Ni Hms 406767

Acetaminophen-induced Liver Injury in Rats and Mice:
Comparison of Protein Adducts, Mitochondrial Dysfunction, and

Oxidative Stress in the Mechanism of Toxicity
Mitchell R. McGill
*
, C. David Williams
*
, Yuchao Xie, Anup Ramachandran, and Hartmut
Jaeschke
Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical
Center, Kansas City, KS, USA
Abstract
Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the West. In
mice, APAP hepatotoxicity can be rapidly induced with a single dose. Because it is both clinically
relevant and experimentally convenient, APAP intoxication has become a popular model of liver
injury. Early data demonstrated that rats are resistant to APAP toxicity. As a result, mice are the
preferred species for mechanistic studies. Furthermore, recent work has shown that the
mechanisms of APAP toxicity in humans are similar to mice. Nevertheless, some investigators
still use rats. New mechanistic information from the last forty years invites a reevaluation of the
differences between these species. Comparison may provide interesting insights and confirm or
exclude the rat as an option for APAP studies. To this end, we treated rats and mice with APAP
and measured parameters of liver injury, APAP metabolism, oxidative stress, and activation of the
c-jun N-terminal kinase (JNK). Consistent with earlier data, we found that rats were highly
resistant to APAP toxicity. Although overall APAP metabolism was similar in both species,
mitochondrial protein adducts were significantly lower in rats. Accordingly, rats also had less
oxidative stress. Finally, while mice showed extensive activation and mitochondrial translocation
of JNK, this could not be detected in rat livers. These data support the hypothesis that
mitochondrial dysfunction is critical for the development of necrosis after APAP treatment.
Because mitochondrial damage also occurs in humans, rats are not a clinically relevant species for
studies of APAP hepatotoxicity.
Keywords
Acetaminophen; hepatotoxicity; protein adducts; mitochondria; oxidant stress; c-jun-N-terminal
kinase
2012 Elsevier Inc. All rights reserved.
For Correspondence: Dr. Hartmut Jaeschke, University of Kansas Medical Center, Department of Pharmacology, Toxicology &
Therapeutics, 3901 Rainbow Blvd, MS 1018, Kansas City, KS 66160 USA, hjaeschke@kumc.edu, Tel: +1 913 588 7969, Fax: +1 913
588 7501.
*
These authors contributed equally to this study.
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CONFLICT OF INTEREST STATEMENT
The authors do not have any conflict of interest to disclose.
NIH Public Access
Author Manuscript
Toxicol Appl Pharmacol. Author manuscript; available in PMC 2013 November 01.
Published in final edited form as:
Toxicol Appl Pharmacol. 2012 November 1; 264(3): 387394. doi:10.1016/j.taap.2012.08.015.
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INTRODUCTION
When used as directed, acetaminophen (APAP) is a safe and effective analgesic and fever
reducer. However, large doses of APAP can cause serious liver injury. In fact, APAP
overdose is the primary cause of acute liver failure in many countries throughout the West
(Bernal, 2003; Gow et al., 2004; Larson et al., 2005), responsible for more than 70,000
hospitalizations each year in the U.S. alone (Budnitz et al., 2011). Research on the
mechanism of APAP-induced liver injury began four decades ago, following the first
published report of this toxicity in humans (Davidson and Eastham, 1966). Though many
important questions have yet to be answered, the mechanism of APAP toxicity has been well
investigated in rodents (Jaeschke et al., 2011; 2012) and progress is now being made in
humans (Antoine et al., 2012; Atoniades et al., 2012; Davern et al., 2006; McGill et al.,
2012) and with in vitro human models (McGill et al., 2011). This profusion of data likely
makes APAP the best characterized hepatotoxicant.
Because APAP-induced liver injury is clinically relevant, well studied, and can be rapidly
induced in vivo with a single dose, it has become a standard model in the pharmacology and
toxicology literature. In particular, APAP overdose in rodents is frequently used to test the
hepatoprotective potential of herbal therapeutics. While this can be a valid approach, a
number of concerns have been raised (Jaeschke et al., 2010, 2011). For example, one of the
most common issues in the complementary and alternative medicine literature is the use of
rats to evaluate protection against APAP injury. It has been known since the early 1970s that
rats are resistant to the liver-damaging effects of APAP (Mitchell et al., 1973). Doses which
far exceed the LD
50
for mice cause only minimal necrosis in rat liver. The reason for this
difference in susceptibility is not well understood. In mice, APAP hepatotoxicity begins
with metabolism of the parent compound to the reactive electrophile N-acetyl-p-
benzoquinone imine (NAPQI). NAPQI depletes glutathione (GSH) and binds to proteins,
primarily to the amino acid cysteine (Nelson, 1990; Cohen et al., 1997). Differences in
APAP metabolism and protein binding could account for the difference between mice and
rats. However, while protein binding appears to be a necessary first step toward injury, it is
not sufficient to directly cause cell death (Jaeschke et al., 2012). 3-hydroxyacetanilide
(AMAP), a non-hepatotoxic isomer of APAP, also binds to proteins (Tirmenstein and
Nelson, 1989). Moreover, toxicity develops only after the onset of oxidative stress and
mitochondrial dysfunction, and preventing these phenomena protects against APAP (Kon et
al., 2004; Cover et al., 2005; Ramachandran et al., 2011a,b). Moreover, activation and
mitochondrial translocation of c-Jun N-terminal kinase (JNK) have repeatedly been shown
to play a role in APAP toxicity in the liver (Gunawan et al., 2006; Latchoumycandane et al.,
2007; Hanawa et al., 2008; Saito et al., 2010). Thus, mitochondrial dysfunction, oxidative
stress, and/or JNK activation may also be different between the two species.
A better understanding of the differences between rats and mice will not only aid future
researchers in selection of the best model for their experiments, it may provide important
new mechanistic insights into APAP toxicity. Therefore, the objective of the present study
was to investigate potential differences in the mechanism of APAP-induced liver injury
between rats and mice with emphasis on protein adduct formation, oxidative stress, and JNK
activation.
METHODS
Animals
C57Bl/6 mice (Jackson Laboratories, Bar Harbor, ME), Fischer 344 and Sprague-Dawley
rats (Harlan Laboratories, Indianapolis, IN) between 812 weeks of age were kept in a
temperature controlled facility with a 12 hour light/dark cycle and free access to food and
McGill et al. Page 2
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water. For all experiments, food was withdrawn 1215 hours prior to treatment with APAP.
The drug was administered i.p. or p.o. at the indicated doses in one of two metabolically
inert vehicles: warm saline (mice) or 20% Tween-80 (rats). Saline and solutions of Tween
are popular vehicles for the mouse and rat models, respectively, and have been shown to not
interfere with APAP toxicity (Kelava et al., 2010). At various times, the animals were
sacrificed by cervical dislocation (mice) or exsanguination (rats) under anesthesia. Blood
was drawn from the caudal vena cava and centrifuged to obtain serum. After taking blood,
livers were excised and portions were flash frozen for determination of glutathione (GSH),
APAP-cysteine adducts on proteins (APAP-CYS), and western blotting, or fixed in 10%
phosphate-buffered formalin for histology. For organelle isolation, fresh liver tissues were
minced and gently homogenized on ice with 2030 passes using a tight-fitting motor-driven
Teflon pestle in a glass mortar. Subcellular fractions were collected by differential
centrifugation. Briefly, cell debris was removed with 2,500 g spin for 10 min. The
supernatant was then centrifuged at 20,000 g for 10 min to collect mitochondria and again at
110,000 g for 1 h to pellet the mixed microsomes and light membranes. The new supernatant
was then collected as the cytosolic fraction.
Biochemical assays
Serum alanine aminotransferase (ALT) was measured using a kit (Pointe Scientific) and
glutamate dehydrogenase (GDH) activity was determined as described (McGill et al., 2012).
Liver GSH levels were measured using a modified Tietze assay as described (Jaeschke and
Mitchell, 1990).
APAP-CYS
APAP-CYS protein adducts were measured using the high pressure liquid chromatography
with electrochemical detection (HPLC-ED) method of Muldrew et al. (2002) with
previously described modifications (Ni et al., 2012). For total liver adducts, tissues were
homogenized with a blade-type homogenizer in 10 mM sodium acetate buffer (pH 6.5),
filtered through Bio-Spin 6 columns (Bio-Rad) to remove low molecular weight compounds
with the potential to interfere in the assay, and digested overnight with proteases to free the
APAP-CYS. For measurement of protein adducts in the mitochondrial fraction, the pellets
were resuspended in small volumes of sodium acetate buffer and subjected to 3 cycles of
freeze-thaw to homogenize. The debris was then pelleted by centrifugation and supernatants
were filtered and digested as above. Protein was measured using the bicinchoninic acid
assay (BCA).
Histology and immunohistochemistry
Liver sections were stained with hematoxylin and eosin for assessment of tissue necrosis.
Nitrotyrosine staining was performed as previously described (Knight et al., 2002) using a
rabbit polyclonal anti-nitrotyrosine antibody (Life Technologies, Grand Island, NY) and the
Dako LSAB peroxidase kit (Dako, Carpinteria, CA).
Statistical methods
One-way analysis of variance (ANOVA) was used to assess statistical significance between
three or more groups. When a difference was detected, the Student Newman-Keuls test was
used for multiple comparisons. For non-normally distributed data, the Kruskal-Wallis test
was used. p < 0.05 was considered significant.
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RESULTS
APAP toxicity in rats
For our initial studies, two strains of rats were chosen based on previously published data
reporting liver injury after APAP overdose: Fischer (F344) and Sprague-Dawley rats. The
rats were treated orally with 1, 1.5, or 2 g APAP per kg body weight and sacrificed 24 h
later. Doses were chosen based on the literature and on the limit of solubility of APAP
(Mitchell et al., 1973). Though Fischer rats did have significantly elevated plasma ALT
activity after the 1.5 g/kg dose, both strains showed considerable resistance (Table 1).
Administering the drug i.p. at 1 g/kg in Fischer (F344) rats did not change these findings
(Figure 1A). For direct comparison, time course studies with mice (300 mg/kg) and rats (1 g/
kg) were executed in parallel. While mice had a dramatic increase in both plasma ALT and
GDH over time, rat liver enzyme levels in plasma remained low despite receiving a much
higher dose (Figure 1A,B). Moreover, we observed large areas of necrosis in mouse liver by
histology, but little or no injury could be seen in rat samples (Figure 1C). These results are
consistent with earlier reports (Mitchell et al., 1973) and show that rats are highly resistant
to APAP-induced liver injury.
APAP metabolism in mice and rats
APAP toxicity depends upon the formation of the reactive metabolite NAPQI, which will
deplete GSH and bind to proteins (Nelson, 1990). Protein binding is known to be the
initiating event in the mechanism of injury. To determine whether or not there is a difference
in metabolism in the liver between mice and rats, we measured liver GSH and total liver
APAP-protein binding (Figure 2A, 3A). GSH concentrations were significantly reduced
after 1 h in the livers of mice treated with the toxic dose of 300 mg/kg and remained low
until 3 h and then started to recover (Figure 2A). In rats treated with 1 g/kg, there was a
modest delay in GSH depletion so that the lowest concentrations were achieved at 3 h.
Interestingly, there was no recovery of liver GSH in the rats even as late as 24 h after APAP
treatment (Figure 2A). This is partially because the much higher dose took longer to clear,
but it is likely also due to differences in GSH resynthesis. In mice, there was significant
induction of the glutamyl-cysteine ligase catalytic subunit (Gclc) after APAP treatment, but
this was not seen in rats (Figure 2C).
Consistent with the delayed GSH depletion, the concentration of APAP-protein adducts
increased more slowly in rat liver homogenates than in mouse samples, though similar levels
were measured in both species by 6 h (Figure 3A). The fact that similar overall protein
binding was achieved in rats given a dose of APAP more than threefold higher shows that
formation of NAPQI occurs much less readily in these animals. Further, the fact that rats
given this much higher dose were resistant to injury despite similar levels of protein binding
when compared with mice suggests that there are factors other than total protein binding to
consider.
Mitochondrial protein adducts, oxidative stress and JNK activation in mice and rats
Mitochondrial dysfunction is known to play a role in APAP hepatotoxicity in both mice
(Meyers et al., 1988; Jaeschke, 1990) and humans (McGill et al., 2012). It is well-
established that NAPQI binds to mitochondrial proteins (Tirmenstein and Nelson, 1989) and
it is generally accepted that this is an important early event in the mitochondrial dysfunction
and associated oxidative stress seen after APAP overdose. For this reason, although there
was little difference in total liver protein binding, mitochondrial APAP-protein adducts were
evaluated (Figure 3B). The increase in mitochondrial adduct levels from rats paralleled the
increase seen in mice. Importantly, however, at all time points the levels were significantly
lower in the samples from rats. This was observed even at 6 h when the total adduct levels
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between the two species were similar (Figure 3A). It is possible that the lower mitochondrial
protein binding is responsible for the resistance to injury. Cyp2e1 is the major P450 enzyme
responsible for APAP metabolism in rodents (Lee et al., 1996) and there is evidence that
mitochondrial Cyp2e1 can contribute to, and may itself be sufficient for, the formation of
mitochondrial APAP-protein adducts and oxidative stress (Knockaert et al., 2011). To
determine whether or not this species difference in mitochondrial protein binding could be
due to differences in mitochondrial P450 protein expression we immunoblotted for Cyp2e1
in mitochondria from livers of both species before and after APAP treatment (Figure 3C).
There was no difference in basal mitochondrial Cyp2e1 between mice and rats. In fact, we
observed an increase in rats at later time points after APAP treatment. Thus, the lower
mitochondrial APAP-protein adduct levels are likely not due to differences in mitochondrial
Cyp2e1.
Mitochondrial protein binding after APAP treatment is thought to lead to downstream
mitochondrial oxidative stress. The major reactive oxygen species in the mechanism of
APAP toxicity in mice is superoxide (O
2
), which dismutates to molecular oxygen and

hydrogen peroxide or reacts with nitric oxide (NO) to form peroxynitrite (ONOO
), a potent
oxidant and nitrating species. To determine whether or not oxidative stress developed in
either species, tissue levels of GSSG were measured and the GSSG-to-GSH ratio calculated.
Hepatic GSSG levels in mice were 0.5% of the total in controls and increased significantly
after 3 h, reflecting an oxidant stress in mouse livers (Figure 2B). In contrast, GSSG levels
in rats were 0.2% in controls and remained <0.1% at all time points after APAP
administration. These data indicate a substantial oxidant stress after APAP in mice but not in
rats. These data were confirmed by immunostaining for nitrotyrosine protein adducts (Figure
4), which are a footprint of peroxynitrite formation. Extensive centrilobular nitrotyrosine
staining was evident in mouse livers but not in rats, suggesting that peroxynitrite formation
occurred only in mice (Figure 4).
JNK activation has been shown to be critical in the mechanism of APAP-induced liver
injury (Gunawan et al., 2006; Latchoumycandane et al., 2007; Hanawa et al., 2008; Saito et
al., 2010). Because JNK activation may be the result of oxidative stress after APAP
treatment in mice (Nakagawa et al., 2008; Saito et al., 2010), we next tested whether or not
we could detect JNK phosphorylation (p-JNK) and mitochondrial translocation in liver
samples from these two species (Figure 5A,C). Very little phosphorylated JNK could be
detected in mitochondrial fractions from rats after APAP, though there was a clear increase
in mouse samples at 1 and 3 h post- APAP. The difference in the mitochondrial p-JNK
results could have been due to lack of activation or to lack of translocation. To test the latter
possibility, we also blotted for p-JNK in the cytosol fractions (Figure 5B,C), as well as for
total (non-phosphorylated + phosphorylated) JNK in both fractions (Figure 5C).
Interestingly, non-phosphorylated JNK appears to be constitutively present in mitochondria
from rat liver but not from mice. The reason for this is not yet known. In any case, very little
p-JNK could be detected in rat samples when compared with samples from mice. Together,
these data show that there is no relevant oxidative stress or JNK activation in rats after
APAP overdose.
DISCUSSION
The objective of this study was to evaluate potential mechanistic differences of APAP
hepatotoxicity between rats and mice. Although it is known that rats are resistant to APAP
hepatotoxicity (Mitchell et al., 1973), many investigators continue to choose this species for
their studies of potentially hepatoprotective compounds. This is especially true in the area of
herbal therapeutics and natural products (Jaeschke et al., 2011) but it is not limited to this
field (Laskin et al., 1995; Miyamoto et al., 2008; Ahmed et al., 2011). Initially, species
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differences in APAP toxicity were thought to result from different rates of APAP
metabolism (Davis et al., 1974). Consistent with this, our data revealed that APAP protein
binding in rats was similar to a standard mouse model of toxicity when a threefold higher
dose was administered. Moreover, we observed a delay in hepatic GSH depletion and
APAP-protein adduct formation in rats compared with mice. In this study, the rats and mice
were fasted for approximately the same amount of time (1215 h) before APAP treatment. It
is possible that this was simply insufficient for the rat model due to a difference in GSH
turnover between species. Indeed, the starting GSH levels in livers from fasted rats were on
average 1 mol/g liver higher than in livers from control mice. However, optimization of
this value would be difficult due to the fact that the rate of GSH synthesis in rat liver is
actually increased during fasting (Lauterburg and Mitchell, 1981). In any case, induction of
cytochrome P450 enzymes with phenobarbital or a similar compound is one strategy that has
been used to compensate for the apparent difference in metabolism (Mitchell et al., 1973).
However, this usually requires several days of pretreatment. More importantly, many of
these compounds are nuclear receptor activators and the effect of these treatment regimens
on the mechanism of toxicity has not been well investigated. The mouse model is more
convenient and better characterized. Interestingly, despite the delayed metabolism in rats, at
later time points APAP protein binding was similar in both species at the doses used. Thus,
it is likely that there are other downstream factors responsible for the difference in
susceptibility. After several decades of research on the mechanisms of APAP toxicity, it is
now possible to compare some of these downstream events between mice, rats, and even
humans, in greater detail.
Mitochondrial dysfunction and oxidative stress in mice and rats
Mitochondrial dysfunction is known to occur after APAP overdose in mice (Jaeschke and
Bajt, 2006). Protein adducts in mitochondria are higher after APAP treatment compared
with the non-hepatotoxic isomer 3-hydroxyacetanilide (Tirmenstein and Nelson, 1989). It is
generally accepted that this increased mitochondrial protein binding leads to mitochondrial
dysfunction and oxidative stress (Jaeschke and Bajt, 2006). APAP overdose inhibits
mitochondrial respiration (Meyers et al., 1988) and causes a decrease in hepatic ATP levels
in the liver (Jaeschke, 1990). Using electron microscopy, swelling and lysis of mitochondria
were also observed (Placke et al., 1987). Evidence for superoxide and peroxynitrite
formation selectively in mitochondria has also been found (Jaeschke, 1990; Cover et al.,
2005) and it was later discovered that mitochondrial depolarization occurs in primary mouse
hepatocytes treated with high concentrations of APAP (Kon et al., 2004; Reid et al., 2005).
Importantly, well-characterized inhibitors of the mitochondrial permeability transition
(MPT) were protective in this model (Kon et al., 2004), and mice deficient for the MPT pore
regulator cyclophilin D had reduced liver injury in vivo (Ramachandran et al., 2011a).
However, the MPT is only regulated by cyclophilin D after low but not high overdoses of
APAP (LoGuidice and Boelsterli, 2011). Similar to the results with AMAP mentioned
above, we saw reduced mitochondrial APAP-protein adducts in rats. Together with the
absence of GSSG, nitrotyrosine protein adducts, p-JNK formation or p-JNK translocation to
the mitochondria in this species, these data strongly suggest that no mitochondrial
dysfunction or oxidative stress occurs in rats after APAP overdose. Moreover, there was no
elevation of serum GDH activity, which has been used as a marker of mitochondrial damage
(McGill et al., 2012), though this could be due to the lack of necrosis and enzyme release.
Protein binding, especially mitochondrial protein binding, is necessary for initiation of
APAP toxicity (Tirmenstein and Nelson, 1989). A large number of compounds (extracts
from natural products) have been claimed to protect against APAP through antioxidant
effects or through prevention of mitochondrial damage. However, the metabolic activation
of APAP is rarely evaluated. Any reduction in APAP-protein adducts by inhibition of
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metabolism or scavenging of NAPQI will be protective against APAP-induced liver injury.
Without protein binding, downstream events in the mechanism of toxicity (e.g.
mitochondrial dysfunction, oxidative stress, JNK activation) will not occur and one could
mistakenly conclude that the compound of interest protects by blocking one or more of these
events. For this reason, measurement of GSH or APAP-CYS should be the first experiment
performed in any test of potentially hepatoprotective compounds relying on the APAP
model. In both cases, an early time point (0.5 1 h post-APAP) should be used.
Observations later than 1 h may miss early differences in protein adduct formation, and in
mice GSH levels begin to recover by 4 6 h (Jaeschke et al., 2011).
JNK activation in mice and rats
JNK is phosphorylated and translocates to mitochondria early in APAP hepatotoxicity in
mice (Gunawan et al., 2006; Hanawa et al., 2008; Ramachandran et al., 2011b) and this is
thought to occur at least partly as a result of an initial oxidative stress (Nakagawa et al.,
2008; Saito et al., 2010; Ramachandran et al., 2011a). Our results confirmed these findings
(Figure 5). Importantly, inhibition of JNK activation in mice reduces ALT activity in plasma
as well as the appearance of necrosis in liver sections, reduces nuclear DNA fragmentation,
and prevents the further development of mitochondrial oxidative stress after APAP. In
contrast, we could not detect JNK phosphorylation in mitochondria from rats. This supports
the conclusion that these animals did not develop mitochondrial dysfunction or oxidative
stress. The exact relationship between JNK activity and oxidative stress after APAP
intoxication is not fully understood. However, there is evidence that the early oxidant stress
is involved in JNK activation, which appears to amplify the mitochondrial oxidant stress
(Saito et al., 2010). Interestingly, non-phosphorylated JNK was present in mitochondria
from control rat liver but not from control mice (Figure 5A). This may suggest that liver
injury involving JNK requires both mitochondrial localization and phosphorylation.
Localization or translocation alone is insufficient.
Mechanisms of APAP toxicity in humans
Progress is now being made in the study of APAP toxicity in humans (McGill et al., 2012;
Antoine et al., 2012; Antoniades et al., 2012). Data from the human cell line HepaRG and
from human samples have provided evidence that mitochondrial damage also occurs in
humans (McGill et al., 2011, 2012). APAP selectively causes necrosis of hepatocyte-like
cells in HepaRG cultures, and this is preceded by loss of mitochondrial membrane potential
and the development of mitochondrial oxidative stress (McGill et al., 2011). In humans,
glutamate dehydrogenase (GDH) and mitochondrial DNA (mtDNA) are detectable in
plasma during APAP-induced liver injury but are low or nondetectable in samples from
overdose patients without serious liver injury or from healthy controls (McGill et al., 2012).
These biomarkers were also elevated in mice after treatment with high doses of APAP but
not after treatment with furosemide, a diuretic which can cause similar centrilobular necrosis
but without the antecedent mitochondrial dysfunction. These data suggest that GDH and
mtDNA in plasma are specific biomarkers for mitochondrial injury and that humans develop
this injury after APAP overdose. Because there was lower mitochondrial protein binding in
rats and they were resistant to mitochondrial damage in our study, this species is probably
not a clinically relevant model for APAP-induced liver injury and the mouse is preferred.
Potential issues with the mouse model
A caveat to our interpretation of these data is that APAP toxicity in some strains of mice has
not been as thoroughly studied as in others and there is wide variation in sensitivity to APAP
(Harrill et al., 2009). It is tempting to speculate that, in addition to differences in expression
of cell death genes (Harrill et al., 2009), this may be due in part to variation in mitochondrial
protein binding and/or mitochondrial dysfunction and oxidative stress. For our experiments,
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we chose C57Bl/6 mice. This strain is most commonly used for studies of APAP
hepatotoxicity and the mechanism of toxicity in these animals is well understood.
Nevertheless, other susceptible strains such as ICR mice (Jaeschke, 1990), C3Heb/FeJ mice
(Knight et al., 2001; Cover et al., 2005) and B6C3F1 mice (Agarwal et al., 2011)
consistently show evidence of mitochondrial oxidant stress and peroxynitrite formation after
APAP overdose.
While the mouse, in general, appears to be more clinically relevant than the rat model, there
may still be important differences between mice and humans. For example, although
Cyp2e1-deficient mice are protected against APAP-induced liver injury (Lee et al., 1996),
another study found that recombinant human CYP3A4 was much more active than human
2E1 in converting APAP to APAP-GSH (Laine et al., 2009). Further, CYP2E1 activity is
low in the metabolically competent human liver cell line HepaRG (Anthrieu et al., 2010),
but these cells metabolize APAP and develop toxicity (McGill et al., 2011). Thus, the
enzymes responsible for APAP metabolism may be different in mice and humans.
Conclusions
Rats are much more resistant to APAP hepatotoxicity than mice. This is likely the result of
reduced mitochondrial protein binding, which limits mitochondrial dysfunction and prevents
the oxidative stress and peroxynitrite formation in rats (Figure 6). These data support the
already well-established role of mitochondria in the mechanism of APAP toxicity.
Furthermore, because mitochondrial dysfunction occurs in humans and probably leads to the
necrosis observed after APAP overdose, rats are not a human-relevant species for studies
using the APAP liver injury model.
Acknowledgments
This investigation was supported in part by National Institutes of Health Grants AA12916 and DK070195 and by
grants from the National Center for Research Resources (5P20RR021940-07) and the National Institute of General
Medical Sciences (8 P20 GM103549-07) from the National Institutes of Health. M.R. McGill and C.D. Williams
were supported by the Training Program in Environmental Toxicology (T32 ES007079-26A2) from the National
Institute of Environmental Health Sciences.
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Highlights
Acetaminophen overdose causes severe liver injury only in mice but not in rats
APAP causes hepatic GSH depletion and protein adduct formation in rats and
mice
Less protein adducts were measured in rat liver mitochondria compared to
mouse
No oxidant stress, peroxynitrite formation or JNK activation was present in rats
The limited mitochondrial adducts in rats are insufficient to trigger cell necrosis
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Figure 1.
Serum enzymes in mice and rats treated with APAP. Mice and Fischer 344 rats were treated
i.p. with 300 mg APAP/kg body weight or 1 g APAP/kg, respectively. At various times, the
animals were sacrificed and serum was collected. (A) Time course of ALT activity in serum
from mice and rats after APAP. (B) Time course of glutamate dehydrogenase (GDH)
activity in serum from mice and rats. (C) Representative H&E stained liver sections from
mice (top row) and rats (bottom row) treated with APAP. Data are expressed as mean
SEM for n = 34 animals per group. *P < 0.05 (compared to t=0).
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Figure 2.
Liver glutathione (GSH) and glutathione disulfide (GSSG) in mice and Fischer 344 rats after
APAP treatment. Mice and rats were treated i.p. with 300 mg APAP/kg body weight or 1 g
APAP/kg, respectively. At the indicated times, the animals were sacrificed and liver samples
were flash frozen for later analysis of GSH and GSSG. (A) Total GSH levels. (B) GSSG-to-
GSH ratio shown as a percentage. (C) mRNA levels of glutamate-cysteine ligase (gclc).
Data are expressed as mean SEM for n = 34 animals per group. *P < 0.05 (compared to
t=0).
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Figure 3.
Total liver and mitochondrial APAP-protein adducts in mice and rats. Mice and rats were
treated i.p.with 300 mg APAP/kg body weight or 1 g APAP/kg, respectively. At various
times, the animals were sacrificed and livers were excised. One lobe from each was
immediately homogenized for subcellular fractionation by differential centrifugation. The
remaining tissue was flash frozen for later analysis of total liver adducts. (A) Total liver
APAP-CYS time courses. (B) Liver mitochondria APAP-CYS time courses. Data are
expressed as mean SEM for n = 34 animals per group. *P < 0.05 (compared to t=0)
#
P <
0.05 vs. rats.
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Figure 4.
Nitrotyrosine staining in mice (top row) and rats (bottom row) after APAP treatment. Mice
and rats were treated i.p. with 300 mg APAP /kg body weight or 1 g APAP/kg, respectively.
At the indicated times, the animals were sacrificed and livers were fixed in phosphate-
buffered formalin. Sections were stained using an anti-3-nitrotyrosine antibody.
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Figure 5.
JNK phosphorylation and mitochondrial translocation in livers from mice and rats after
APAP treatment. P-JNK was measured by western blotting in mitochondrial and cytosolic
fractions from mice and rats after treatment with 300 mg APAP /kg body weight or 1 g
APAP/kg, respectively for the indicated times (C). Densitometric analysis of P-JNK and
total JNK in the cytosol (A) and the mitochondria (B).
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Figure 6.
Diagram of APAP metabolism and downstream events in mice and rats. The reactive
metabolite binds to mitochondrial proteins more in the mouse than in the rat. This leads to
an initial mitochondrial oxidative stress with JNK activation and the amplification of the
mitochondrial oxidant stress and the membrane permeability transition (MPT) pore opening
in the mouse, which do not occur in the rat.
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Table 1
APAP Hepatotoxicity in Rats
Strain Dose ALT
Fischer (F344) Control 35 11
Fischer (F344) 1 g/kg 39 4
Fischer (F344) 1.5 g/kg
101 7
*
Sprague-Dawley Control 30 1
Sprague-Dawley 1 g/kg 41 11
Sprague-Dawley 2 g/kg 31 4
Two strains of rats were treated p.o. with the indicated doses of APAP for 24 h. The animals were then sacrificed and ALT activities were
measured in serum. Data represent mean SE of n = 4 animals per group.
*
P < 0.05 (compared to control).

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Acetaminophen-induced Liver Injury in Rats and Mice:

Comparison of Protein Adducts, Mitochondrial Dysfunction, and

), which dismutates to molecular oxygen and

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