Induction of Cytochrome P450 2E1 Increases
Hepatotoxicity Caused by Fas Agonistic Jo2
Antibody in Mice
Xiaodong Wang, Yongke Lu, and Arthur I. Cederbaum
Cytochrome P450 2E1 (CYP2E1) may be a central pathway in generating oxidative stress,
This study evaluated the ability of CYP2E1 to potentiate or synergize the hepatotoxicity of
Fas in vivo. C57BL/6 mice were injected intraperitoneally with pyrazole (Pyr) to induce
CYP2E1. Then, 16-hour fasted mice were administered agonistic Jo2 anti-Fas antibody ip.
Other mice were treated with Pyr or Jo2 alone. Levels of serum aminotransferase were 8.3-
evaluation of liver showed more extensive acidophilic necrosis and severe pathological
changes in the Pyr/Jo2-treated mice. DNA fragmentation and caspase-8 and -3 activities
were more elevated in the Pyr/Jo2 group compared with Jo2 alone. CYP2E1 activity and
protein levels were higher in the Pyr/Jo2 group than in Jo2 alone. Levels of inducible nitric
oxide synthase, 3-nitrotyrosine protein adducts, malondialdehyde, and protein carbonyls
were also higher in the Pyr/Jo2 group compared with Jo2 alone. Glutathione and activities
of catalase and Cu-Zn superoxide dismutase were decreased in the Pyr/Jo2 group. Admin-
istration of chlormethiazole, an inhibitor of CYP2E1, to the Pyr/Jo2-treated mice caused a
with a decrease in CYP2E1 protein and activity. In conclusion, enhanced hepatotoxicity of
Fas was found in mice with elevated levels of CYP2E1. We speculate that overexpression of
CYP2E1 might synergize and increase the susceptibility to Fas induced-liver injury.
lestasis, hepatocellular carcinoma, and iron overload.1
Alcoholic liver disease is a result of complex pathophysi-
ological events involving various types of liver cells and
lcoholic liver disease includes alcoholic fatty liver
(steatosis), alcoholic hepatitis, and alcoholic cir-
rhosis and its complications: intrahepatic cho-
apoptotic-inducing factors, endotoxins, and cytokines.2
The liver is an important target of the toxicity of drugs,
toxins, xenobiotics, and oxidative products. The ethanol-
in metabolism and activation of toxic substrates, such as
ethanol, carbon tetrachloride, acetaminophen, and N-ni-
trosodimethylamine.3-6CYP2E1 may be an important
determinant of human susceptibility to toxicity and car-
cinogenicity of industrial and environmental chemicals.
CYP2E1 may be a central pathway in oxidative stress,
production of reactive oxygen species, and hepatotoxic
injury, especially in the presence of CYP2E1 inducers.7-9
Hepatic apoptosis has been shown to occur in both
experimental and clinical alcoholic liver injury and acute
liver failure.10,11There are two major pathways leading to
hepatic apoptosis: (1) the exogenous pathway, which is
mediated by death signal and receptor systems including
Fas/Fas ligand and tumor necrosis factor (TNF)/TNF
receptor; and (2) the endogenous pathway mediated by
intracellular stress signals including mitochondrial cyto-
Abbreviations: CYP2E1, cytochrome P450 2E1; Pyr, pyrazole; TNF, tumor
necrosis factor; Sal, saline; ALT, alanine aminotransferase; AST, aspartate amino-
nal deoxynucleotidyl transferase–mediated dUTP nick-end labeling; GSH,
glutathione; SOD, superoxide dismutase; CMZ, chlormethiazole.
From the Department of Pharmacology and Biological Chemistry, Mount Sinai
School of Medicine, New York, NY.
Received April 12, 2005; accepted May 21, 2005.
Supported by United States Public Health Service grant AA-03312 from the
National Institute on Alcohol Abuse and Alcoholism.
Address reprint requests to: Dr. A.I. Cederbaum, Department of Pharmacology
and Biological Chemistry, Mount Sinai School of Medicine, One Gustave L. Levy
Place, New York, NY 10029. E-mail: email@example.com; fax:
Copyright © 2005 by the American Association for the Study of Liver Diseases.
Published online in Wiley InterScience (www.interscience.wiley.com).
Potential conflict of interest: Nothing to report.
chrome c release.12,13The Fas/Fas ligand system plays a
central role in ethanol-induced hepatic apoptosis.14-16
Chronic ethanol feeding to rats for 7 weeks did not alter
with lipopolysaccharide increased Fas ligand content to
similar extents in hepatocytes and Kupffer cells of the
by low concentrations of ethanol in HepG2 cells was as-
sociated with Fas-receptor activation and subsequent
the contributions that CYP2E1 plays in alcoholic liver
disease and other drug-induced liver injury, including
nonalcoholic steatohepatitis,19it is of interest to evaluate
whether CYP2E1 contributes or potentiates Fas-medi-
explore the susceptibility and possible synergistic effect of
CYP2E1 overexpression to Fas antibody hepatotoxicity
and assess the involvement of CYP2E1 in the increase of
hepatotoxicity of Fas antibody-induced liver injury fol-
lowing pyrazole pretreatment in mice. These studies may
provide an experimental model to better understand the
mechanism of ethanol-induced liver damage.
Materials and Methods
Animals and Treatments. Experimental animals
were male C57BL/6 mice (20-23 g, 6-8 weeks of age)
purchased from Charles River Breeding Laboratory (Bos-
ton, MA) and housed in a facility approved by the Amer-
ican Association for Accreditation of Laboratory Animal
Care. Experiments were performed with approval of
Mount Sinai’s Animal Use and Committee. Mice were
divided into saline (Sal), Jo2 alone (Jo2), pyrazole (Pyr)
Jo2) groups, respectively (four groups of 8-10 mice
each). Mice were injected intraperitoneally with Pyr
(Sigma, St. Louis, MO), 120 mg/kg body weight, once
a day for 2 days to induce CYP2E1. After 16-hour
fasting (for maximal CYP2E1 induction), mice were
administered intraperitoneally with Sal or with agonis-
tic Jo2 hamster anti-mouse Fas monoclonal antibody
(BD Pharmingen, San Diego, CA), 0.2 ?g/g body
weight.20A Jo2 concentration of 0.2 ?g per gram body
weight or about 4 ?g per mouse was found to initiate
mild toxicity, so this was the dose of Jo2 chosen for the
At 8 hours after administration of Jo2 or saline, mice
ment of serum aminotransferases and TNF-?. The liver
was rapidly excised, and specimens were immediately cut
logical and immunohistochemical assessment. The re-
maining liver samples were immediately frozen in liquid
nitrogen and stored at ?70°C in aliquots for preparation
of homogenates and further use. Serum alanine amino-
transferase (ALT) and aspartate aminotransferase (AST)
levels were measured using a diagnostic kit (Ther-
moDMA, Louisville, CO) and kinetically following
changes in absorbance at 340 nm. The serum TNF-?
Biotechnology, Rockford, IL).21
Liver Histopathology and Immunohistochemistry.
Small liver fragments were immediately cut into 1-mm3
blocks, fixed in ice-cold 3% glutaraldehyde in phosphate-
buffered saline for transmission electron microscopy
processed into paraffin sections for hematoxylin-eosin
staining and histopathological observation. The morpho-
logical changes of liver tissues were observed by two pa-
thologists who were blinded from the experimental
information. All changes of degeneration and necrosis
were graded as none (0), mild (?25%), moderate (25%-
50%), and severe (?75%).
Immunohistochemical staining was performed with
the ImmunoCruz Rabbit ABC Staining System Kit
with polyclonal rabbit–anti-CYP2E1 antibody (1:200) (a
gift from Dr. Jerome Lasker, Hackensack Biomedical Re-
search Institute, Hackensack, NJ), for 3-nitrotyrosine (3-
NT) protein adducts with polyclonal rabbit–anti–3-NT
antibody (1:100) (Upstate USA Inc., Lake Placid, NY),
and for inducible NO synthase (iNOS) with polyclonal
rabbit–anti-iNOS antibody (1:200) (Chemicon, Te-
mecula, CA). Slides were visualized with 3,3-diamino-
benzidine, and positive staining was reflected by a
brownish-yellow color. Immunofluorescence staining of
an anti-rabbit–fluorescein–immunoglobulin G (1:10)
(Biomeda, Foster City, CA). Positive staining was re-
flected by a yellowish-green fluorescence. In each case, a
uation of a specific positive reaction was marked as nega-
tive (?), weakly positive (?), moderately positive (??),
and strongly positive (???).
Cytochrome P450 2E1 Activity and Lipid Peroxi-
dation. Liver homogenates were freshly prepared in a
5-10 volume of ice-cold 150 mmol/L KCl. Microsomes,
mitochondria, and the cytosol fractions were prepared
using differential centrifugation. The protein concentra-
tion of the different fractions was determined using a
protein assay kit obtained from BioRad (Hercules, CA).
HEPATOLOGY, Vol. 42, No. 2, 2005 WANG, LU, AND CEDERBAUM401
CYP2E1 activity was measured in liver microsomes by
assaying the oxidation of p-nitrophenol to p-nitrocat-
The production of thiobarbituric acid reactive sub-
stances, expressed as malondialdehyde equivalents, was
assayed in liver mitochondria, microsomes and total liver
homogenate fractions by the spectrophotometric analysis
of malondialdehyde was calculated using an extinction
coefficient of 1.56 ? 105mol/L/cm and expressed as pi-
comoles per milligram of protein.
Western Blot Analysis of Protein Expression and
Carbonyls. Levels of CYP2E1, iNOS, and Fas receptor
protein in 10-50 ?g of protein samples from freshly pre-
pared microsome or cytosol fractions were determined
using Western blot analysis with anti-CYP2E1 antibody
(1:10,000), anti-iNOS antibody (1:2,000), and anti-Fas
antibody (1:200) (Santa Cruz Biotechnology), respec-
tively, followed by incubation with horseradish peroxi-
dase conjugated to goat anti-rabbit immunoglobulin G
(1:5,000) (Sigma). Chemiluminscence reaction using an
enhanced chemiluminescence kit (Amersham Bio-
sciences, Buckinghamshire, England) was performed for
1 minute followed by exposure to Kodak BioMax film
assayed in liver homogenates using 20 ?g of protein sam-
ples and the OxyBlot Protein Oxidation Detection Kit
(Chemicon). The sample loading was controlled by addi-
bands of protein adducts detected via Western blot anal-
ysis were quantitated with the Automated Digitizing Sys-
tem (UN-SCAN-IT gel programs, version 5.1, Silk
Scientific Corp., Orem, UT).
Terminal Deoxynucleotidyl Transferase–Mediated
dUTP Nick-End Labeling and DNA Fragmentation
Analysis. Apoptosis was assessed via terminal deoxynu-
cleotidyl transferase–mediated dUTP nick-end labeling
(TUNEL) assay using the ApopTag in situ apoptosis de-
tection kit (Serological Corp., Atlanta, GA). The labeled
DNA was visualized with horseradish peroxidase–conju-
gated anti-digoxigenin antibody. For a negative control,
the terminal deoxynucleotidyl transferase enzyme was
omitted from the reaction mixtures. Slides were counter-
stained with 0.5% methyl green. The quantitative analy-
sis of positive nuclei with DNA fragmentation or
?400 [25 visual fields]). The formation of a DNA frag-
sis. DNA was loaded onto a 1.5% agarose gel containing
ethidium bromide, electrophoresed in Tris-acetate–
[pH 8.0]) at 80 V and photographed under UV illumina-
Caspase Activities. Caspase-8, -3, -9, and -6 activity
was determined in liver tissue homogenates by measuring
proteolytic cleavage of the proluminescent substrates (0.1
mmol/L) Z-IETD-AFC, AC-DEVD-AMC, AC-LEHD-
AFC (Calbiochem, La Jolla, CA), and AC-VEID-AFC
(MP Biomedicals, Aurora, OH), which are cleaved by
caspase-8, caspase-3, caspase-9, and caspase-6, respec-
tively. Fluorescence was determined with a spectroflu-
orometer (PerkinElmer, Wellesley, MA) based on the
400, ?em? 505) or AMC (caspase-3 activity, ?ex? 380,
?em? 460). The results were expressed as arbitrary units
of fluorescence per milligram of protein.
Antioxidant Levels. Glutathione (GSH) was ana-
lyzed in liver tissue homogenates via fluorescence assay
with the proluminescent substrate o-phthalaldehyde (1
mg dissolved in 1 mL of 50 mmol/L sodium phosphate).
Fluorescence was determined with ?ex? 350 and ?em?
420 nm. Catalase activity was assayed by measuring the
decomposition of H2O2at 240 nm.25The activity of Cu/
Zn-superoxide dismutase (SOD) was determined by the
kinetic measurement of the absorbance at 525 nm from
the Vs/Vc ratio of the autoxidation rates as a function of
Cu/Zn-SOD in cytosol (Calbiochem).
Evaluation of Chlormethiazole Inhibition In Vivo.
Chlormethiazole (CMZ), a drug used for alcohol with-
drawal and agitation, and an efficient inhibitor of
CYP2E1 activity,26was used to try to prevent the Jo2
agonist Fas antibody–induced liver injury found in Pyr-
treated mice. Mice were divided into Sal (n ? 3), Pyr/Jo2
(n ? 6), and Pyr/Jo2 plus CMZ (n ? 16) groups, respec-
tively. CMZ was injected intraperitoneally at a dose of 75
mg/kg body weight 30 minutes after Jo2 injection. At 8
the retro-orbital venous sinus for measurement of serum
imens were immediately cut into small fragments and
placed in a fixative for histopathological observation. The
Statistical Analysis. Values reflect the mean ? SEM.
For statistical analysis, one-way ANOVA (and subse-
data analysis toolpac. The Student t test was used for the
TUNEL assays after square root calculation of positive
cell counts. P values of less than .05 were considered sta-
402WANG, LU, AND CEDERBAUMHEPATOLOGY, August 2005
Liver Pathological Changes. In initial experiments,
we first tested the effect of various doses of the Jo2 anti-
body alone on liver injury to select a concentration that
would cause only a mild toxic effect, thereby setting the
stage to try to potentiate this mild injury (via Pyr treat-
were slightly increased 8 hours after administration of 2
and 4 ?g Jo2, but were more dramatically elevated after
giving 6 and 8 ?g of the Jo2 anti-Fas antibody per mouse
(Fig. 1A). The low toxic dose of 4 ?g per mouse (0.2 ?g
per g body weight) was chosen for all the following stud-
ies. Mice were treated with Sal, Pyr alone, Jo2 alone, or
AST activities were significantly higher in the combined
Jo2/Pyr treatment than that in the mice given only Jo2 or
only Pyr (P ? .001) (Fig. 1B). These concentrations of
Jo2 and Pyr were chosen because they did not produce
light microscopy, severe pathological changes were de-
tected in the combined Jo2/Pyr treatment group as many
hepatocytes appeared to display extensive acidophilic ne-
crosis (apoptosis) and focal hemorrhages in the hepatic
there was no marked infiltration of inflammatory cells
(Fig. 2B-C compared with Fig. 2A). Electron microscopy
ulum dilation, and numerous lipid droplets in hepato-
cytes (Fig. 2E-F compared with Fig. 2D). Some mild
Fig. 1. Levels of serum ALT and AST after treatment with Jo2, Pyr, or
Jo2 plus Pyr. (A) Effects of increasing doses of Jo2 anti-Fas antibody on
serum ALT and AST activity. Mice fasted for 16 hours were treated with
saline or the Jo2 antibody (1, 2, 4, 6, and 8 ?g per mouse). Serum ALT
and AST were measured at 8 hours after Jo2 administration (mean ?
SEM for 8-10 mice). *P ? .05; **P ? .01 Jo2 vs. saline control. (B)
Mice were pretreated with pyrazole once a day for 2 days (120 mg/kg
body weight) or saline, fasted for 16 hours, and treated with Jo2 anti-Fas
antibody (0.2 ?g/g body weight) or saline. Serum ALT and AST were
measured at 8 hours after the treatment with Jo2 anti-Fas antibody or
saline. ***P ? .001 for Pyr/Jo2 vs. the other groups. ALT, alanine
aminotransferase; AST, aspartate aminotransferase; Sal, saline; Pyr,
Fig. 2. Morphological changes of liver tissues observed under (A-C) light microscopy and (D-F) transmission electron microscopy. Panels A and
D are from the Jo2-treated mice; panels B, C, E, and F are from the Pyr/Jo2-treated mice. (A) Slight sinusoid dilation and congestion, swelling, and
focal necrosis of hepatocytes in centrilobular zone (arrows) (hematoxylin-eosin stain; original magnification ?200). (B-C) Many hepatocytes show
extensive acidophilic necrosis (apoptosis) and severe hemorrhages but no marked infiltration of inflammatory cells in hepatic centrilobular zone or
intermediate lobular zone (arrows) (hematoxylin-eosin stain; original magnification ?200). (D) Limited mitochondrial swelling and endoplasmic
reticulum dilation, and no marked lipid droplets in hepatocytes (arrows) (transmission electron microscopy; original magnification ?12,000). (E-F)
Severe mitochondrial swelling, endoplasmic reticulum dilation, and numerous lipid droplets in hepatocytes (arrows) (panel E, transmission electron
microscopy, original magnification ?12,000; panel F, transmission electron microscopy, original magnification ?3,000).
HEPATOLOGY, Vol. 42, No. 2, 2005WANG, LU, AND CEDERBAUM 403
lesions were observed in the mice administrated only Jo2,
mainly including dilation and congestion in the sinusoid,
swelling and focal necrosis of hepatocytes in the centri-
lobular area, and slight mitochondrial swelling and endo-
plasmic reticulum dilation in hepatocytes (Fig. 2A,D).
Only mild swelling of hepatocytes was found after Pyr
administration alone (data not shown) while the mor-
phology of Sal control was normal (data not shown). The
expression in situ of the Fas receptor was increased in the
Pyr/Jo2 group compared with the other groups (Fig. 3A,
panels 3 and 4 compared with panels 1 and 2). The pro-
tein levels of Fas receptor as detected via Western blot
analysis were also higher in the Pyr/Jo2 group than in the
other groups (Fig. 3B). These results suggest that Fas re-
ceptor expression gradually increased with increased
pathological changes. The content of TNF-? in serum
was higher in both the Pyr/Jo2 and Jo2 alone group com-
significant difference between the Pyr/Jo2 and Jo2 alone
groups (P ? .05) (Fig. 3C).
CYP2E1 Protein Expression and Catalytic Activity.
There was an approximately twofold increase in CYP2E1
activity in the Pyr alone group compared with the Sal
Fig. 4. Activity and protein expression of CYP2E1. (A) CYP2E1 activity
was measured by evaluating the oxidation of p-nitrophenol as described
in Materials and Methods. **P ? .01 Pyr vs. saline-treated, *P ? .05
Pyr/Jo2 vs. saline-treated. (B) Immunohistochemical staining showing in
situ expression of CYP2E1 in hepatocytes. Panel B1 shows Sal controls;
panel B2 shows Jo2 alone; panel B3 shows Pyr alone; panel B4 shows
Pyr/Jo2 administration (arrows indicate the centrilobuar zone of the liver)
(original magnification ?200). (C) The levels of CYP2E1 in 10- or 30-?g
microsome fractions were determined via Western blot analysis. CYP2E1,
cytochrome P450 2E1; Sal, saline; Pyr, pyrazole.
Fig. 3. Fas receptor expression and serum TNF-? contents. (A) Im-
munofluorescence staining of Fas receptor in liver sections. Panel A1 is
from the Jo2-treated mice, panel A2 is from the Pyr-treated mice, and
panels A3 and A4 are from the Pyr/Jo2-treated mice. Staining intensity
was ??? for panels A3 and A4; ?? for panel A1; and ? for panel
A2 (arrows) (original magnification ?200). (B) Western blot analysis for
expression of the Fas receptor. Numbers under the blots refer to the ratio
of Fas/?-actin. (C) Serum TNF-? level was assayed using a mouse TNF-?
ELISA kit. **P ? .01 for Pyr/Jo2 or for Jo2 vs. saline control; #P ? .05
for Pyr/Jo2 vs. Jo2-treated. TNF, tumor necrosis factor; Sal, saline; Pyr,
404WANG, LU, AND CEDERBAUMHEPATOLOGY, August 2005
in the Pyr/Jo2 group (P ? .05) compared with the Sal
controls (Fig. 4A). Increases of 2.9- and 2.5-fold of
CYP2E1 protein expression were detected via Western
blot analysis in the Pyr/Jo2 and Pyr alone groups com-
nohistochemistry confirmed that expression of CYP2E1
in situ was markedly higher in the Pyr/Jo2 and Pyr alone
groups (??-???) compared with the Sal controls
(0-?) (Fig. 4B). The highest level of CYP2E1 was in the
centrilobular zone of the liver acinus, the zone in which
ity and protein expression of CYP2E1 were also found in
the Jo2 alone group. The mechanism for this will require
Apoptotic Liver Injury. To assess apoptosis, caspases
activities, DNA fragmentation, and DNA ladder forma-
tion were determined. Both caspase-8 and caspase-3 ac-
tivity was significantly higher in the Pyr/Jo2 group and
the Jo2 alone group than that in Sal controls, and there
was a significant difference in the Pyr/Jo2 group com-
1). The activities of caspase-9 and caspase-6 were also
significantly higher in both the Pyr/Jo2 and Jo2 alone
group than that in Sal controls, but there was no signifi-
cant difference between the two (P ? .05) (Table 1). Pyr
treatment had no significant effect on any of the caspase
activities. TUNEL results showed that there were more
hepatocytes with positive staining nuclei in the Pyr/Jo2
group than in the Jo2 group (Fig. 5A). After quantitative
increase in the Pyr/Jo2 group (27.20 ? 5.76) compared
with the Jo2 alone group (19.8 ? 3.54) (P ? .01) (Fig.
5B). Pyr treatment alone caused a small increase in
TUNEL-positive nuclei compared with Sal control.
DNA ladder analysis showed that a DNA ladder was ob-
served only in the Jo2 alone and Pyr/Jo2 groups, but not
the Pyr alone group (Fig. 5C).
Liver Lipid Peroxidation and Protein Carbonyls.
carbonyls, levels of thiobarbituric acid reactive substances
homogenates were analyzed. The amount of malondial-
dehyde, an end product of lipid peroxidation, was signif-
icantly higher in the mitochondrial and microsomal
alone group than that in Sal controls or the Jo2 alone
group (Table 2). The levels of protein carbonyls were also
controls or Jo2 alone group (Fig. 6B). Jo2 alone did not
increase either lipid peroxidation or protein carbonyls.
Expression of iNOS and 3-NT Protein Adducts.
NO and NO-derived products such as peroxynitrite
(ONOO?) may play a key role in stress-induced injuries
and certain liver diseases,27including alcoholic liver dis-
ease.28Immunohistochemical observation showed that
3-NT protein adducts were mainly expressed in situ in
hepatocytes in the central lobular zone of the liver or in
the area subject to injury (Fig. 6A). The positive expres-
sion of 3-NT adducts was higher in the Pyr/Jo2 group
(???) than in the Jo2 alone group (?) (Fig. 6A). Sim-
ilarly, expression of iNOS was mainly found in the cen-
trilobular zone and nearby areas showing injury in the
Pyr/Jo2 group, and iNOS levels were higher in the Pyr/
Jo2 group (???) than in the Jo2 alone or Pyr alone
groups (?) (Fig. 7A). The expression of iNOS protein in
liver cytosol fractions was also increased in the Pyr/Jo2
group and the Pyr alone group compared with the Sal
control, or the Jo2 alone group (Fig. 7B). Jo2 had little
effect on iNOS levels or 3-NT protein adducts.
Changes of Antioxidant Levels. GSH levels were
slightly increased in the Jo2 and Pyr alone groups com-
pared with Sal controls but were markedly decreased in
the Pyr/Jo2 group (P ? .01) (Fig. 8A). Catalase activity
gradually decreased in all experimental groups compared
with Sal controls; the lowest activity was observed in the
slightly but not significantly decreased in the Jo2 or Pyr
alone groups; the lowest Cu/Zn-SOD activity was in the
these changes were not considered significant (P ? .05)
Protective Effect of CMZ. The hypothesis being
evaluated in this study was that induction of CYP2E1 by
treatment with Pyr may potentiate or synergize with Fas
to promote liver injury. It was therefore important to
provide evidence that inhibition of CYP2E1 would pre-
vent the enhanced toxicity found in the Pyr/Jo2 group.
shown to prevent alcoholic liver disease in the intragastric
Table 1. Activities of Caspase-8, -3, -9, and -6
in Liver Homogenates
506 ? 172
254 ? 48
512 ? 99
1,592 ? 210
3,484 ? 1,017**
2,940 ? 1,267*
3,476 ? 765*
7,605 ? 1,484*
783 ? 113
516 ? 230
452 ? 96
1,729 ? 599
6,767 ? 692**‡
5,458 ? 1,498*†
4,911 ? 1,652*
8,594 ? 673*
NOTE. Caspase-8, -3, -9, and -6 activity was determined in liver tissue
homogenates by measuring proteolytic cleavage of the specific substrate as
described in Materials and Methods. The results are expressed as arbitrary units
of fluorescence per milligram of protein.
*P ? .01, **P ? .05 Jo2 vs. saline control; pyrazole/Jo2 vs. saline control.
†P ? .01, ‡P ? .05 pyrazole/Jo2 vs. Jo2 alone.
HEPATOLOGY, Vol. 42, No. 2, 2005WANG, LU, AND CEDERBAUM405
infusion model.29Serum ALT was significantly lower in
the Pyr/Jo2/CMZ group compared with the Pyr/Jo2
group (P ? .001) (Fig. 9A). The activity of CYP2E1 was
also lower in the Pyr/Jo2/CMZ group compared with the
CYP2E1 protein level after treatment with CMZ (Fig.
9B-C). Histopathological evaluation showed that there
was only restricted focal necrosis and slight congestion in
(mild or moderate changes), while extensive acidophilic
necrosis and severe degeneration was observed in the he-
patic centrilobular zone of the Pyr/Jo2 group (Fig. 9D).
The biochemical and toxicological effects of CYP2E1
have been studied in HepG2 cells engineered to express
this cytochrome P450 and in cultured hepatocytes from
pyrazole-treated rats with high levels of CYP2E1.30To
further explore these biological effects in vivo, the current
potential susceptibility of mice induced to express
CYP2E1 to Fas antibody-induced liver damage.31Hepa-
totoxicity significantly increased in the Pyr/Jo2 group
compared with the other groups. There were also low
hepatotoxic levels in the Pyr alone group. Subtoxic ad-
ministration of Pyr was able to potentiate the hepatotox-
icity caused by suboptimal administration of Jo2
tion with the least toxicity of the inducer itself, we ad-
justed the concentration of Pyr administration to 120
mg/kg body weight in this experimental model.
in receptor cross-linking and apoptosis of Fas-positive
cells. Growing evidence suggests that the Fas/Fas ligand
system is one of the most important signaling pathways
The Fas receptor is constitutively expressed in hepato-
Table 2. Lipid Peroxidation in Different Fractions
of Liver Tissues
163 ? 39173 ? 31377 ? 80*221 ? 112
690 ? 1061,232 ? 4252,549 ? 601** 3,184 ? 1,609*†
2,090 ? 579 2,569 ? 2795,833 ? 439** 3,704 ? 118**
NOTE. The production of thiobarbituric acid-reactive substances, expressed as
malondialdehyde equivalents, was assayed as described in Materials and Meth-
ods. The results are expressed as picomoles per milligram of protein.
*P ? .01, **P ? .05 pyrazole vs. saline control; pyrazole/Jo2 vs. saline
†P ? .01, pyrazole/Jo2 vs. Jo2 alone.
Fig. 5. DNA fragmentation analysis. (A) Apoptosis of liver was assessed based via TUNEL assay using the ApopTag in situ apoptosis detection
kit as described in Materials and Methods. Panel A1 shows Jo2 alone; panel A2 shows Pyr alone; panel A3 shows Pyr/Jo2 administration (arrows)
(original magnification ?200). (B) The quantitative analysis of positive nuclei with DNA fragmentation was performed by counting the average number
of apoptotic nuclei per visual field (original magnification ?400, 25 visual fields). **P ? .01 Pyr/Jo2 vs. Jo2- or Pyr-treated, #P ? .01 Jo2- vs.
Pyr-treated. (C) The observation of a DNA fragmentation ladder was performed by agarose gel electropherosis as described in Materials and Methods.
Sal, saline; Pyr, pyrazole.
406WANG, LU, AND CEDERBAUMHEPATOLOGY, August 2005
cytes. Fas-mediated apoptosis may occur in an autocrine
or paracrine fashion via a soluble form of the ligand. Fas-
mediated killing is not restricted to lymphocytes but may
also occur in nonlymphoid cells such as hepatocytes (e.g.,
after bile duct ligation).32-34The intracellular pathway of
apoptosis includes receptor oligomerization and recruit-
ment of the Fas-associated protein with death domain,
which eventually leads to the activation of caspase 8 and
downstream caspases.35-37Cell death stimulated by ago-
nist Fas antibodies is characteristic of apoptosis, suggest-
ing that the lethal effects are a result of interaction of the
antibody with a functional Fas antigen as opposed to
complement-mediated lysis.21,38Apoptosis was produced
in the Jo2-treated mice, and the apoptosis was signifi-
assays of DNA fragmentation and caspase-8 and -3 activ-
ities. The Pyr treatment did not cause DNA fragmenta-
tion or increase activities of caspases. The toxicity found
after Pyr/Jo2 treatment did not appear to reflect an in-
flammatory response as significant infiltration of inflam-
were elevated in the Pyr/Jo2 and Jo2 groups, so this cyto-
kine may be important for the Jo2 toxicity and the Jo2
contribution to the overall toxicity in the Pyr/Jo2 group.
Toxic interactions between CYP2E1 and TNF-? have
been reported.39,40Further studies on Kupffer cell activa-
tion and cytokine production are planned in these mod-
els. That Jo2 causes apoptosis and increases TNF-? but
does not evoke an inflammatory response is in agreement
with a previous report.41These results suggest that Fas
antibody was able to synergize with CYP2E1 to directly
result in the death of hepatocytes expressing the Fas re-
CYP2E1 plays a role in ethanol or hepatotoxin-in-
duced oxidative stress and lipid peroxidation.42,43In-
Fig. 6. Levels of 3-NT and protein carbonyl adducts. (A) Immunohistochemical staining was performed using the ImmunoCruz ABC kit for 3-NT
protein adducts. In each case, a negative control (nonimmune serum) was used. Panel A1 shows Sal control; panel A2 shows Jo2 alone; panel A3
shows Pyr alone; panel A4 shows Pyr/Jo2 administration (arrows) (original magnification ?200). (B) The protein carbonyl adducts level was assayed
in liver homogenates as described in Materials and Methods. In each case, a negative control (derivatization–control solution instead of the DNPH
solution) and a positive control (dinitrophenylated standard protein) were used. Sal, saline; Pyr, pyrazole; NT-SM, nontreated standard molecule.
Fig. 7. Levels of iNOS. (A) Immunohistochemical observation showed
that the iNOS protein was mainly expressed in situ in hepatocytes in the
centrilobular zone or in the surrounding vessel area. Panel A1 shows Sal
control; panel A2 shows Jo2 alone; panel A3 shows Pyr alone; panel A4
shows Pyr/Jo2 administration (arrows) (original magnification ?200).
(B) The levels of iNOS protein in 50 ?g of protein samples from freshly
prepared cytosol fractions were determined via Western blot analysis as
described in Materials and Methods. Sal, saline; Pyr, pyrazole; iNOS,
inducible NO synthase.
HEPATOLOGY, Vol. 42, No. 2, 2005 WANG, LU, AND CEDERBAUM 407
be critical features in ethanol-induced liver injury.8-10,44,45
Nitrosative stress also causes cellular dysfunction, DNA
damage, and cell death.46,47Peroxynitrite, formed by the
rapid reaction of NO and superoxide, produces nitrated
tyrosine protein adducts, eventually resulting in the de-
nitration of tyrosine.48,49These data suggest that per-
oxynitrite may play a critical role in the development of
hepatotoxicity. The levels of iNOS, 3-NT protein ad-
ducts, malondialdehyde, and protein carbonyl formation
were higher in the Jo2/pyr group than in the Jo2 alone
group. This suggests that oxidative and nitrosative stress
occur to a greater extent in the Pyr/Jo2 group. Future
studies will evaluate the possible protection by antioxi-
dants, ONOO?scavengers, and inhibitors of iNOS on
the liver toxicity found in the Pyr/Jo2 group. We specu-
late that the increase in iNOS, which produces NO, and
the increase in CYP2E1, which produces superoxide, sets
formation of 3-NT protein adducts.
Modulation and adaptation of antioxidants and anti-
oxidant enzymes is critical to protect cells against oxidant
stress. Levels of several antioxidants were elevated in
to be an important adaptation to protect the cells against
CYP2E1-dependent oxidant stress.30Levels of antioxi-
dants and antioxidant enzymes are gradually decreased
stress and scavenging of activated reactive oxygen species.
Levels and activities of GSH and Cu-Zn-SOD did not
controls but did decrease 2- to 5-fold in the Jo2/Pyr
group, which may reflect antioxidant mechanisms to re-
Fig. 8. Changes in antioxidant levels. (A) GSH was analyzed in liver tissue homogenates. The concentration of GSH was calculated from a GSH
standard curve and results were expressed as nmol per mg of protein. *P ? .05 for Pyr/Jo2 vs. Jo2- or Pyr-treated. (B) Catalase activity was assayed
by measuring the decomposition of H2O2at 240 nm. The activity of catalase was calculated using a formula of activity (units/mg protein)
?OD240/43.6/mg protein ? 103. *P ? .05 for Jo2 vs. saline. **P ? .01 for Pyr vs. saline. ***P ? .001 for Pyr/Jo2 vs. saline. ##P ? .01 for
Pyr/Jo2 vs. Jo2. (C) The activity of SOD was determined as the kinetic measurement of the 525 nm absorbance from the Vs/Vc ratio of the
autoxidation rates as a function of Cu/Zn-SOD in the cytosol. Results are expressed as units per mg of protein. *P ? .05 for Pyr/Jo2 vs. saline control.
#P ? .05 for Pyr/Jo2 vs. Jo2-treated. GSH, glutathione; Sal, saline; Pyr, pyrazole; SOD, superoxide dismutase.
Fig. 9. Protective effect of CMZ against Jo2 plus pyrazole-induced liver
damage. (A) CMZ was injected intraperitoneally at a dose of 75 mg/kg
body weight 30 minutes after Jo2 injection into Pyr-treated mice. Serum
ALT was measured at 8 hours after the addition of Jo2 anti-Fas antibody.
***P ? .001 Pyr/Jo2/CMZ vs. Pyr/Jo2. (B) CYP2E1 activity was mea-
sured in liver microsome fractions via oxidation of p-nitrophenol. **P ?
.001 Pyr/Jo2/CMZ vs. Pyr/Jo2. (C) The levels of CYP2E1 in 50 ?g of
microsomal protein were determined via Western blot analysis. (D) Liver
pathology. Panel 1 shows extensive acidophilic necrosis and severe
degeneration of hepatocytes in the hepatic centrilobular or intermedia-
telobular zone in mice 8 hours after Jo2 administration following Pyr
pretreatment for 2 days (arrows) (hematoxylin-eosin stain, original mag-
nification ?200). Panel 2 shows restricted focal necrosis and slight
congestion in the hepatic centrilobular zone in Jo2/Pyr-treated mice after
administration of CMZ (arrows) (hematoxylin-eosin stain, original mag-
408 WANG, LU, AND CEDERBAUMHEPATOLOGY, August 2005
move CYP2E1 plus Jo2-derived oxidants. Such decreases
may contribute to the developing toxicity in the Pyr/Jo2-
treated mice. Future experiments will evaluate the ability
of antioxidants and iNOS inhibitors to prevent this tox-
CYP2E1 levels and activities were increased in the Pyr
and Pyr/Jo2 mice, and the Fas-induced liver pathology
correlates with CYP2E1 levels and elevated lipid peroxi-
dation. The histopathological localizations of liver injury
were in agreement with the distributions of CYP2E1,
iNOS, and 3-NT protein adducts. CMZ protected the
liver from free radical damage by inhibiting CYP2E1 ac-
tivity, thereby ameliorating pathological changes and at-
tenuating fibrosis.50There was a significant decrease in
ALT, CYP2E1 activity, and protein level, as well as pre-
vention of the liver pathology in the Pyr/Jo2 mice treated
Jo2 Fas antibody-induced hepatotoxicity following pre-
treatment with Pyr, most probably by inhibiting the ac-
tivity and overexpression of CYP2E1 in the liver.
In conclusion, the CYP2E1 inducer Pyr could poten-
tiate the hepatotoxicity caused by the Jo2 agonist Fas
antibody, suggesting that overexpression of CYP2E1
might contribute to the synergy and susceptibility of the
liver to Fas-induced injury. Potential mechanisms in-
volved in the potentiated toxicity include elevated oxida-
tive stress, nitrosative stress, lipid peroxidation, and
other hepatotoxins such as Fas to promote liver injury. It
is interesting to speculate that some of the interactions
between alcohol and Fas may involve the alcohol-induc-
ible CYP2E1, but this has yet to be validated.
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