STEATOHEPATITIS/METABOLIC LIVER DISEASE
Cytochrome P450 2E1 Contributes to Ethanol-Induced
Fatty Liver in Mice
Yongke Lu, Jian Zhuge, Xiaodong Wang, Jingxiang Bai, and Arthur I. Cederbaum
Cytochrome P450 2E1 (CYP2E1) is suggested to play a role in alcoholic liver disease, which
includes alcoholic fatty liver, alcoholic hepatitis, and alcoholic cirrhosis. In this study, we
investigated whether CYP2E1 plays a role in experimental alcoholic fatty liver in an oral
ethanol-feeding model. After 4 weeks of ethanol feeding, macrovesicular fat accumulation
and accumulation of triglyceride in liver were observed in wild-type mice but not in
CYP2E1-knockout mice. In contrast, free fatty acids (FFAs) were increased in CYP2E1-
knockout mice but not in wild-type mice. CYP2E1 was induced by ethanol in wild-type
mice, and oxidative stress induced by ethanol was higher in wild-type mice than in CYP2E1-
knockout mice. Peroxisome proliferator-activated receptor ? (PPAR?), a regulator of fatty
in CYP2E1-knockout mice. Chlormethiazole, an inhibitor of CYP2E1, lowered macrove-
wild-type mice fed ethanol. The introduction of CYP2E1 to CYP2E1-knockout mice via an
adenovirus restored macrovesicular fat accumulation. These results indicate that CYP2E1
contributes to experimental alcoholic fatty liver in this model and suggest that CYP2E1-
derived oxidative stress may inhibit oxidation of fatty acids by preventing up-regulation of
PPAR? by ethanol, resulting in fatty liver. (HEPATOLOGY 2008;47:1483-1494.)
ical events involving various types of liver cells and
injurious factors such as oxidative/nitrosative stress, lipo-
polysaccharide (LPS), and cytokines.2Fatty liver is a uni-
form and early response of the liver to alcohol
consumption, and it was previously considered to be be-
nign. However, now it is known that fatty livers and re-
lated disorders like obesity and chronic ethanol treatment
lcoholic liver disease includes alcoholic fatty liver
(steatosis), alcoholic hepatitis, and alcoholic cir-
rhosis1and is a result of complex pathophysiolog-
cause increased sensitivity to hepatotoxins such as LPS.3-5
There is an increased interest in and need to understand
the mechanisms by which ethanol induces steatosis.
atosis is also related to oxidative stress, as shown by the pio-
neering studies of Diluzio, who found that antioxidants can
mation and necrosis but also reduces steatosis by 50% in
pathways have been suggested to play a key role in how
cytochrome P450 2E1 (CYP2E1).10In this study, we used
CYP2E1-knockout mice, the CYP2E1 inhibitor chlorme-
thiazole (CMZ), and adenovirus-mediated expression of
CYP2E1 to evaluate whether CYP2E1 plays a role in alco-
holic fatty liver.
Materials and Methods
Animals and Ethanol Treatment.
ground CYP2E1-knockout and wild-type mice were
kindly provided by Dr. Frank J. Gonzalez (Laboratory of
Abbreviations: ACC, acetyl CoA carboxylase; AMPK, AMP-activated protein
kinase; AOX, acyl-CoA oxidase; CMZ, chlormethiazole; CYP2E1, cytochrome
P450 2E1; FFAs, free fatty acids; L-FABP, L-fatty acid binding protein; FAS, fatty
acyl synthase; HNE, 4-hydroxy-2-nonenal; LPS, lipopolysaccharide; 3-NT, 3-ni-
dismutase; SREBP, sterol regulatory element-binding protein.
From the Department of Pharmacology and Systems Therapeutics, Mount Sinai
School of Medicine, New York, NY
Received July 23, 2007; accepted January 2, 2008.
Supported by United State Public Health Service Grants AA03312 and
AA06610 (to A.I.C.) and AA015697 (to J.B.) from the National Institute on
Alcohol Abuse and Alcoholism.
Address reprint requests to: Arthur I. Cederbaum, Department of Pharmacology
E-mail: email@example.com; fax: 212-996-7214.
Copyright © 2008 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.
Metabolism, National Cancer Institute, Bethesda,
MD),11and the female offspring of these mating pairs
were used in this study. All mice were housed in temper-
ature-controlled animal facilities with 12-hour light/12-
hour dark cycles and were permitted consumption of tap
water and Purina standard chow ad libitum until being
fed the liquid diets. The mice received humane care, and
lined in the Guide for the Care and Use of Laboratory
Animals and with approval of the Mount Sinai Animal
Care and Use Committee.
Female mice were initially fed the control liquid dex-
trose diet (Bio-Serv, Frenchtown, NJ) for 3 days to accli-
mate them to the liquid diet. Afterward, knockout and
wild-type mice were fed either the liquid ethanol diet
diet, as described by Lieber and DeCarli.12For experi-
ments involving 2 weeks of feeding (CMZ and adenovi-
rus), the mice were directly subjected to the diet
containing ethanol as 35% of total calories (equivalent to
6.2% [vol/vol]). In initial experiments, we observed that
the mice could not tolerate ethanol at 35% of total calo-
ries for more than 2 weeks; therefore, for experiments
involving 3 or 4 weeks of feeding, the content of ethanol
was gradually increased every 3-4 days from 10% (1.77%
(4.42% [vol/vol]), 30% (5.31% [vol/vol]), and finally
35% of total calories. The wild-type and knockout con-
trol mice were pair-fed the control dextrose diet on an
the dextrose or ethanol liquid diets for 4 weeks as above,
LPS (Sigma, serum type 055:B5; single dose, 4 mg/kg
body weight, ip) was injected, and 8 hours later, the mice
were sacrificed. For CMZ experiments, during the 2
weeks of feeding with 35% ethanol or dextrose liquid
diets, CMZ (Sigma; 50 mg/kg body weight, ip) was in-
jected every other day into the mice. For adenovirus-
adenovirus. The ethanol-fed mice had access to their ra-
tions ad libitum, and the conditions of knockout and
wild-type mice were comparable. The amount of food
consumed by CYP2E1-knockout mice and wild-type
mice was approximately the same, and CMZ or adenovi-
shown). Whole liver was removed and liver weight mea-
sured; then the liver was rapidly excised into fragments
placed in 10% formalin solution for paraffin blocking.
Another aliquot was placed into 50 mM sodium phos-
phate containing 18% sucrose at 4°C overnight and then
was frozen and cut into 10-?m frozen sections for oil red
for further assays. Liver homogenates were prepared in
Liver Histology and Immunohistochemistry. Liver
sections were stained with H&E for pathological evalua-
tion. Steatosis was quantified as the percentage of cells
containing fatty droplets. Necroinflammation was quan-
examined (one 200? field area ? 0.95 mm2). The pa-
thologists were unaware of the treatment groups when
evaluating the slides.
Immunohistochemical staining for CYP2E1, 3-nitro-
tyrosine (3-NT), and 4-hydroxy-2-nonenal (HNE) ad-
ducts was performed by using anti-CYP2E1 antibody (a
gift from Dr. Jerome Lasker, Hackensack Biomedical Re-
search Institute, Hackensack, NJ), anti-3-NT adducts
IgG (Upstate, Lake Placid, NY), and anti-HNE Michael
adducts IgG (Calbiochem, La Jolla, CA), followed by a
rabbit ABC staining system (Santa Cruz Biotechnology,
Santa Cruz, CA).
Serum ALT, Ethanol, and Tumor Necrosis Factor
Serum alanine aminotransferase
(ALT) and ethanol were assayed using an Infinity kit
(Thermo Electron, Melbourne, Australia) and an ethanol
assay kit (Biovision, Mountain View, CA), respectively.
a TNF? ELISA kit (Biosource, Camarillo, CA).
Liver Triglyceride, Free Fatty Acids, and TBAR
using an Infinity kit (Thermo Electron, Melbourne, Aus-
an Infinity kit (Wako, Richmond, VA). Liver TBAR de-
termination was measured as described in Lu et al.13
Cytochrome P450 2E1 Activity. CYP2E1 activity
was measured by the rate of oxidation of p-nitrophenol to
p-nitrocatechol by isolated hepatic microsomes.14
Western Blotting. Western blotting was performed
to evaluate the levels of CYP2E1, peroxisome prolifera-
tor-activated receptor ? (PPAR?), sterol regulatory ele-
ment-binding protein 1 (SREBP-1), AMP-activated
protein kinase (AMPK), acetyl CoA carboxylase (ACC),
phosphorylated acetyl CoA carboxylase (p-ACC), and L-
fatty acid binding protein (L-FABP) using antibodies
against CYP2E1, PPAR?. (Rockland, Gilbertsville, PA),
acyl-CoA oxidase (AOX; a gift from Professor Paul Van
Veldhoven, K.U. Leuven, Belgium),15SREBP-1 (mouse
anti-SREBP-1 monoclonal antibody, clone 2A4; Santa
and L-FABP (Santa Cruz Biotechnology, Santa Cruz,
1484 LU ET AL.HEPATOLOGY, May 2008
ondary antibody. Blots were quantified using the UN-
SCAN-IT automated digitizing system version 5.1 (Silk
Scientific), and the results were expressed as the ratio of
protein to ?-actin.
RNA Isolation and RT-PCR. Total RNA was pre-
pared from fresh liver using TRIzol Reagent (Invitrogen,
Carlsbad, CA). Reverse-transcription polymerase chain
reaction (RT-PCR) to assay messenger RNA (mRNA)
levels of PPAR?, AOX, carnitine palmitoyltransferase
formed as described,16-18except for using ?-actin as the
Recombinant Adenovirus Production. Ad5 adeno-
viral vector with compensating deletions in the early re-
(Ontario, Canada). An adenovirus containing CYP2E1
and ?-galactosidase was developed using an Ad5 adeno-
viral vector as described previously.19Purified recombi-
nant adenovirus (3 ? 109plaque-forming units)
possessing CYP2E1 (Ad-2E1) and ?-galactosidase (Ad-
LacZ) were diluted in 0.2 mL of normal saline and in-
jected into the tail vein of CYP2E1-knockout mice.
Statistics. Results are expressed as means ? SDs.
followed by the Student-Newman-Keuls post hoc test.
Ethanol-Induced Liver Injury and Steatosis in
Wild-Type and CYP2E1-Knockout Mice. Mice body
weight did not change during the first 2 weeks of ethanol
feeding in wild-type or knockout mice (Fig. 1A). In the
third week, ethanol-fed wild-type mice had lost weight
compared with the dextrose-fed mice. The ethanol-fed
knockout mice lost a slight but not significant amount of
body weight, compared with the dextrose-fed knockout
weight of both the knockout and the wild-type mice fur-
ther decreased (Fig. 1A). After 1 or 2 weeks of ethanol
feeding, serum ALT levels in both wild-type and knock-
out mice had not changed (Fig. 1B). After 3 weeks of
ethanol feeding, serum ALT levels had increased 2.2-fold
in wild-type mice and 1.6-fold in knockout mice; there
was no statistical difference between the knockout and
wild-type mice (Fig. 1B). After 4 weeks of ethanol feed-
ing, serum ALT levels further increased up to 3-fold in
wild-type mice and 2.2-fold in knockout mice; again,
there was no statistical difference between the knockout
and wild-type mice (Fig. 1B). After 4 weeks of ethanol
feeding, serum ethanol levels did not differ significantly
between the knockout and wild-type mice (data not
levels by ethanol feeding in the wild-type and knockout
Pathology examination showed no morphological
change in the wild-type and CYP2E1-knockout mice af-
ter 1 week of ethanol feeding (Fig. 1C). After 2 weeks of
ethanol feeding, small lipid droplets were observed in the
wild-type mice but not in the knockout mice. After 3
weeks of ethanol feeding, much bigger and more lipid
droplets were observed, mainly around the central vein of
the wild-type mice; however, no lipid droplets were seen
in the knockout mice (Fig. 1C). After 4 weeks of ethanol
feeding, extensive lipid droplets were observed in the
wild-type mice, but only a small number of tiny lipid
found both in wild-type and knockout mice without sig-
nificant difference between the two genotypes (Fig. 1D).
Oil red O staining showed that prominent lipid droplets
were observed in frozen liver sections from wild-type
CYP2E1-knockout liver sections (Fig. 2A). Similarly, in
CYP2E1-knockout mice, the liver triglyceride content
did not increase after 4 weeks of ethanol feeding, whereas
in the wild-type mice, triglycerides increased more than
2-fold (Fig. 2C). The liver–to–body weight ratio in-
a small, nonsignificant increase was observed in the
knockout mice (Fig. 2B). Interestingly, in the wild-type
mice liver FFA content did not increase after 4 weeks of
ethanol feeding, whereas in the knockout mice, FFAs in-
creased 2-fold (Fig. 2D).
Levels of CYP2E1 and Oxidative Stress in Wild-
Type and CYP2E1-Knockout Mice.
CYP2E1 protein was absent in CYP2E1-knockout mice
fed either dextrose or ethanol (Fig. 3A). In the wild-type
mice, CYP2E1 protein was induced by ethanol feeding
(Fig. 3A). CYP2E1 activity was increased about 4-fold
after ethanol feeding in wild-type mice, but in CYP2E1-
knockout mice fed dextrose or ethanol, rates of PNP ox-
idation were low (Fig. 3B). Induction of CYP2E1 can
induce oxidative stress.6After feeding with ethanol,
TBAR levels, a marker of oxidative stress, increased about
9-fold in wild-type mice but only 3-fold in CYP2E1-
knockout mice (Fig. 3C).
PPAR? Is Up-regulated after Ethanol Feeding in
Knockout But Not in Wild-Type Mice.
of alcoholic fatty liver17,20,21; their expression was exam-
ined by western blotting. As shown in Fig. 4A,B, protein
dextrose-fed wild-type and CYP2E1-knockout mice. We
only detected the mature, active 68-kDa form of
HEPATOLOGY, Vol. 47, No. 5, 2008LU ET AL.1485
SREBP-1, not the 125-kDa precursor form (Fig. 4A).
PPAR?, SREBP-1, AMPK, and p-AMPK levels were not
altered by ethanol feeding in the wild-type mice. Simi-
larly, SREBP-1, AMPK, and p-AMPK did not signifi-
cantly change in the ethanol-fed knockout mice.
However, PPAR? was up-regulated 2-fold by ethanol
feeding in CYP2E1-knockout mice. PPAR?-regulated
acyl-CoA oxidase (AOX) was decreased more than 2-fold
Fig. 1. Mouse body weight (A), serum ALT (B), and H&E staining (C; arrows showing lipid droplets) after varying weeks of oral ethanol feeding.
(D) Steatosis and necroinflammation scores after 4 weeks of oral ethanol feeding.*P ? 0.05 and**P ? 0.01, compared with WT DEX group;#P ?
0.05 and##P ? 0.01, compared with KO Dex group;&&P ? 0.01, compared with WT ethanol (ETOH) group.
1486 LU ET AL.HEPATOLOGY, May 2008
after ethanol feeding in the wild-type mice but was not
decreased in the knockout mice (Fig. 4A,B). There were
no differences in levels of L-FABP (Fig. 4A) or ACC or
p-ACC (data not shown) between the dextrose- and eth-
CYP2E1-knockout mice. RT-PCR analysis showed that
after ethanol feeding, PPAR? and AOX mRNA were up-
regulated in the CYP2E1-knockout mice but down-reg-
ulated in the wild-type mice (Fig. 4C,D). Although in
some cases there appeared to be good agreement in
there was poor correlation of effects on the protein versus
mRNA levels. For example, ethanol lowered AOX pro-
tein and mRNA in wild-type mice and elevated PPAR?
protein and mRNA in CYP2E1-knockout mice. How-
ever, ethanol increased AOX mRNA but not protein in
the knockouts. Reasons for this are not clear, but it is
suggested that mRNA level does not always reflect the
corresponding protein level. RT-PCR analysis did not
detect any significant change in L-FABP, CPT1a, or FAS
among the 4 groups (Fig. 4A,C).
Wild-Type But Not CYP2E1-Knockout Mice Are
Vulnerable to LPS Liver Damage After Chronic Eth-
anol Feeding. Vulnerability to LPS-induced damage
is increased by fatty liver induction3,4and chronic eth-
anol treatment.5We examined whether this ethanol-
promoted vulnerability to LPS-induced damage was
potentiated by CYP2E1. After chronic ethanol or dex-
trose feeding for 4 weeks, 4 mg/kg body weight of LPS
was injected, and 8 hours later, the mice were sacri-
ficed. Ethanol alone caused 3-fold and 2.2-fold in-
creases in serum ALT levels in the wild-type and
CYP2E1-knockout mice, respectively (Fig. 5A). LPS
treatment caused a further increase in serum ALT level
in the ethanol-fed wild-type mice but not in the knock-
out mice (Fig. 5A). LPS treatment had no effect in
dextrose-fed mice. Pathological examination showed
that more necroinflammatory foci were found in etha-
nol-fed wild-type mice than in ethanol-fed knockout
mice (3.2 ? 1.7 versus 0.8 ? 0.5, P ? 0.01) after being
treated with LPS (Fig. 5B). As in the absence of LPS
(Fig. 2A), considerable fat accumulation occurred in
the ethanol-plus-LPS-treated wild-type mice but not in
the ethanol-plus-LPS-treated CYP2E1-knockout mice
(Fig. 5B). Triglyceride content was elevated compara-
bly by ethanol or by ethanol plus LPS treatment in
wild-type mice but not in knockout mice (Fig. 5C). Thus,
LPS promotes injury but does not increase steatosis beyond
that caused by ethanol feeding alone in the wild-type mice.
LPS does not promote injury or induce steatosis in ethanol-
fed CYP2E1-knockout mice.
Fig. 2. Effects of oral ethanol feeding on hepatic steatosis in mice. (A) Oil red O staining after 4 weeks of oral ethanol feeding (arrows showing
positive staining). (B) Liver weight.*P ? 0.05, compared with WT dextrose group. (C) Hepatic triglyceride content after 4 weeks of oral ethanol
feeding.**P ? 0.01, compared with WT dextrose group;##P ? 0.01, compared with KO ethanol group. (D) Hepatic FFA content.**P ? 0.01,
compared with KO dextrose group;#P ? 0.05, compared with KO ethanol group.
HEPATOLOGY, Vol. 47, No. 5, 2008LU ET AL.1487
CMZ, an Inhibitor of CYP2E1, Lowers Ethanol
Fatty Liver and Oxidative Stress in Wild-Type Mice.
The CYP2E1 inhibitor CMZ was used to further assess
the feeding time of ethanol was shortened to 2 weeks.
Feeding of ethanol for 2 weeks did not change ALT levels
(Fig. 1B) but induced steatosis in wild-type mice (Fig.
1C). After a 2-week feeding with ethanol, moderate ste-
atosis was observed in the wild-type mice (Fig. 6A), and
lipid droplets were mainly in the area where elevated
CYP2E1 was present (Fig. 6B). Liver triglyceride content
increased about 2-fold (Fig. 6C). CMZ treatment low-
ered ethanol-induced steatosis (Fig. 6A) and decreased
liver triglyceride content (Fig. 6C). Ethanol-induced
CYP2E1 was decreased by CMZ (Fig. 6B,D). Interest-
ingly, PPAR? protein levels were increased by the etha-
nol-plus-CMZ treatment (Fig. 6D), which is consistent
with the results showing an increase in PPAR? level with
ethanol feeding did not promote triglyceride accumula-
tion in the knockout mice, and CMZ had little effect on
triglyceride levels in the knockouts (Fig. 6C).
Ethanol-induced oxidative stress is partially mediated
by CYP2E1,6and it would be expected that CMZ could
inhibit oxidative stress by inhibiting CYP2E1.22Indeed,
feeding ethanol to wild-type mice caused an increase in
TBAR levels (Fig. 7A), which was inhibited by CMZ
treatment (Fig. 7A). To further substantiate the effect of
chemical analysis of 3-NT and HNE protein adduct for-
mation was evaluated. Ethanol feeding caused an increase
in formation of 3-NT and HNE adducts, markers of ox-
idative stress, and CMZ treatment inhibited this increase
in 3-NT and HNE adducts (Fig. 7B)
Adenovirus-Mediated Expression of CYP2E1 Pro-
duces Fatty Liver in CYP2E1-Knockout Mice.
prove that the absence of fatty liver in ethanol-fed
CYP2E1-knockout mice was a result of the lack of
CYP2E1, an adenovirus containing CYP2E1 (Ad-2E1)
and ?-galactosidase (Ad-LacZ) was used. This Ad-2E1
toxicological properties of CYP2E1.19,23The adenovirus
was injected into CYP2E1-knockout mice, and 5 days
later expression of CYP2E1 was detected in the livers of
8A). No expression of CYP2E1 was detected in the Ad-
positive (Fig. 8A). In pilot experiments, after 3 weeks of
mice died. Therefore, like the CMZ experiments, the
2 weeks of ethanol feeding, serum ALT level was elevated
in the Ad-2E1-injected knockout mice but not in the
Ad-LacZ-injected knockout mice (Fig. 8B). Thus, partial
fed CYP2E1-knockout mice. Similarly, moderate steato-
sis was observed in the Ad-2E1-injected knockout mice
but not in the Ad-LacZ-injected knockout mice (Fig.
8C). Tiny lipid droplets were observed in the Ad-2E1
mice but not the Ad-LacZ mice (Fig. 8C), and these lipid
droplets were mainly in the hepatic areas where CYP2E1
was expressed (Fig. 8D).
CYP2E1 is elevated in many pathophysiological con-
CYP2E1 has been reported to be elevated in chronically
Fig. 3. Effects of 4 weeks of oral ethanol feeding on hepatic CYP2E1
induction and MDA levels in mice. (A) CYP2E1 protein (western blotting
analysis). (B) Hepatic CYP2E1 activity.**P ? 0.01, compared with WT
dextrose group;##P ? 0.01, compared with KO ethanol group;&&P ?
0.01, compared with KO dextrose group. (C) Hepatic oxidative stress as
reflected by TBAR levels. Effects of 4 weeks of oral ethanol feeding on
hepatic TBAR levels.*P ? 0.05, compared with KO dextrose group;
##P ? 0.01, compared with WT dextrose group;$$P ? 0.01, compared
with KO ethanol group.
1488 LU ET AL. HEPATOLOGY, May 2008
obese, overfed rats,27by prolonged starvation,28,29and in
rats during ketosis induced by a high-fat diet.30CYP2E1
expression in HepG2 cells is down-regulated by insulin
and up-regulated by the thyroid hormone triiodothyro-
nine.31Hepatic CYP2E1 was increased in humans with
nonalcoholic steatohepatitis.32In rats, there is a strong
correlation between the degree of steatosis and ethanol
induction of CYP2E1,33and an inhibitor of CYP2E1,
CMZ, not only blunts ethanol-induced inflammation,
necrosis, and fibrosis but also lowers liver fat.34These
experiments support the possibility that CYP2E1 plays a
role in alcoholic fatty liver. In this study, we used a
chronic oral ethanol feeding model to evaluate whether
In this oral feeding model, we observed obvious steatosis
in wild-type mice but not in CYP2E1-knockout mice
after 2, 3, or 4 weeks of ethanol feeding. Administration
of CYP2E1 to the knockout mice restored steatosis. An-
other approach, use of CMZ to inhibit CYP2E1, blocked
show that CYP2E1 contributes to experimental alcoholic
On the other hand, in the intragastric infusion model,
Kono et al.35reported that after the feeding of ethanol for
4 weeks, there was no difference in ethanol-induced ste-
atosis between CYP2E1-knockout mice and wild-type
mice. Similarly, in this model, Wan et al.36reported that
ethanol infusion for 21 days promoted fat accumulation
in CYP2E1-knockout mice but not in wild-type mice.
it is not clear whether the mode of ethanol delivery, oral
role of CYP2E1 in ethanol-induced fatty liver. Using the
intragastric infusion model, Yin et al.37reported that
TNF? plays an important role in alcoholic fatty liver.
ethanol-induced steatosis in the oral feeding model. We
speculate that because oxidative stress and lipid peroxida-
tion are involved in ethanol-induced steatosis,7,8the ab-
sence of CYP2E1 lowers oxidative stress and lipid
Fig. 4. Effects of 4 weeks of oral ethanol feeding on fatty acid metabolism regulating molecules. (A) PPAR?, AOX, L-FABP, SREBP-1, p-AMPK, and
AMPK protein (western blotting analysis). (B) PPAR?, AOX, SREBP-1 (68-kDa mature form), L-FABP/?-actin ratios.**P ? 0.01, compared with KO
DEX group;##P ? 0.01, compared with WT DEX group. (C) PPAR?, AOX, CPT1a, L-FABP, and FAS mRNA (RT-PCR analysis). (D) PPAR?, AOX, L-FABP,
FAS, and CPT1a mRNA/?-actin ratios.*P ? 0.05, compared with KO DEX group;#P ? 0.05, compared with WT DEX group.
HEPATOLOGY, Vol. 47, No. 5, 2008LU ET AL. 1489
peroxidation in the oral liquid diet model used in our
study (for example, Fig. 3C), which lowers ethanol-in-
duced steatosis. Other sources of oxidative stress, for ex-
ample, endotoxemia, TNF? production, and CYP4A,
may occur in the intragastric model to promote ethanol-
induced steatosis in the absence of CYP2E1 in that
model. This will require further study but is consistent
with the liver injury that occurs in the intragastric model
in CYP2E1-knockout mice.35
Recent studies have focused on the effects of ethanol
on transcription factors regulating fatty acid metabo-
lism.20,21,38Several enzymes involved in fatty acid oxida-
tion are predominantly controlled by PPAR?.17We
observed that PPAR? was up-regulated after chronic oral
feeding with ethanol in the CYP2E1-knockout mice but
not in wild-type mice. FFAs are endogenous ligands for
nuclear hormone receptors such as PPAR.39FFAs were
elevated 2-fold after 4 weeks of feeding ethanol to the
despite this accumulation of FFAs, hepatic triglyceride
levels were not elevated in the livers of the CYP2E1-
knockout mice fed ethanol, and the accumulated FFAs
were not being incorporated into triglycerides. L-FABP
and FAS did not change after ethanol treatment either in
the knockout or the wild-type mice, so they do not seem
to be responsible for the FFA elevation in the knockout
mice. Another possible mechanism in addition to low
levels of oxidative stress by which CYP2E1-knockout
mice might have failed to develop alcoholic fatty liver
would be because PPAR?-dependent oxidation of fatty
acids was maintained in the knockout mice but decreased
in the wild-type mice. PPAR?-regulated AOX is the first
enzyme of peroxisomal ?-oxidation of fatty acids.15In
wild-type mice, AOX protein was decreased by ethanol
feeding in association with failure to up-regulate PPAR?
protein. In the knockouts, AOX protein was not lowered
appears that in the knockouts, up-regulated PPAR?
maintained AOX levels and prevented the decrease pro-
duced by ethanol in the wild-type mice. Future studies to
this issue, especially because another target of PPAR?,
CPT1a, was the same in wild-type and knockout mice.
role in experimental alcoholic liver injury because of the
oxidative stress it generates,40,41although some studies
injury.35It has been reported that there is a strong associ-
Fig. 5. Effects of 4 weeks of oral ethanol feeding on LPS liver injury. (A) Serum ALT levels. (B) H&E staining (circles showing inflammatory cell
invasion and arrows showing lipid droplets). (C) Liver triglyceride levels.**P ? 0.01, compared with DEX Sal group;##P ? 0.01, compared with DEX
LPS group;&&P ? 0.01, compared with WT ethanol (ETOH) LPS group;$P ? 0.05 and$$P ? 0.01, compared with WT ETOH Sal group.
1490LU ET AL. HEPATOLOGY, May 2008
ation between increased liver steatosis and systemic oxi-
dative alterations in metabolic syndrome patients,42and
lipid peroxidation is significantly increased among pa-
tients with nonalcoholic fatty liver disease.43,44Antioxi-
dants have been shown to prevent alcohol-induced fatty
liver.7The lipid peroxidation marker malondialdehyde
(MDA), assayed via TBAR levels, was increased after 4
weeks of ethanol feeding in the CYP2E1-knockout mice,
which may be a result of elevated FFAs. However, TBAR
the knockout mice, consistent with the concept that
CYP2E1 plays an important role in ethanol-induced ox-
idative stress. Furthermore, the elevated TBAR levels and
HNE adducts) by ethanol feeding were inhibited by
CMZ in wild-type mice in association with a decrease in
CYP2E1 by the CMZ treatment. Interestingly, PPAR?
was up-regulated when oxidative stress was inhibited by
CMZ treatment. Differences in ethanol-induced oxida-
tive stress in wild-type versus CYP2E1-knockout mice may
in the latter. It is possible that oxidative stress inhibits fatty
liver. This remains to be further evaluated.
With respect to liver injury beyond steatosis, only
modest liver injury is observed in the liquid oral models,
and small elevations in ALT were observed in the wild-
type and knockout mice. These results show that chronic
ethanol produces some toxicity in CYP2E1-knockout
mice. Elevated FFAs might be one of the reasons for this
CYP2E1-independent toxicity. FFAs might evoke hepa-
tocyte damage by ROS generated from oxidation by mi-
crosomal enzymes such as CYP4A other than CYP2E1.45
Indeed, CYP4A is up-regulated in CYP2E1-knockout
mice fed with a choline-methionine-deficient diet.45Ox-
idized FAs themselves become sources for lipid peroxida-
tion reactions that are directly cytotoxic.46It was recently
suggested that accumulated triglycerides may be a protec-
Fig. 6. Effects of CMZ on hepatic steatosis and levels of CYP2E1 and PPAR? in wild-type and CYP2E1-knockout mice. (A) H&E and oil red O
staining (arrowheads show oil red–positive staining, and arrows show lipid droplets). (B) Immunohistochemical staining for CYP2E1 (arrows show lipid
droplets). (C) Liver triglyceride content.*P ? 0.05, compared with dextrose group;#P ? 0.05 and##P ? 0.01, compared with ethanol group;$P ?
0.05, compared with the dextrose or CMZ dextrose group. (D) CYP2E1 and PPAR? (western blotting analysis); bottom numbers show CYP2E1/?-actin
or PPAR?/?-actin ratios.
HEPATOLOGY, Vol. 47, No. 5, 2008LU ET AL. 1491
tive mechanism to prevent progressive liver injury from
FFA lipotoxicity by “buffering the accumulated FFA.”39
The reason for FFA elevation in the knockout mice is not
make only a small contribution to overall fatty acid oxi-
dation. Similarly, ethanol is a substrate of CYP2E1, but
subjected to CYP2E1 oxidation.47
LPS toxicity is increased in fatty liver,4and chronic
ethanol exposure also enhances LPS liver injury.5Is
chronic ethanol exposure–enhanced LPS liver injury a
knockout mice were challenged with LPS; ALT levels
mice. This result is consistent with our previous observa-
tions that CYP2E1-knockout mice showed less LPS liver
injury after pyrazole treatment than did wild-type mice
treated with pyrazole to induce CYP2E1.22We have sug-
gested that the induction of CYP2E1 primes or sensitizes
mice or rats to LPS-induced injury.13,22LPS treatment
did not change hepatic fat accumulation in either wild-
Fig. 7. Effects of CMZ on hepatic oxidative/nitrosative stress as reflected by (A) MDA (TBAR) levels and (B) 3-NT and HNE adduct formation.*P ?
0.05, compared with control group;##P ? 0.01, compared with ethanol group.
Fig. 8. Effect of adenovirus-mediated CYP2E1 expression in CYP2E1-knockout mice on ethanol steatosis and ALT levels. (A) CYP2E1 and
?-galactosidase expression after adenovirus injection into CYP2E1-knockout mice. (B) Serum ALT levels.**P ? 0.01, compared with LacZ group. (C)
H&E staining after 2 weeks of feeding ethanol to CYP2E1-knockout mice treated with either Ad-LacZ or Ad-CYP2E1 (arrows show lipid droplets). (D)
Immunohistochemical staining for CYP2E1 (arrows show lipid droplets).
1492 LU ET AL. HEPATOLOGY, May 2008
levels in ethanol-fed wild-type mice might be potentiated
extent of injury in the wild-type mice that exhibit fatty
liver and a lesser extent of injury in the CYP2E1-knock-
out mice that do not display fatty liver. This is consistent
with the “two-hit” theory, with the first hit being hepatic
fat accumulation and the second hit LPS. Thus, either
CYP2E1 directly synergizes with LPS to promote liver
injury, or CYP2E1 plays a role in fat accumulation in the
liver, and the latter increases LPS toxicity.
(Laboratory of Metabolism, National Cancer Institute, Be-
thesda, MD) for CYP2E1-knockout and wild-type mice;
Professor Paul Van Veldhoven (K.U. Leuven, Belgium) for
AOX antibody; and Drs. Stephen Ward and Swan Thung
(Department of Pathology, Mount Sinai School of Medi-
cine) for help with necroinflammation evaluation.
We thank Dr. Frank J. Gonzalez
1. Baptista A, Bianchi L, De Groote J, Besmet VJ, Gedigk P, Korb G, et al.
2. Rao RK, Seth A, Sheth P. Recent advances in alcoholic liver disease I. Role
of intestinal permeability and endotoxemia in alcoholic liver disease. Am J
Physiol Gastrointest Liver Physiol 2004;286:G881-G884.
3. Yang SQ, Lin HZ, Lane MD, Clemens M, Diehl AM. Obesity increases
sensitivity to endotoxin liver injury: implications for the pathogenesis of
steatohepatitis. Proc Natl Acad Sci U S A 1997;94:2557-2562.
4. Yang S, Lin H, Diehl AM. Fatty liver vulnerability to endotoxin-induced
damage despite NF-kappaB induction and inhibited caspase 3 activation.
Am J Physiol Gastrointest Liver Physiol 2001;281:G382-G392.
potentiates lipopolysaccharide liver injury despite inhibiting Jun N-termi-
nal kinase and caspase 3 activation. J Biol Chem 2002;277:13037-13044.
6. Kessova I, Cederbaum AI. CYP2E1: biochemistry, toxicology, regulation
and function in ethanol-induced liver injury. Curr Mol Med 2003;3:509-
7. Diluzio NR. Prevention of the acute ethanol-induced fatty liver by the
simultaneous administration of antioxidants. Life Sci 1964;3:113-118.
8. Wheeler MD, Kono H, Yin M, Rusyn I, Froh M, Connor HD, et al.
early alcohol-induced liver injury in rats. Gastroenterology 2001;120:
in mice lacking Cu, Zn-superoxide dismutase. HEPATOLOGY 2003;38:
10. Cederbaum AI. Introduction-serial review: alcohol, oxidative stress, and
cell injury. Free Radic Biol Med 2001;31:1524-1526.
11. Lee SS, Buters JT, Pineau T, Fernandez-Salguero P, Gonzalez FJ. Role of
CYP2E1 in the hepatotoxicity of acetaminophen. J Biol Chem 1996;271:
ods Enzymol 1994;233:585-594.
rats treated with the CYP2E1 inducer pyrazole. Am J Physiol Gastrointest
Liver Physiol 2005;289:G308-G319.
14. Reinke LA, Moyer MJ. p-Nitrophenol hydroxylation. A microsomal oxi-
15. Van Veldhoven PP, Van Rompuy P, Fransen M, De Bethune B, Man-
naerts GP. Large scale purification and further characterization of rat
pristanoyl-CoA oxidase, Eur J Biochem 1994;222:795-801.
16. Fischer M, You M, Matsumoto M, Crabb DW. Peroxisome proliferator-
activated receptor alpha (PPARalpha) agonist treatment reverses PPARal-
fed mice. J Biol Chem 2003;278:27997-28004.
17. Bing C, Russell S, Becket E, Pope M, Tisdale MJ, Trayhurn P, Jenkins JR.
adipose tissue in tumour-bearing mice. Br J Cancer 2006;95:1028-1037.
18. Ng DS, Xie C, Maguire GF, Zhu X, Ugwu F, Lam E, Connelly PW.
Hypertriglyceridemia in lecithin-cholesterol acyltransferase-deficient mice
is associated with hepatic overproduction of triglycerides, increased lipo-
genesis, and improved glucose tolerance. J Biol Chem 2004;279:7636-
19. Bai J, Cederbaum AI. Adenovirus mediated overexpression of CYP2E1
increases sensitivity of HepG2 cells to acetaminophen induced cytotoxic-
ity. Mol Cell Biochem 2004;262:165-176.
20. You M, Fischer M, Deeg MA, Crabb DW. Ethanol induces fatty acid
synthesis pathways by activation of sterol regulatory element-binding pro-
tein (SREBP). J Biol Chem 2002;277:29342-29347.
21. You M, Matsumoto M, Pacold CM, Cho WK, Crabb DW. The role of
AMP-activated protein kinase in the action of ethanol in the liver. Gastro-
22. Lu Y, Cederbaum AI. Enhancement by pyrazole of lipopolysaccharide-
induced liver injury in mice: role of cytochrome P450 2E1 and 2A5.
23. Bai J, Cederbaum AI. Adenovirus-mediated expression of CYP2E1 pro-
duces liver toxicity in mice. Toxicol Sci 2006;91:365-371.
24. Caro AA, Cederbaum AI. Oxidative stress, toxicology, and pharmacology
of CYP2E1. Annu Rev Pharmacol Toxicol 2004;44:27-42.
25. Woodcroft KJ, Hafner MS, Novak RF. Insulin signaling in the transcrip-
tional and posttranscriptional regulation of CYP2E1 expression. HEPA-
26. Ohkuwa T, Sato Y, Naoi M. Hydroxyl radical formation in diabetic rats
induced by streptozotocin. Life Sci 1995;56:1789-1798.
27. Raucy JL, Lasker JM, Kraner JC, Salazar DE, Lieber CS, Corcoran GB.
Induction of cytochrome P450IIE1 in the obese overfed rat. Mol. Phar-
28. Johansson I, Lindros KO, Eriksson H, Ingelman-Sundberg M. Transcrip-
tional control of CYP2E1 in the perivenous liver region and during star-
vation. Biochem Biophys Res Commun 1990;173:331-338.
29. Johansson I, Ekstrom G, Scholte B, Puzycki D, Jornvall H, Ingelman-
Sundberg M. Ethanol-, fasting-, and acetone-inducible cytochromes
P-450 in rat liver: regulation and characteristics of enzymes belonging to
the IIB and IIE gene subfamilies. Biochemistry 1988;27:1925-1934.
30. Yun Y, Casazza JP, Sohn DH, Veech RL, Song BJ. Pretranslational acti-
vation of cytochrome P450IIE during ketosis induced by a high fat diet.
Mol Pharmacol 1992;41:474-479.
31. Peng HM, Coon MJ. Regulation of rabbit cytochrome P4502E1 expres-
sion in HepG2 cells by insulin and thyroid hormone. Mol Pharmacol
32. Weltman M, Farrell G, Hall P, Ingelman-Sundberg M, Liddle C. Hepatic
cytochrome P450 2E1 is increased in patients with nonalcoholic steato-
hepatitis. HEPATOLOGY 1998;27:128-133.
33. Jarvelainen HA, Fang C, Ingelman-Sundberg M, Lukkari TA, Sippel H,
Lindros KO. Kupffer cell inactivation alleviates ethanol-induced steatosis
and CYP2E1 induction but not inflammatory responses in rat liver.
J Hepatol 2000;32:900-910.
of ethanol-induced liver disease in the intragastric feeding rat model by
chlormethiazole. Proc Soc Exp Biol Med 2000;224:302-308.
35. Kono H, Bradford BU, Yin M, Sulik KK, Koop DR, Peters JM, et al.
36. Wan YY, Cai Y, Li J, Yuan Q, French B, Gonzalez FJ, et al. Regulation of
peroxisome proliferator activated receptor alpha-mediated pathways in al-
HEPATOLOGY, Vol. 47, No. 5, 2008 LU ET AL.1493
37. Yin M, Wheeler MD, Kono H, Bradford BU, Gallucci RM, Luster MI, Download full-text
Thurman RG. Essential role of tumor necrosis factor alpha in alcohol-
induced liver injury in mice. Gastroenterology 1999;117:942-952.
38. You M, Crabb DW. Recent advances in alcoholic liver disease II. Minire-
view: molecular mechanisms of alcoholic fatty liver. Am J Physiol Gastroi-
ntest Liver Physiol 2004;287:G1-G6.
39. Yamaguchi K, Yang L, McCall S, Huang J, Yu XX, Pandey SK, et al.
Inhibiting triglyceride synthesis improves hepatic steatosis but exacerbates
liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis.
Physiol Rev 1997;77:517-544.
41. Bradford BU, Kono H, Isayama F, Kosyk O, Wheeler MD, Akiyama TE,
et al. Cytochrome P450 CYP2E1, but not nicotinamide adenine dinucle-
otide phosphate oxidase, is required for ethanol-induced oxidative DNA
damage in rodent liver. HEPATOLOGY 2005;41:336-344.
42. Palmieri VO, Grattagliano I, Portincasa P, Palasciano G. Systemic oxida-
tive alterations are associated with visceral adiposity and liver steatosis in
patients with metabolic syndrome. J Nutr 2006;136:3022-3026.
43. Konishi M, Iwasa M, Araki J, Kobayashi Y, Katsuki A, Sumida Y, et al.
Increased lipid peroxidation in patients with non-alcoholic fatty liver dis-
ease and chronic hepatitis C as measured by the plasma level of 8-isopros-
tane. J Gastroenterol Hepatol 2006;21:1821-1825.
antioxidant status among patients with nonalcoholic fatty liver disease
(NAFLD). J Clin Gastroenterol 2006;40:930-935.
45. Leclercq IA, Farrell GC, Field J, Bell DR, Gonzalez FJ, Robertson GR.
CYP2E1 and CYP4A as microsomal catalysts of lipid peroxides in murine
nonalcoholic steatohepatitis. J Clin Invest 2000;105:1067-1075.
46. Penumetcha M, Khan N, Parthasarathy S. Dietary oxidized fatty acids: an
atherogenic risk? J Lipid Res 2000;41:1473-1480.
47. Lieber CS. Metabolism of alcohol. Clin Liver Dis 2005;9:1-35.
1494LU ET AL. HEPATOLOGY, May 2008