Toxicology 185 (2003) 79 /87
www.elsevier.com/locate/toxicol
Late administration of COX-2 inhibitors minimize hepatic
necrosis in chloroform induced liver injury
Carmen K. Begay, A. Jay Gandolfi Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, 1703 E. Mabel St., Tucson, AZ 85721-0207,
USA
Received 2 July 2002; received in revised form 4 October 2002; accepted 8 October 2002
Abstract
Our previous studies have described the protective effects of hepatoprotective agents against liver injury elicited by
chloroform even when given 24 h after the toxicant, at a time when the liver injury is taking place and rapidly
developing. However, the mechanisms involved in this protection remain unknown. The cytoprotective mechanism of
these hepatoprotectants such as DMSO, may be due to a dramatic shift in the production of prostaglandins that are
responsible for controlling the degree of inflammatory response that can affect blood flow in the liver. In this study, NS398, a specific COX-2 inhibitor, and indomethacin, a COX-1 and COX-2 inhibitor, were administered 24 h after
chloroform dosing to determine their effect on liver injury in Sprague /Dawley rats. The extent of necrosis was
evaluated by H&E staining, while injury to hepatocytes was evaluated by measuring plasma levels of alanine
transaminase (ALT). Both COX inhibitors, indomethacin and NS-398, prevented an increase in (ALT) at 48 h after
initial toxicant insult and attenuated further liver necrosis. No changes in cellular proliferative activity occurred in all
the treatment groups, which indicates that protection from the Cyclooxygenase (COX) inhibitors did not have an effect
on regeneration of cells at 32 and 48 h. These results indicate COX inhibitors provide a significant protective effect on
liver cells against CHCl3 injury and may provide further insight into therapeutic interventions against hepatotoxicants.
# 2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Sprague /Dawley rats; Cyclooxygenase-1; Cyclooxygenase-2; Hepatotoxicity; Chloroform
1. Introduction
Injury to hepatocytes by a toxicant stimulates
the release of a cascade of factors by the cells in the
liver. Immediately following injury, formation of
Corresponding author. Tel.: /1-520-626-6696; fax: /1520-626-2466
E-mail address: gandolfi@pharmacy.arizona.edu (A.J.
Gandolfi).
arachidonic acid (AA) metabolites in the liver
contributes to the initiation of acute inflammation.
Cyclooxygenase (COX) enzymes are key inflammatory mediators which convert AA to prostaglandin H2, the precursor of inflammatory
mediators such as prostaglandin E2 (PGE2), prostacyclin and thromboxane A2 (Fu et al., 1990;
Hawkey, 1999). Two COX isoforms have been
identified (Xie et al., 1991; Kujubu et al., 1991).
COX-1 is associated with prostaglandin produc-
0300-483X/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 3 0 0 - 4 8 3 X ( 0 2 ) 0 0 5 9 4 - 2
80
C.K. Begay, A.J. Gandolfi / Toxicology 185 (2003) 79 /87
tion required for normal physiological organ
function and is expressed in a variety of cells and
tissues (O’Neill and Ford-Hutchinson, 1993). In
contrast, COX-2, is induced at sites of inflammation or other cellular stresses by endotoxin, mitogens, and cytokines (Vane et al., 1994; Gilroy et
al., 1998). Overexpression of COX-2 has been
observed in various inflammatory diseases, such
as rheumatoid arthritis, and ulcerative colitis
(Kang et al., 1996; Singer et al., 1998).
The COX pathway may be a major factor in the
attenuation of liver injury. Selective inhibitors
were used to investigate the contribution of COX
isoforms on hepatoprotection. Indomethacin, an
nonsteroidal anti-inflammatory drug (NSAID)
inhibits both COX isoforms. In contrast, NS-398
is highly selective for COX-2 (Futaki et al., 1994).
The inhibition of COX-2 can block inflammatory
responses and protect disruption of the blood /
nerve barrier by reducing prostaglandin synthesis
(Miyamoto et al., 1999). Selective COX-2 inhibitors have been shown to suppress inflammation in
rheumatoid arthritis (Katori et al., 1998). Inhibition of COX with indomethacin and NS-398
blocks the downstream production of prostanoid
vasodilatory metabolites. In the liver, endothelial
and Kupffer cells are considered the major cellular
sources of prostaglandins, while hepatocytes rapidly metabolize COX products (Billiar et al.,
1990; Decker, 1991).
In this study, in vivo experiments were conducted to determine if inhibition of COX-2 was
involved in the attenuation of liver necrosis after
initial treatment with CHCl3. The COX inhibitors
tested in Sprague/Dawley rats were indomethacin,
a dual COX-1 and COX-2 inhibitor, and NS-398,
a specific COX-2 inhibitor. Nordihydroguaiaretic
acid (NDGA), a LOX inhibitor is responsible for
another pathway of AA metabolism and was used
to compare the effect of a LOX inhibitor with
COX inhibitors.
The hepatoprotective effects of COX inhibitors
against CHCl3 in Sprague /Dawley rats were
compared with previous studies where DMSO
and aminobenzotriazole (ABT) were shown to
protect against further liver injury induced by
CHCl3 (Lind and Gandolfi, 1999a,b; Lind et al.,
2000). Thus DMSO and ABT were included to
give a reference point for the potency of the COX
and LOX inhibitors.
Previous studies have shown that DMSO and
ABT suppressed the hepatotoxic effect of several
different xenobiotic compounds (e.g. chloroform,
bromobenzene, halothane) in Sprague/Dawley
rats when given 24 h after the chemical exposure,
a time when liver necrosis is progressing (Siegers,
1978; Lind and Gandolfi, 1997, 1999a,b; Lind et
al., 2000). Chloroform was selected for these
subsequent studies as a model compound since it
produces a very reproducible, time-dependent liver
injury in our rats. As liver injury develops,
microcirculation through the narrow sinusoidal
channels of the liver is reduced. However, treatment with DMSO and ABT, re-established blood
flow in the liver by decreasing the number of
activated Kupffer cells (Ito et al., 2000).
Acute chloroform (CHCl3) administration in
Sprague /Dawley rats results in hepatic damage,
which peaks at 48 h after dosing. Based on a
previous time course of injury study, measurements were selected at 32 and 48 h after CHCl3
dosing as this is the time during which DMSO
begins to attenuate the developing injury (Lind
and Gandolfi, 1999a). Initial injury to hepatocytes
by a toxicant involves the release of various
mediators by activated Kupffer cells including
cytokines, reactive oxygen intermediates, and AA
metabolites (Laskin, 1990). Evidence of hepatoprotection against chemically induced injury includes decreases in both ALT activity and the
extent of hepatic centrilobular necrosis (Lind and
Gandolfi, 1999b).
The study provides information for evaluating
the role of the COX inhibitors to intervene in the
development of liver injury at such a late time in
the process of liver injury recovery. These studies
may provide potential therapeutic agents for liver
injury induced by toxicants.
2. Materials and methods
2.1. Chemicals
Chloroform, dimethyl sulfoxide, indomethacin,
NDGA, ABT, and ALT ten reagent were pur-
C.K. Begay, A.J. Gandolfi / Toxicology 185 (2003) 79 /87
chased from Sigma-Aldrich (St. Louis, MO). NS398 (N -[-2-cyclohexyloxy]-4-nitrophenyl methanesulphonamide) was obtained from Cayman Chemical Company (Ann Arbor, MI).
2.2. Animals
Male Sprague /Dawley rats (250 /300 g) were
purchased from Harlan Sprague /Dawley, Inc.
(Indianapolis, IN) and acclimated to our animal
facility for at least 7 days prior to use. The animals
were maintained on a 12 h day:12 h night cycle and
received Rat/Mouse Diet and water ad libitum. All
protocols were approved by the University of
Arizona Animal Care and Use Committee in
accordance with the NIH Guide for the Care and
Use of Laboratory Animals guidelines.
2.3. CHCl3-induced liver injury and
hepatoprotectants
Chloroform was administered to groups of
animals (N /8) via oral gavage at 0.75 ml/kg
CHCl3, 20% in corn oil. The hepatoprotective
agents (DMSO and ABT) and the inhibitors
(indomethacin, NS-398 and NDGA) were administered via intraperitoneal (i.p.) injection 24 h
after CHCl3 dosing. As reported previously, the
DMSO treatment was 2 ml/kg DMSO in 50%
saline the ABT dose was 30 mg/kg in saline (Lind
and Gandolfi, 1999a,b; Lind et al., 2000). The
doses of the COX and LOX inhibitors were
selected from literature (Katori et al., 1998;
Tsugawa et al., 1999; Bandeira-Melo et al., 2000;
Mikuni et al., 1998; Miyamoto et al., 1999).
Preliminary studies (unpublished data) were performed to determine the optimal dose of NS-398,
indomethacin, and NDGA for effective protection
against CHCl3. The selected doses of indomethacin and NS-398 inhibited plasma PGE2 production
up to 24 h after dosing by 83 and 57%, respectively, while NDGA inhibited plasma LTB4 production up to 8 h after dosing by 64%. At these
dose levels no toxic side effects of the COX or
LOX inhibitors were seen and there are no
reported effects in the literature.
For the eicosanoid inhibition studies, each
group (N /8) received either 10 mg/kg NDGA
81
in saline, 20 mg/kg indomethacin or 1 mg/kg NS398 (dissolved in ethanol; diluted with saline).
Indomethacin was dissolved in 0.1 N NaOH
buffered with Tris and neutralized with 0.1 N
HCl as previously described (Bandeira-Melo et al.,
2000). The CHCl3 only-‘saline’ group (N /8)
received saline, i.p. in place of the hepatoprotective
agent at 24 h after CHCl3.
2.4. Sample collection
Previous studies indicated that ALT values for
the treatment groups began to diverge approximately 8 h after DMSO or ABT administration
(Lind and Gandolfi, 1999a). Therefore, sample
collection times for assessing the status of liver
injury were selected at 32 and 48 h after CHCl3
dosing. The rats were killed with a lethal (200 mg/
kg) dose of i.p. pentobarbital. Cardiac blood was
obtained while under deep anesthesia at 32 and 48
h after CHCl3 dosing and centrifuged to obtain
plasma. Plasma samples were immediately analyzed for ALT activity. Livers were removed and a
section was cut longitudinally into strips and
placed in neutral buffered formalin. Blood and
liver tissue was also obtained from a group of
untreated control animals. ALT activity was
expressed as Units/liter (U/l).
2.5. Histopathology
All liver sections were fixed in formalin solution
(10% neutral buffered, Sigma). These were later
embedded in paraffin and stained with hematoxylin and eosin (H&E). The extents of liver necrosis
produced by CHCl3 and subsequent protection by
the hepatoprotectants were examined at 32 and 48
h. The morphologic changes produced in the liver
by CHCl3 were examined during the time course of
the experiment. Necrosis scoring was performed
using the SIMPLEPCI software (C Imaging, Inc.).
Digital images were acquired with a SPOT Jr
camera mounted on a Nikon E400 microscope
using a 10x objective. The score was determined by
dividing the measured necrotic area by the total
area of the field.
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C.K. Begay, A.J. Gandolfi / Toxicology 185 (2003) 79 /87
2.6. Mitotic index
The proportion of cells undergoing mitosis was
determined with H&E slides. Cells in the prophase,
metaphase, anaphase or telophase of the cell cycle
were identified manually. The mitotic index was
assayed as the number of mitoses observed per ten
high power fields chosen at random as previously
described (Theocharis et al., 2001). On each slide,
ten fields were viewed by light microscopy (400 /)
and 20 cells were counted (i.e. 200 hepatocytes per
slide). In each pre-determined-field a ‘zig-zag’
pattern was used to select the cells. The mitotic
index was determined as the percentage of the 200
hepatocytes in various phases of the cell cycle.
3. Statistical analysis
All data were analyzed using STATAQUEST
version 4.0 for Windows (College Station, TX).
Test of significance of differences between treated
and saline-treated groups were calculated using the
nonparametric Kruskal/Wallis one way analysis
of variance and using Wilcoxon rank-sum tests
Bonferroni-corrected for multiple comparisons as
post hoc significance tests. Data are expressed as
mean/S.E.M. for the animals in each treatment
group. P B/0.05 was considered to be statistically
significant.
et al., 2000), at the 32 h time point, the ALT levels
and necrosis were not significantly different.
Therefore, this time point was not used in the
hepatoprotection studies. However, for the mitotic
index evaluation, the 32 h time point was included
since by 48 h the hepatoprotection was evident and
all relevant cell proliferation should have occurred
prior to 48 h.
4.1. Hepatoprotection against CHCl3 liver
toxicity */ALT levels
ALT levels were measured in the plasma of all
rats at 48 h after CHCl3 (Fig. 1). CHCl3-induced
liver injury in rats resulted in maximal plasma
enzymes (ALT) and peak hepatic necrosis at 48 h.
For comparative purposes, ALT levels were measured in rats given DMSO or ABT at 24 h after
chloroform. The extent and location of hepatoprotection by DMSO and ABT was identical to
that previously reported (Lind and Gandolfi,
1999b; Lind et al., 2000). Both DMSO and ABT,
significantly reduced the ALT levels by 86% and
84%, respectively (data not shown). Of the three
eicosanoid inhibitors tested, both the nonselective
COX inhibitor, indomethacin, and NS-398, the
COX-2 specific inhibitor displayed significant
4. Results
To determine the hepatoprotective effects of
COX inhibitors, Sprague/Dawley rats received
CHCl3 p.o. and 24 h later received DMSO, ABT,
NDGA, indomethacin, NS-398 or saline i.p. Based
on previous studies (Lind and Gandolfi, 1999b;
Lind et al., 2000) the dose of chloroform (0.75 ml/
kg) was selected to achieve maximal ALT levels at
48 h without causing death in Sprague/Dawley
rats. In this study 98% of the animals survived.
The ‘saline’ treated rats (CHCl3 treated but no
inhibitor) as well as the control (untreated rats)
were killed at 48 h.
As previously reported in a more detailed time
course of CHCl3-induced liver injury study (Lind
Fig. 1. COX inhibitors protect against CHCl3-induced liver
injury. ALT levels were measured after CHCl3 dosing. DMSO,
ABT, COX and LOX inhibitors were administered 24 h after
CHCl3 dosing. Rats received 0.75 ml/kg CHCl3 p.o. and 24 h
later received DMSO, ABT, NDGA, indomethacin, NS-398, or
saline i.p. At 48 h after CHCl3 challenge, ALT levels peak. Data
is shown as mean9/S.E.M. (N/8). , Indicates P B/0.05 vs.
saline group.
C.K. Begay, A.J. Gandolfi / Toxicology 185 (2003) 79 /87
protection against further liver injury. Indomethacin and NS-398 reduced ALT levels by 50.6% and
49.0%, respectively, when measured at 48 h after
CHCl3 ingestion. In contrast, NDGA, a LOX
inhibitor did not show any protective effects
against CHCl3 induced toxicity.
4.2. Hepatoprotection against CHCl3 liver
toxicity */histopathology
To confirm that the reduction in ALT reflected
a reduction in liver damage, liver sections stained
by H&E were examined to determine the extent of
inflammation, the number of necrotic cells, and
hepatocyte proliferation. The extent of liver necrosis as measured by histopathological analysis
confirmed the plasma enzyme levels. Substantial
necrosis (48%9/7.11) was noted in the liver with
peak ALT levels of 32879/524 at 48 h after CHCl3.
Both indomethacin and NS-398 were more effective at protecting against further liver damage than
the LOX inhibitor, NDGA. Extensive hepatic
necrosis was observed at 48 h in the CHCl3 treated
rats followed by saline treatment (Fig. 2b). In these
rats, the necrotic areas included ballooned hepatocytes due to macrovesicular steatosis and the
extent of necrosis bridged to other centrilobular
vein. Additionally, mesenchymal cells accumulated
in the centrilobular area and infiltration of neutrophils was clearly visible. During peak necrosis,
the necrosis score for CHCl3-treated rats was
48%9/7.11 (Fig. 3). Substantial areas of centrilobular injury were present in the rats receiving
lipoxygenase inhibitor, NDGA as the protective
agent (37%9/5.9; Fig. 2e). However, the rats,
treated with indomethacin or NS-398 after the
CHCl3 dosing, showed a substantial decrease in
necrosis score to 18.2%9/4.9 and 12.9%9/1.9,
respectively (Fig. 3). The injury was limited to
just a few cells around the central vein (Fig. 2c and
d).
4.3. Liver regeneration
To examine the possibility that the hepatoprotective effects of COX inhibitors may have resulted
from an increase in acceleration of recovery
through cell division, the effects of COX inhibitors
83
on the mitotic index of CHCl3-induced liver cells
were examined. For the CHCl3-induced rats, there
were no significant changes in the mitotic index at
32 or 48 h after initial toxicant dose (Table 1).
Neither DMSO nor ABT nor the eicosanoid
inhibitors had an effect on hepatocyte regeneration when given at 24 h after CHCl3. This results
suggest that hepatoprotection by these agents did
not occur by accelerated cell division.
5. Discussion
The study was designed to evaluate whether
inhibition of COX activity would have a heptoprotective effect against CHCl3-induced liver injury. The principle findings of this study show that
administration of COX inhibitors, indomethacin
or NS-398, at 24 h after initial CHCl3 dose, was
beneficial in this model in protecting against
progressing liver injury.
The administration of CHCl3 in rats caused
severe liver injury, evident through both histopathology and increased ALT activities. The
mechanism responsible for CHCl3 induced hepatotoxicity is believed to be mediated by reactive
intermediate metabolites formed in the course of
CHCl3 biotransformation in the liver (Pohl et al.,
1980; Hewitt et al., 1980). The development of
hepatic necrosis in rats becomes prominent as
early as 10 h after administration of CHCl3 and
peaks at 48 h (Lind et al., 2000). After 48 h, a
hepatic regenerative process occurs, as tissue
repair is a biological response that accompanies
chemically-induced liver injury.
In response to liver injury, inflammation reactions are activated and inflammatory cytokines,
such as tumor necrosis factor-a, interleukin-1, and
AA metabolites, such as thromboxane A2, prostaglandin I2, PGE2, and leukotrienes are released
(Ljungman et al., 1991). AA is metabolized to
bioactive eicosanoids by three major pathways,
COX, LOX, and NADPH-dependent cytochrome
P-450 monooxygenase enzymatic pathways
(Smith, 1992). The formation of these eicosanoids
in the liver contributes to the initiation of the acute
phase response to inflammation. COX enzymes
catalyze the conversion of AA to active prosta-
84
C.K. Begay, A.J. Gandolfi / Toxicology 185 (2003) 79 /87
Fig. 2
C.K. Begay, A.J. Gandolfi / Toxicology 185 (2003) 79 /87
Fig. 3. Assessment of hepatic necrosis in rats administered
CHCl3 then treated with COX inhibitors. Results represent area
of necrosis over total area at 10/. Late administration of COX
inhibitors induced a significant decrease in liver necrosis
compared with rats treated with saline only (, indicates P B/
0.05 vs. saline group). There were no statistically differences
between NDGA treated group and saline group at P B/0.05.
Data is shown as mean9/S.E.M. (N/8).
Table 1
Mitotic indices (mitotis/10HPF) of liver regeneration in CHCl3treated rats examined at different time points post-toxin
administration
Treatment
Control
Saline
NS-398
Indomethacin
NDGA
DMSO
ABT
32 h after CHCl3
B/0.01
0.069/0.01
0.089/0.02
0.099/0.02
0.099/0.03
0.079/0.01
0.079/0.01
48 h after CHCl3
B/0.01
0.109/0.01
0.099/0.01
0.099/0.01
0.109/0.03
0.079/0.01
0.069/0.01
There were no statistically differences between treatment
groups. The amount of cells undergoing mitotic activity did not
change. Data is shown as mean9/S.E.M. (N/8).
85
glandin mediators (Fu et al., 1990; Hawkey, 1999).
PGE2 is a major product of COX activity and
contributes to inflammation by causing vasodilation and potentiating the effects of mediators such
as bradykinin and IL-1 (Davies et al., 1984). In
addition, AA metabolites produced by COX play
a role in apoptotic cell death (Iida et al., 1998).
COX-1 is involved in physiological prostaglandin
formation and is expressed in most types of cells
and tissues (O’Neill and Ford-Hutchinson, 1993).
COX-2, is induced and activated at sites of
inflammation by endotoxin, mitogens, and cytokines (Vane et al., 1994; Gilroy et al., 1998). Thus,
the inhibition of COX enzyme activities by our
hepatoprotective agents is essential and may have
dramatic effects in protecting against liver injury
and preserving the liver structure.
We examined the effects of COX inhibitors on
hepatotoxicity using indomethacin and NS-398.
Inhibition of COX with indomethacin and NS-398
block the downstream production of vasodilator
prostanoid metabolites (Poyser, 1999). Indomethacin has been shown to inhibit the conversion of
AA to PGE2 and thus prevents exacerbation of the
existing injury (Rufer et al., 1982). Indomethacin
was effective in our model by suppressing both
ALT levels and further liver necrosis when measured at a time when peak necrosis should occur.
In this study, we employed NS-398, to investigate the role of COX-2 in liver injury. NS-398 was
reported to attenuate the increase in tumor necrosis factor-a, a cytokine involved in inflammation
(Araki et al., 2001). The overexpression of inducible COX-2 has been associated with vascular
inflammation and cellular proliferation. In the
present study, NS-398 was able to reduce ALT
levels and prevent further liver necrosis, when
given 24 h after initial CHCl3 dose. The reduction
in ALT levels may again be a result of reduced
PGE2. A reduction in prostaglandin synthesis is
Fig. 2. The effects of late administration of COX inhibitors on progressing liver necrosis. (a) Rat liver showing normal histology, (b)
CHCl3-treated rat liver, at 48 h post toxin administration. Extensive centrilobular necrosis was observed in these liver section. Large
cells surrounding the necrotic zone are hepatocytes ballooned due to macrovesicular steatosis. Extent of necrosis bridges to other
centrilobular veins. (c) Late administration of specific COX-2 inhibitor, NS-398, necrosis is limited around centrilobular vein and does
not extend to another central vein. (d) Late administration of indomethacin, a COX-1 and COX-2, liver necrosis was limited to cells
around the centrilobular vein. (e) Liver of rat given NDGA, 24 h after CHCl3 dosing, necrosis extend to another central vein (H&E,
magnification/ 40).
86
C.K. Begay, A.J. Gandolfi / Toxicology 185 (2003) 79 /87
known to protect the blood /nerve barrier, which
is disrupted during liver injury (Miyamoto et al.,
1999). NS-398 can also have an effect on cell
proliferation. In a previous report, NS-398 treatment resulted in suppressed cell proliferation by
inducing a G0/G1 cell-cycle arrest (Nakanishi et
al., 2001). Since there were no changes in the
mitotic index in our hepatoprotection model, we
are not sure if NS-398 had an effect on cell
proliferation in our system.
Although NDGA was able to alleviate Fe-NTAmediated nephro- and hepatotoxicity and tumor
promotion in a prior study (Ansar et al., 1999), the
concentration of NDGA used in this study was not
sufficient to attenuate liver injury at the 48 h time
point. It is possible that NDGA participates in
mediating the reduction of LTB4 at time points
earlier than we tested. NDGA inhibits LOX as
well as monooxygenase activity and provides a
possible mechanism for the protective role of
NDGA against the onset of carcinogenesis, which
is induced by the chemicals that are metabolized
by the cytochrome P-450-dependent enzyme system.
It is important to determine if there are changes
in cellular regeneration rates in the liver following
injury and therapeutic treatment. The mitotic
index was used to measure changes in cellular
growth rate. At the time of peak ALT levels and
maximal necrosis, CHCl3 alone did not alter the
mitotic index. None of the hepatoprotective agents
(DMSO, ABT, indomethacin, NS-398) caused an
increase in mitotic index either at the 48 h time
point or earlier (32 h) when increased mitosis
would need to be present if proliferation the
mechanism of protection. Thus the protective
effect must result from limiting the initial injury
rather than replacing damaged cells.
In conclusion, NS-398 and indomethacin suppressed ALT levels and necrosis in response to
CHCl3-induced liver injury, at a time when liver
necrosis is occurring and progressing. The prevention is the result of interfering with the lesion and
not a stimulation of cell regeneration. Therefore,
our study may provide insight into therapeutic use
of COX inhibitors for toxicant induced liver injury
at such a late time.
Acknowledgements
The authors are grateful to Anne Dahlgren for
her technical assistant and to Dr James RangerMoore for his assistance with statistics. The
Experimental Pathology Core of the Southwest
Environmental Health Science Center (ES-006694)
prepared liver histology (H&E) slides. Douglas W.
Cromey assisted with the quantification of the liver
pathology. This work was supported by a grant
from the National Institutes of Health (ES 08414).
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