquots were centrifuged at 140g for 2 min and supernatants
discarded. Cells were resuspended with DCF (100 M) in 3 ml of
phosphate-buffered saline and incubated for 25 min at 37°C in atmosphere
of 95% O2-5% CO2. Following incubation, cells were centrifuged
and washed free of excess dye, resuspended in 2 ml of
phosphate-buffered saline, and excited at 488 nm in a Hitachi F-2500
Fluorescence Spectrophotometer. Oxidative stress was indicated by
Hitachi Fluorescence Spectrophotometer FL Solutions software used
to measure the 53- nm peak to determine DCF production.
Confocal Microscopy Assays. MPT was observed on a Zeiss
LSM 410 confocal microscope following the methods of Nieminen et
al. (1995) with modifications. Briefly, freshly isolated hepatocytes
were placed in media on collagen/fibronectin-coated dishes containing
acetaminophen (1 mM) for treated cells and media alone for
control cells and allowed to adhere for approximately 2 h. Following
cell adhesion, the cells were subsequently washed free of acetaminophen
and reincubated in media alone, media plus cyclosporine A (10
M), or media plus trifluoperazine (10 M). Hepatocytes were subsequently
labeled as previously described (Nieminen et al., 1995)
with 500 nM TMRM for 15 min followed by TMRM plus 1 MCalcein
AM for 15 min. Following cell labeling, media were washed from cells
to remove fluorescent tracers, and fresh media were placed back on
the cells. The plated cells were then placed on the confocal microscope
and mitochondria observed with images taken at h 4. The color
intensity was adjusted in Adobe Photoshop using the auto contrast
and auto level functions. Some experiments used APAP-treated and
control cells that were labeled with 2 ,7 -dichlorodihydrofluorescin
diacetate as an oxidative stress determinant in the confocal microscopy
studies. The color intensity of the auto contrast and auto levels
was adjusted in Adobe Photoshop.
Statistical Analyses. Analysis of variance was performed with a
Tukey post hoc test using the SPSS 9.0 program (SPSS, Chicago, IL).
Statistical significance was defined as experimental being p 0.05
from control.

Results
Acetaminophen Toxicity in Freshly Isolated Mouse
Hepatocytes. Davies, Boobis, and coworkers (Boobis et al.,
1986; Tee et al., 1986) and Racz and coworkers (Grewal and
Racz, 1993; Rafeiro et al., 1994) previously showed that incubation
of acetaminophen with freshly isolated hepatocytes
followed by washing the hepatocytes to remove acetaminophen
and subsequent reincubation of the hepatocytes with
media alone resulted in significant toxicity during the reincubation
phase. We have used this as one approach to study
the role of MPT, alteration of mitochondrial membrane potential,
and production of oxidative stress in acetaminophen
toxicity.
In initial experiments, acetaminophen (1 mM) was incubated
with freshly isolated hepatocytes (Fig. 1). At the end of
1 and 2 h, hepatocytes were centrifuged and washed twice to
remove acetaminophen followed by resuspension in media
alone. The hepatocytes were subsequently reincubated. Acetaminophen
was added back to other hepatocyte incubations
for the full length of the incubation. Control hepatocytes were
incubated with media alone for 2 h, washed twice, and resuspended
in media alone. Cell death was determined by measuring
increased propidium iodide fluorescence, an event
that occurs as a result of membrane damage and permits
propidium iodide to bind DNA (Nieminen et al., 1992). Toxicity
was also determined by measuring the presence of ALT
in the media that occurs as a result of hepatocyte lysis or
leakage. As shown in Fig. 1, incubation of acetaminophen for
2 h followed by washing the cells resulted in toxicity at 5 h,
and the relative amount of toxicity was not significantly
different from the amount of toxicity when acetaminophen
was added back to the hepatocytes. However, when the cells
were washed at 1 h and reincubated, toxicity did not significantly
increase. Incubation in media alone did not result in
a significant increase in toxicity. We have previously shown
in these mice that 2 h is a critical window for in vivo toxicity.
GSH depletion and covalent binding to proteins are maximal
at this time, and this time is immediately before toxicity
occurs (James et al., 2003c). The effect of acetaminophen on
GSH depletion and covalent binding to proteins was determined
in the hepatocytes. In the isolated hepatocytes, we
found that GSH was depleted by approximately 50% at 0.5 h
and maximally (93%) at 1 h. Covalent binding had occurred
by 2 h (data not shown). From these data, we chose 2 h as the
optimal time to wash the hepatocytes free of acetaminophen
and determine the effect of potential inhibitors of toxicity.
Acetaminophen-Induced Mitochondrial Permeability
Transition in Mouse Hepatocytes. To determine the
role of MPT in acetaminophen-induced toxicity in mouse
hepatocytes, cyclosporine A and trifluoperazine were utilized.
Cyclosporine A and trifluoperazine are known inhibitors
of MPT (Nieminen et al., 1995, 1997; Crompton et al.,
1999; Lemasters, 1999). Acetaminophen was incubated with
hepatocytes, and at 2 h, the hepatocytes were washed free of
acetaminophen. The hepatocytes were subsequently resuspended
in media containing the MPT inhibitors. As shown in
Fig. 1. Time course for development of acetaminophen
toxicity. Freshly isolated mouse hepatocytes
were incubated with acetaminophen (1 mM)
throughout the course of the experiment (APAP) for
1 h (W1H) or 2 h (W2H). At the indicated time, the
hepatocytes were washed twice by centrifugation
followed by resuspension in media containing 1mM
APAP or in media alone (arrow) (Control, W1H,
W2H). Toxicity was determined at the designated
time. Controls were incubated with media alone,
washed twice at 2 h, and resuspended in media
alone. A, toxicity determined by measurement of
ALT into the media. B, toxicity determined by increased
fluorescence following addition of propidium
iodide. , samples significantly different
from control (p 0.05).
MPT in Acetaminophen Hepatotoxicity 511
Fig. 2, A through D, both cyclosporine A (10 M) and trifluoperazine
(10 M) inhibited further development of toxicity
in the hepatocytes as determined by ALT release (Fig. 2, A
and C) and by propidium iodide fluorescence (Fig. 2, B and
D). Likewise, addition of the dithiol reagent, DTT (0.5 mM),
completely eliminated toxicity (Fig. 2, E and F). Dithiothreitol
has been previously reported to reduce dithiols at the
MPT pore and eliminate MPT (Petronilli et al., 1994).
To further evaluate the role of MPT in acetaminophen
toxicity, confocal microscopy studies were pursued using the
dye calcein AM. This dye enters the hepatocytes and is hydrolyzed
by esterases to yield a nondiffusible ionized species.
The green dye labels the cytosol green and is normally excluded
from mitochondria, but following MPT, the dye enters
the mitochondria (Lemasters, 1999). Also, TMRM associates
with mitochondria possessing high membrane potential,
thus, labeling the mitochondria. Figure 3A shows mitochondria
(dark red spheres) in the control hepatocytes, incubated
with media alone, excluding the calcein. Figure 3B shows
hepatocytes that have been incubated with acetaminophen
for 4 h. As shown, the dye has entered the mitochondria, and
the mitochondria are no longer visible, nor are they compart-
Fig. 2. Effect of cyclosporine A, trifluoperazine, dithiothreitol,
and N-acetylcysteine on acetaminophen
toxicity. Freshly isolated mouse hepatocytes were
incubated with 1 mM acetaminophen. At the end of
2 h, the hepatocytes were washed twice by centrifugation
followed by resuspension in media. Subsequently,
10 M cyclosporine A (A and B), 10 M
trifluoperazine (C and D), 0.5 mM dithiothreitol (E
and F), or 1.0 mM N-acetylcysteine (G and H) were
added to the media (arrow), and the incubation continued.
Control samples were treated identically to
experimental samples except that they were incubated
with media only. Toxicity was determined at
the indicated times. A, C, E, and G, effects on toxicity
as determined by release of ALT into the media.
B, D, F, and H, effects on toxicity as determined by
increase in fluorescence following the addition of
propidium iodide. , samples significantly increased
from the same 2-h incubation (p 0.05).
512 Reid et al.
mentalized from the cytosol. These data indicate that acetaminophen
produced MPT in the hepatocytes. Figure 3D
shows that the MPT inhibitor trifluoperazine (10 M) inhibited
the entrance of the dye into the mitochondria and maintained
compartmentalization. Figure 3C shows that cyclosporine
A (10 M) also inhibited this effect.
Acetaminophen-Induced Alterations in Mitochondrial
Membrane Potential. MPT is accompanied by decreases
in mitochondrial membrane potential. A time course
for loss of mitochondrial membrane potential ( m) was
determined using the dye JC-1. JC-1 is a cationic dye that
exhibits potential-dependent accumulation and formation of
red fluorescent J-aggregates in mitochondria that have high
m. In contrast, the JC-1 exists as a monomer that produces
green fluorescence in the cytoplasm and mitochondria
that have low m. Formation of J-aggregates in the mitochondria
is indicated by a fluorescence emission shift from
green (535 nm) to red (590 nm). Mitochondrial depolarization
is indicated by a decrease in the red/green fluorescence intensity
ratio (Reers et al., 1995). In this experiment, hepatocytes
were incubated with acetaminophen or media, and at
2 h, the cells were washed and reincubated in media alone. At
the indicated time, JC-1 was added to an aliquot of cells, and
the relative fluorescence was determined as described under
Materials and Methods. Figure 4 shows that mitochondrial
membrane potential remained high for the 5-h incubation
time in the control hepatocytes that were incubated in media
alone. In hepatocytes initially incubated with acetaminophen
and subsequently incubated with media alone, mitochondrial
membrane potential in the hepatocytes was decreased by
80% at 5 h. Other hepatocytes were incubated with acetaminophen
in the preincubation phase and incubated with the
MPT inhibitor cyclosporine A (10 M) in the reincubation
phase. As shown in Fig. 4, cyclosporine A inhibited the loss of
mitochondrial membrane potential. Thus, the inhibitor of
MPT decreased the loss of membrane potential.
Acetaminophen-Induced Oxidative Stress. To determine
the role of oxidative stress in acetaminophen toxicity,
mouse hepatocytes were incubated with acetaminophen for
2 h followed by washing the cells to remove acetaminophen.
The antioxidant NAC (1 mM) was then added in the late
phase (after 2 h). NAC completely eliminated toxicity (Fig. 2,
G and H) and also eliminated the loss of mitochondrial membrane
potential (Fig. 4).
The role of oxidative stress in acetaminophen toxicity was
further evaluated using the dye DCF. This dye is converted
by esterases in the cell to an impermeable product. Upon
oxidation, it is converted to the green fluorescent product
dichlorofluorescein (Myhre et al., 2003). In Fig. 5A, confocal
microscopy was utilized to show that incubation of hepatocytes
with acetaminophen resulted in increased fluorescence,
whereas fluorescence was not observed in control hepatocytes.
Thus, acetaminophen toxicity is accompanied by increased
oxidative stress. In Fig. 5B, the relative increased
fluorescence in the hepatocytes was quantified using fluorometric
analysis. Hepatocytes were incubated with acetaminophen
for 2 h followed by washing to remove acetaminophen.
Reincubation of hepatocytes in the presence of the dye DCF
for 0.5 h resulted in an increase in fluorescence over time.
Addition of cyclosporine A (10 M) or N-acetylcysteine (1
mM) to hepatocytes in this reincubation phase significantly
attenuated the large increase in fluorescence. There was no
increase in fluorescence in control hepatocytes that were not
incubated with acetaminophen.
Discussion
This study examines the hypothesis that the toxicity of
acetaminophen occurs as a result of MPT. MPT is an important
mechanism in various hepatotoxicities. MPT is an
abrupt increase in the permeability of the inner mitochondrial
membrane to small molecular weight solutes. It occurs
with membrane depolarization, uncoupling of oxidative phosphorylation,
release of intramitochondrial ions and metabolic
intermediates, and mitochondrial swelling. MPT is a lethal
event for the cell (Crompton et al., 1999; Lemasters, 1999;
Kim et al., 2003) and occurs with release of superoxide. Thus,
MPT can be mediated by oxidant stress and cause increased
oxidant stress. It was previously postulated to be the source
of superoxide leading to peroxynitrite and tyrosine nitration
in acetaminophen-induced hepatotoxicity (Jaeschke et al.,
2003; James et al., 2003a,b).
In this study, we utilized freshly isolated mouse hepatocytes
in suspension to determine the role of MPT in acetaminophen
toxicity because these hepatocytes have high levels of cytochrome
P-450 enzymes necessary for acetaminophen metabolism
and subsequent toxicity (James et al., 2003a). Cultured
hepatocytes (cells that have been isolated and plated for more
Fig. 3. Effect of acetaminophen on MPT in individual hepatocytes.
Freshly isolated mouse hepatocytes were incubated with media containing
or absent of APAP (1 mM) for 2 h. Cells were subsequently washed to
remove excess APAP and reincubated with either media alone or with
media containing 10 M cyclosporin A or 10 M trifluoperazine. Cells
were subsequently labeled with 500 nM tetramethylrhodamine methyl
ester for 30 min and 1 M calcein AM for 15 min. The hepatocytes were
subsequently washed to remove excess dye and visualized by confocal
microscopy at 4 h with a 63 objective. A, hepatocytes from control
incubations. B, hepatocytes incubated with acetaminophen. C, hepatocytes
incubated with acetaminophen followed by cyclosporine A. D, hepatocytes
incubated with acetaminophen followed by trifluoperazine.
MPT in Acetaminophen Hepatotoxicity 513
than 18–24 h) have significantly less cytochrome P-450 (Steward
et al., 1985; Wu et al., 1990) and are not highly sensitive to
the toxic effects of acetaminophen (Harman et al., 1991). We
followed the approach of Davies and Boobis (Boobis et al., 1986;
Tee et al., 1986), in which hepatocytes were incubated with
acetaminophen for 2 h, subsequently washed free of acetaminophen,
and reincubated in media alone. The washing step removes
greater than 99% of the acetaminophen (Tee et al., 1986),
thereby permitting an analysis of events occurring after the
metabolic phase. As shown in Fig. 1, incubation of the hepatocytes
with acetaminophen for 2 h produced an insignificant
increase in toxicity. Subsequent reincubation of the washed
hepatocytes with media alone resulted in significant toxicity
during the reincubation phase. Addition of acetaminophen in
the reincubation did not alter toxicity (Fig. 1). Incubating hepatocytes
with acetaminophen for only 1 h did not result in increased
toxicity (Fig. 1). These data indicate acetaminophen
toxicity occurs in two phases (Fig. 6). The first phase depends
upon the presence of acetaminophen for at least 2 h and involves
acetaminophen metabolism to the reactive metabolite
NAPQI leading to GSH depletion and covalent binding of
NAPQI to proteins. The second phase does not depend upon the
presence of acetaminophen. Consistent with previous reports
(Boobis et al., 1986; Tee et al., 1986; Grewal and Racz, 1993;
Rafeiro et al., 1994), we found that GSH was depleted maximally
(93%) by 1 h, and Western blot studies indicated covalent
binding by 2 h (data not shown).
To determine the role of MPT in the second phase, the
effects of inhibitors and the fluorescence of specific dyes were
monitored. The addition of inhibitors at the beginning of the
reincubation phase eliminated the possibility that the effects
were related to inhibition of cytochrome P-450 metabolism of
acetaminophen. Elimination of toxicity by both cyclosporine
A (Fig. 2, A and B) and trifluoperazine (Fig. 2, C and D)
strongly suggests involvement of MPT in acetaminophen
hepatotoxicity. Cyclosporine A associates with cyclophilin D
in the MPT pore and has been shown to be a specific and
potent inhibitor of MPT (Petronilli et al., 1994; Crompton et
al., 1999; Lemasters, 1999). Furthermore, dithiothreitol also
completely eliminate acetaminophen toxicity (Fig. 2, E and F). Oxidation of critical thiols at the pore results in MPT, and
dithiothreitol reduces these disulfides thereby preventing
MPT (Petronilli et al., 1994; Nieminen et al., 1997). Thus, the
inhibitor data are consistent with the second phase of acetaminophen
toxicity being mediated by MPT.
Confocal microscopy studies were performed using the dye
calcein AM to visualize MPT during acetaminophen toxicity
(Fig. 3). This dye is normally excluded from mitochondria and
results in the appearance of a fluorescent background. We used
TMRM, a dark red dye that fluoresces in mitochondria with a
high membrane potential, to confirm that these spheres were in
fact mitochondria. Figure 3A shows mitochondria as red
Fig. 4. Time course for effect of acetaminophen on mitochondrial membrane
potential in hepatocyte incubations. Freshly isolated mouse hepatocytes
were incubated with media alone or with 1 mM acetaminophen.
At 2 h, hepatocytes were washed twice and resuspended in media alone.
At the indicated time, 6.5 M JC-1 was added, and relative fluorescence
was determined on an aliquot of hepatocytes. To some incubations, 10 M
cyclosporine A (APAP CSP) was added, and 1 mM N-acetylcysteine
(APAP NAC) was added to other incubations. , samples significantly
decreased from the same 2-h incubation (p 0.05).
Fig. 5. Effect of acetaminophen on oxidative stress in individual hepatocytes.
A, freshly isolated mouse hepatocytes incubated with 100 M DCF
for 30 min followed by washing to remove excess dye. Hepatocytes were
subsequently incubated with 1 mM APAP or media alone (Control).
Individual hepatocytes were visualized for relative fluorescence by confocal
microscopy at 3 h with a 40 objective. B, hepatocytes incubated
with 1 mM acetaminophen for 2 h and subsequently washed and reincubated
in media alone. To some hepatocytes, 10 M cyclosporine A or 1
mM NAC was added. At the indicated time, DCF was added, and fluorescence
was determined 0.5 h later. , samples that significantly increased
from the same 2-h incubation (p 0.05).
Fig. 6. Postulated mechanism of acetaminophen toxicity.
514 Reid et al.
spheres and calcein being excluded from the mitochondria. Following
MPT, mitochondrial membrane potential is lost, and the
mitochondria no longer exclude calcein (Lemasters, 1999). Figure
3B shows that in acetaminophen-treated hepatocytes, the
red spheres are no longer present indicating that calcein has
entered the mitochondria. Figure 3, C and D, show that inhibitors
of MPT blocked this effect and maintained compartmentalization
between mitochondria and cytosol. These data further
suggest that acetaminophen toxicity occurs with MPT.
Since MPT occurs with loss of mitochondrial membrane
potential, a time course for loss of mitochondrial membrane
potential was performed using JC-1. JC-1 is a cationic dye
that exhibits potential-dependent accumulation and formation
of red fluorescent J-aggregates in mitochondria that
have high m. There was a loss of mitochondrial membrane
potential in the acetaminophen-treated hepatocytes (2–5 h)
but not in the control untreated hepatocytes (Fig. 4). Inclusion
of cyclosporine A, an inhibitor of MPT, in the reincubation
phase prevented loss of mitochondrial membrane potential
(Fig. 4). Thus, the finding that cyclosporine A inhibits not
only APAP toxicity but also the accompanying loss of mitochondrial
membrane potential supports the hypothesis that
MPT is a critical event in toxicity.
MPT may occur as a result of oxidative stress and leads to
additional oxidative stress (Lemasters, 1999). Zorov et al.
(2000) have called this ROS-induced ROS release. The role of
oxidative stress in acetaminophen toxicity was determined
using DCF, a dye converted to a fluorescent derivative by
oxidative stress. Confocal microscopy analysis indicated increased
hepatocyte fluorescence (Fig. 5A). Other hepatocytes
were incubated with acetaminophen to show a time-dependent
increase in fluorescence (Fig. 5B). Addition of cyclosporine
A eliminated the increased fluorescence (Fig. 5B). These
data are consistent with MPT being the source of the large
increase in oxidative stress that occurs in acetaminophen
toxicity. These data, coupled with the finding that cyclosporine
A inhibits toxicity and loss of mitochondrial membrane
potential, support the hypothesis that oxidative stress leading
to MPT is the cause of toxicity and that this occurs in a
distinct phase following APAP metabolism to NAPQI.
Further support for oxidative stress and MPT being critical
events in acetaminophen toxicity was determined using
NAC. Addition of NAC in the reincubation phase eliminated
toxicity (Fig. 2, G and H), eliminated loss of mitochondrial
membrane potential (Fig. 4), and eliminated increased oxidative
stress (Fig. 5). This compound is the primary antidote
given to acetaminophen overdose victims. It is believed to
prevent toxicity by increasing detoxification of the reactive
metabolite NAPQI by a direct reaction or through increase in
GSH (Bessems and Vermeulen, 2001; James et al., 2003a).
The finding that NAC completely eliminated acetaminophen
toxicity, the increased oxidative stress, and the loss of mitochondrial
membrane potential suggests that NAC may play a
major role in preventing acetaminophen toxicity in humans
not only by detoxifying NAPQI but also by preventing MPT.
The mechanism of how the metabolic phase leads to the
oxidative phase and the nature of the oxidizing species in
MPT is unclear. MPT is believed to occur with release of
superoxide, and this species may react with nitric oxide to
form peroxynitrite or may be reduced to form peroxide. Both
peroxynitrite and/or peroxide may be important in the initiation
and/or propagation of MPT. Both are detoxified by GSH
(Sies et al., 1997), and GSH is depleted in hepatocytes by
NAPQI in the metabolic phase. We reported previously that
nitrotyrosine occurred in the necrotic centrilobular hepatocytes
of mice treated with acetaminophen (Hinson et al.,
1998), which suggested involvement of peroxynitrite. Peroxynitrite
is not only a nitrating agent, but it is also an
oxidizing agent (Radi et al., 1991) and has been reported to
produce MPT (Packer et al., 1997). Moreover, peroxynitrite
oxidizes DCF to a fluorescent product (Myhre et al., 2003).
Thus, peroxynitrite is an excellent candidate as the toxicant.
Alternatively, peroxide may be important. Peroxide plus ferrous
iron leads to the hydroxyl radical (Fenton mechanism),
a potent oxidant. Iron chelators have been reported to decrease
acetaminophen toxicity in cultured cells and delay
toxicity in mice (Harman et al., 1991; Schnellmann et al.,
1999). Covalent binding to an iron-containing protein in the
metabolic phase could conceivably lead to the release and
accumulation of free iron. Moreover, Fenton mechanisms will
also oxidize DCF to a fluorescent product (Myhre et al.,
2003). Thus, iron may be mechanistically important. Also,
calcium has been implicated in acetaminophen toxicity (Ray
et al., 1993), and calcium causes MPT (Lemasters, 1999).
Boobis et al. (1990) found that the calcium chelator Quin 2
decreased acetaminophen toxicity in hamster hepatocytes
when added in the reincubation phase. Thus, even though
the data indicate MPT in acetaminophen toxicity, understanding
the complex scenario of events by which the metabolic
phase leads to the oxidative phase and toxicity will
require further investigation.
Acknowledgments
We thank LeeAnn MacMillan-Crow and Danielle Cruthirds for
discussions regarding the assay for mitochondrial membrane potential.

References
Bessems JG and Vermeulen NP (2001) Paracetamol (acetaminophen)-induced toxicity:
molecular and biochemical mechanisms, analogues and protective approaches.
Crit Rev Toxicol 31:55–138.
Blazka ME, Wilmer JL, Holladay SD, Wilson RE, and Luster MI (1995) Role of
proinflammatory cytokines in acetaminophen hepatotoxicity. Toxicol Appl Pharmacol
133:43–52.
Boobis AR, Seddon CE, Nasseri-Sina P, and Davies DS (1990) Evidence for a direct
role of intracellular calcium in paracetamol toxicity. Biochem Pharmacol 39:1277–
1281.
Boobis AR, Tee LB, Hampden CE, and Davies DS (1986) Freshly isolated hepatocytes
as a model for studying the toxicity of paracetamol. Food Chem Toxicol
24:731–736.
Cohen SD, Pumford NR, Khairallah EA, Boekelheide K, Pohl LR, Amouzadeh HR,
and Hinson JA (1997) Selective protein covalent binding and target organ toxicity.
Toxicol Appl Pharmacol 143:1–12.
Crompton M, Virji S, Doyle V, Johnson N, and Ward JM (1999) The mitochondrial
permeability transition pore. Biochem Soc Symp 66:167–179.
Grewal KK and Racz WJ (1993) Intracellular calcium disruption as a secondary
event in acetaminophen-induced hepatotoxicity. Can J Physiol Pharmacol 71:26–
33.
Gujral JS, Knight TR, Farhood A, Bajt ML, and Jaeschke H (2002) Mode of cell death
after acetaminophen overdose in mice: apoptosis or oncotic necrosis? Toxicol Sci
67:322–328.
Harman AW, Kyle ME, Serroni A, and Farber JL (1991) The killing of cultured
hepatocytes by N-acetyl-p-benzoquinone imine (NAPQI) as a model of the cytotoxicity
of acetaminophen. Biochem Pharmacol 41:1111–1117.
Hinson JA, Pike SL, Pumford NR, and Mayeux PR (1998) Nitrotyrosine-protein
adducts in hepatic centrilobular areas following toxic doses of acetaminophen in
mice. Chem Res Toxicol 11:604–607.
Jaeschke H, Knight TR, and Bajt ML (2003) The role of oxidant stress and reactive
nitrogen species in acetaminophen hepatotoxicity. Toxicol Lett 144:279–288.
James LP, Mayeux PR, and Hinson JA (2003a) Acetaminophen-induced hepatotoxicity.
Drug Metab Dispos 31:1499–1506.
James LP, McCullough SS, Knight TR, Jaeschke H, and Hinson JA (2003b) Acetaminophen
toxicity in mice lacking NADPH oxidase activity: role of peroxynitrite
formation and mitochondrial oxidant stress. Free Radic Res 37:1289–1297.
James LP, McCullough SS, Lamps LW, and Hinson JA (2003c) Effect of N-acetyl-
MPT in Acetaminophen Hepatotoxicity 515
cysteine on acetaminophen toxicity in mice: relationship to reactive nitrogen and
cytokine formation. Toxicol Sci 75:458–467.
Kim JS, He L, and Lemasters JJ (2003) Mitochondrial permeability transition: a
common pathway to necrosis and apoptosis. Biochem Biophys Res Commun 304:
463–470.
Laskin DL, Gardner CR, Price VF, and Jollow DJ (1995) Modulation of macrophage
functioning abrogates the acute hepatotoxicity of acetaminophen. Hepatology 21:
1045–1050.
Lawson JA, Fisher MA, Simmons CA, 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.
Lemasters JJ (1999) V. Necrapoptosis and the mitochondrial permeability transition:
shared pathways to necrosis and apoptosis. Am J Physiol 276:G1–G6.
Michael SL, Mayeux PR, Bucci TJ, Warbritton AR, Irwin LK, Pumford NR, and
Hinson JA (2001) Acetaminophen-induced hepatotoxicity in mice lacking inducible
nitric oxide synthase activity. Nitric Oxide 5:432–441.
Michael SL, Pumford NR, Mayeux PR, Niesman MR, and Hinson JA (1999) Pretreatment
of mice with macrophage inactivators decreases acetaminophen hepatotoxicity
and the formation of reactive oxygen and nitrogen species. Hepatology
30:186–195.
Mitchell JR, Thorgeirsson SS, Potter WZ, Jollow DJ, and Keiser H (1974) Acetaminophen-
induced hepatic injury: protective role of glutathione in man and rationale
for therapy. Clin Pharmacol Ther 16:676–684.
Myhre O, Andersen JM, Aarnes H, and Fonnum F (2003) Evaluation of the probes
2 ,7 -dichlorofluorescin diacetate, luminol and lucigenin as indicators of reactive
species formation. Biochem Pharmacol 65:1575–1582.
Nieminen AL, Byrne AM, Herman B, and Lemasters JJ (1997) Mitochondrial permeability
transition in hepatocytes induced by t-BuOOH: NAD(P)H and reactive
oxygen species. Am J Physiol 272:C1286–1294.
Nieminen AL, Gores GJ, Bond JM, Imberti R, Herman B, and Lemasters JJ (1992)
A novel cytotoxicity screening assay using a multiwell fluorescence scanner. Toxicol
Appl Pharmacol 115:147–155.
Nieminen AL, Saylor AK, Tesfai SA, Herman B, and Lemasters JJ (1995) Contribution
of the mitochondrial permeability transition to lethal injury after exposure
of hepatocytes to t-butylhydroperoxide. Biochem J 307:99–106.
Packer MA, Scarlett JL, Martin SW, and Murphy MP (1997) Induction of the
mitochondrial permeability transition by peroxynitrite. Biochem Soc Trans 25:
909–914.
Petronilli V, Costantini P, Scorrano L, Colonna R, Passamonti S, and Bernardi P
(1994) The voltage sensor of the mitochondrial permeability transition pore is
tuned by the oxidation-reduction state of vicinal thiols: increase of the gating
potential by oxidants and its reversal by reducing agents. J Biol Chem 269:16638–
16642.
Radi R, Beckman JS, Bush KM, and Freeman BA (1991) Peroxynitrite oxidation of
sulfhydryls: the cytotoxic potential of superoxide and nitric oxide. J Biol Chem
266:4244–4250.
Rafeiro E, Barr SG, Harrison JJ, and Racz WJ (1994) Effects of N-acetylcysteine and
dithiothreitol on glutathione and protein thiol replenishment during acetaminophen-
induced toxicity in isolated mouse hepatocytes. Toxicology 93:209–224.
Ray SD, Kamendulis LM, Gurule MW, Yorkin RD, and Corcoran GB (1993) Ca2
antagonists inhibit DNA fragmentation and toxic cell death induced by acetaminophen.
FASEB J 7:453–463.
Reers M, Smiley ST, Mottola-Hartshorn C, Chen A, Lin M, and Chen LB (1995)
Mitochondrial membrane potential monitored by JC-1 dye. Methods Enzymol
260:406–417.
Roberts DW, Bucci TJ, Benson RW, Warbritton AR, McRae TA, Pumford NR, and
Hinson JA (1991) Immunohistochemical localization and quantification of the
3-(cystein-S-yl)-acetaminophen protein adduct in acetaminophen hepatotoxicity.
Am J Pathol 138:359–371.
Schnellmann JG, Pumford NR, Kusewitt DF, Bucci TJ, and Hinson JA (1999)
Deferoxamine delays the development of the hepatotoxicity of acetaminophen in
mice. Toxicol Lett 106:79–88.
Sies H, Sharov VS, Klotz LO, and Briviba K (1997) Glutathione peroxidase protects
against peroxynitrite-mediated oxidations: a new function for selenoproteins as
peroxynitrite reductase. J Biol Chem 272:27812–27817.
Steward AR, Dannan GA, Guzelian PS, and Guengerich FP (1985) Changes in the
concentration of seven forms of cytochrome P-450 in primary cultures of adult rat
hepatocytes. Mol Pharmacol 27:125–132.
Tee LB, Boobis AR, Huggett AC, and Davies DS (1986) Reversal of acetaminophen
toxicity in isolated hamster hepatocytes by dithiothreitol. Toxicol Appl Pharmacol
83:294–314.
Wu DF, Clejan L, Potter B, and Cederbaum AI (1990) Rapid decrease of cytochrome
P-450IIE1 in primary hepatocyte culture and its maintenance by added 4-methylpyrazole.
Hepatology 12:1379–1389.
Zorov DB, Filburn CR, Klotz LO, Zweier JL, and Sollott SJ (2000) Reactive oxygen
species (ROS)-induced ROS release: a new phenomenon accompanying induction
of the mitochondrial permeability transition in cardiac myocytes. J Exp Med
192:1001–1014.
Address correspondence to: Jack A. Hinson, Department of Pharmacology
and Toxicology, Slot 638, University of Arkansas for Medical Sciences, Little
Rock, AR 72205. E-mail: hinsonjacka@uams.edu
516 Reid et al.