Expression of CYP2E1 increases oxidative stress and
induces apoptosis of cardiomyocytes in transgenic mice
Wei Zhang
1
, Dan Lu
1
, Wei Dong
1
, Li Zhang
1
, Xiaojuan Zhang
1
, Xiongzhi Quan
1
, Chunmei Ma
2
,
Hong Lian
1
and Lianfeng Zhang
1,2
1 Key Laboratory of Human Disease Comparative Medicine, Ministry of Health, Institute of Laboratory Animal Science, Chinese Academy of
Medical Sciences & Comparative Medical Center, Peking Union Medical College, Beijing, China
2 Key Laboratory of Human Diseases Animal Model, State Administration of Traditional Chinese Medicine, Institute of Laboratory Animal
Science, Chinese Academy of Medical Sciences & Comparative Medical Center, Peking Union Medical College, Beijing, China
Introduction
Cytochrome P450 2E1 (CYP2E1) is one of the cyto-
chrome P450 (P450) isoforms. Overexpression of
CYP2E1 is of direct importance to human health and
has been associated with a range of diseases, including
diabetes [1–3], alcoholic liver disease and cancer [4–8].

R141W
transgenic mice. In addition, levels of
H
2
O
2
and malondialdehyde were increased and levels of glutathione and
total antioxidant capability were strongly reduced in CYP2E1 transgenic
mice and cTnT
R141W
transgenic mice. Myocyte apoptosis was significantly
increased by 19-fold in CYP2E1 transgenic mice and by 11-fold in
cTnT
R141W
transgenic mice, respectively, compared to wild-type mice.
Mitochondrial-dependent apoptotic signal transduction events, such as
cytochrome c release from mitochondria into the cytosol and the expres-
sion of cleaved (active) caspases 3 and 9, were significantly increased in
CYP2E1 transgenic mice and cTnT
R141W
transgenic mice. These results
demonstrate that CYP2E1 over-expression produces apoptosis and that the
up-regulation of CYP2E1 in cTnT
R141W
transgenic mice also correlates
with apoptosis in this model.
Abbreviations
Col3a1, collagen types III; CYP2E1, cytochrome P450 2E1; DCM, dilated cardiomyopathy; GSH, glutathione; MDA, malondialdehyde; a-MHC,
a-myosin heavy chain; PNP, p-nitrophenol; ROS, reactive oxygen species; T-AOC, total antioxidant capability; TUNEL, terminal dUTP nick
end-labeling; WT, wild-type.

R141W
transgenic mice at 1, 3 and
5 months of age were up-regulated by one- to four-
fold compared to those measured in wild-type (WT)
mice (Fig. 1B, C). To study the effect of CYP2E1 on
the heart, C57BL ⁄ 6J mice carrying the CYP2E1 trans-
gene were established. The transgenic plasmids were
individually constructed by inserting the mouse
CYP2E1 cDNA downstream of the a-myosin heavy
chain (a-MHC) promoter (Fig. 1D). Two lines of
CYP2E1 transgenic mice (founders #26 and #36) with
a seven- to nine-fold increase in CYP2E1 levels
compared to WT mice were selected from among 12
founders (Fig. 1E, F). Transgene copy numbers
(mean ± SE) were determined in mice from F2 and
F3 generations separately to be 16.5 ± 2.5 for founder
#26 (n = 4) and 8.2 ± 3.1 for founder #36 (n = 4).
The transgene was stable during the propagation of
the transgenic mice. Transgenic expressed CYP2E1
was mainly localized in microsomal compartments
CYP2E1
E16.5
1
week
2
week
4
week
8
week

WT CYP2E1
12
9
6
3
0
WT CYP2E1
CYP2E1 activity
(nmol·min
–1
·mg
–1
)
GAPDH
Cyto
WT #26 #36
CYP2E1 level
(Relative units)
0
A
C
EFGH
D
B
Fig. 1. Expression of CYP2E1 in mouse heart tissue and the generation of transgenic mice. Protein translational levels of CYP2E1 in heart
tissue from mice at embryonic age 16.5 days and at 1, 2, 4 and 8 weeks of age (A) and in the DCM-affected hearts from cTnT
R141W
trans-
genic mice (DCM) at 1, 3 and 5 months of age (B) were detected by western blotting, using GADPH as a normalization standard; bars repre-
sent the relative levels quantified by densitometry using

Overexpression of CPY2E1 in the heart leads
to the DCM phenotype and increases myocyte
disarray and interstitial fibrosis
Left ventricular dimensions and functions were deter-
mined using hearts from WT, cTnT
R141W
, CYP2E1(#26)
and CYP2E1(#36) transgenic mice (Fig. 3A and
Table 1). The cTnT
R141W
transgenic mice and the two
lines of CYP2E1 transgenic mice exhibited the DCM
phenotype (i.e. chamber dilation and dysfunction). On
light microscopy, both myocyte disarray and fibrosis
were observed in the cTnT
R141W
transgenic mice and
the two lines of CYP2E1 transgenic mice, in contrast
to WT mice (Fig. 3B, C). mRNA expression levels of
collagen types III (Col3a1) were increased by two-fold
in CYP2E1 transgenic mice and by three-fold in
cTnT
R141W
transgenic mice compared to WT mice
(Fig. 3D, E). These results suggest that the overexpres-
sion of CYP2E1 enhances the remodeling of the myo-
cardial arrangement.
Overexpression of CPY2E1 in the heart increases
oxidative stress
Levels of H

CYP2E1 transgenic mice and cTnT
R141W
transgenic
mice.
Overexpression of CYP2E1 in the heart increases
the release of mitochondrial cytochrome c,
activates caspases 3 and 9, and causes
cardiomyocyte apoptosis
Mitochondrial lesions may induce the release of mito-
chondrial factors, such as cytochrome c, triggering
cell death pathways. The release of cytochrome c may
activate caspases 3 and 9 and execute the apoptotic
system [14]. By western blotting, we found that
up-regulation of CYP2E1 increased the release of
cytochrome c from mitochondria into the cytosol, as
well as the levels of cleaved active caspases 3 and 9
in both CYP2E1 transgenic mice and cTnT
R141W
transgenic mice compared to these parameters in WT
mice (Fig. 6A–F). A terminal dUTP nick end-labeling
(TUNEL) assay (Fig. 6G, H) indicated that the
up-regulation of CYP2E1 caused myocyte apoptosis
that was 19-fold higher in CYP2E1 transgenic mice
and 11-fold higher in the cTnT
R141W
transgenic mice
compared to WT mice (n =3, P < 0.05). The
expression level of CYP2E1 was correlated with myo-
cyte apoptosis (r = 0.997) in CYP2E1 transgenic
mice and cTnT

and ketones and plays a key role in gluconeogenesis in
ketone bodies released as a result of energy deprivation
[15–17], and also is markedly induced in fasting [18].
WT CYP2E1#26 CYP2E1#36 cTnT
R141W
WT CYP2E1#26 CYP2E1#36 cTnT
R141W
WT CYP2E1#26 CYP2E1#36 cTnT
R141W
6
*
WT CYP2E1 cTnT
R141W
Col3a1
(Relative units)
WT CYP2E1 cTnT
R141W
4
2
0
*
*
Col3a1
GAPDH
A
B
C
DE
Fig. 3. Histopathological observations in transgenic mice. Heart tissue from WT, CYP2E1#26, CYP2E1#36 and cTnT
R141W

LVAWs (mm) 1.27 ± 0.14 1.16 ± 0.13* 1.11 ± 0.14* 1.07 ± 0.12*
Ejection fraction (%) 62.5 ± 7.08 55.4 ± 6.56* 56.3 ± 6.58* 45.8 ± 7.82*
Fractional shortening (%) 33.5 ± 5.23 28.6 ± 4.34* 29.2 ± 4.38* 22.8 ± 4.59*
W. Zhang et al. CYP2E1 causes apoptosis of cardiomyocytes in vivo
FEBS Journal 278 (2011) 1484–1492 ª 2011 The Authors Journal compilation ª 2011 FEBS 1487
The expression of this enzyme is marked in the liver,
heart, lungs, pancreas, brain and intestine [19,20]. We
found that CYP2E1 was expressed immediately after
birth and was maximally transcribed within the first
week (Fig. 1A), similar to previous observations made
in rat liver [21]. The expression of CYP2E1 is increased
in the human ischemic and dilated heart [12] and in left
ventricular tissue of the spontaneously hypertensive rats
[13]. In the present study, we observed that the CYP2E1
expression level was significantly increased in the heart
of cTnT
R141W
transgenic mice (Fig. 1B, C), which was a
model of DCM [22]. Because CYP2E1 is also involved
in the metabolism of endogenous aldehydes and
ketones, and plays a key role in gluconeogenesis associ-
ated with energy deprivation [15–18], it is possible that
the up-regulation of CYP2E1 in the dilated heart might
meet the energy demand for enhancing systolic function,
which is dysfunctional in DCM mice. However,
CYP2E1-catalyzed metabolism may also cause toxicity
or cell damage through the production of toxic meta-
bolites, oxygen radicals and lipid peroxidation. To
understand the effects of CYP2E1 on the heart, we
produced heart tissue-specific CYP2E1 transgenic mice.

tion were found in CYP2E1 transgenic mice and
cTnT
R141W
transgenic mice (Fig. 5). In addition, the
mitochondrial-dependent apoptotic pathway, initiated
by cytochrome c release and followed by caspase
9-dependent caspase 3 activation, was also triggered in
CYP2E1 transgenic mice and cTnT
R141W
transgenic
mice, and the apoptosis of myocytes was increased in
CYP2E1 transgenic mice and cTnT
R141W
transgenic
mice (Fig. 6), suggesting that CYP2E1 increases the
apoptosis of myocytes through the mitochondrial-
dependent pathway. Clinical and experimental studies
support the hypothesis that oxidative stress and
myocyte apoptosis play an important role in the
pathogenesis of cardiovascular diseases such as ische-
mic heart disease, atherosclerosis, cardiomyopathy and
10.0
8.0
6.0
5.0
*
*
*
*
6.0

protein)
0.3
0.0
T-AOC (U·mg
–1
protein)
1.0
0.0
WT CYP2E1 cTnT
R141W
WT CYP2E1 cTnT
R141W
WT CYP2E1 cTnT
R141W
WT CYP2E1 cTnT
R141W
AB
CD
Fig. 4. Measurements of H
2
O
2
, MDA, GSH and T-AOC in the
heart. WT mice (n = 12), CYP2E1 (n = 12) and cTnT
R141W
(n = 12)
transgenic mice were sacrificed at 5 months of age and the total
lysates of heart tissues were collected. Levels of H
2
O

up-regulated expression of CYP2E1 was involved in
the pathogenesis of DCM in cTNT
R141W
transgenic
mice. In the present study, we found that the overex-
pression of CYP2E1 caused a DCM phenotype similar
to that in cTnT
R141W
transgenic mice, and the expres-
sion level of CYP2E1 was correlated with myocyte
apoptosis (r = 0.997) in CYP2E1 transgenic mice and
cTnT
R141W
transgenic mice. In addition, the up-regula-
tion of CYP2E1 was also found in the heart of a
DCM patient [12]. These results suggest that CYP2E1
may be a modulator of DCM subsequent to mutation
of cTnT
R141W
or other factors. The CYP2E1-mediated
myocyte apoptosis may be associated with the progress
of DCM, such that the inhibition of CYP2E1 may
mitigate the progression of DCM.
In summary, we report that the CYP2E1 expression
level is strongly increased in the DCM-affected heart
of cTnT
R141W
transgenic mice. The present study
provides the first evidence indicating that the over-
expression of CYP2E1 causes cardiac oxidative stress,

CYP2E1 activity
CYP2E1 activity was measured by the rate of oxidation of
PNP to p-nitrocatechol in the presence of NADPH and O
2
,
as described previously [40], using 200 lg of homogenate in a
3
2
1
Cyto
Mito
*
*
*
*
A
Cyt-c
in cyto
Cyt-c
B
Cytochrom c
(Relative units)
0
C
Cyt c
in mito
Cleaved
D
3
2

8
6
*
*
TUNEL-positive
cell (%)
4
2
0
WT CYP2E1 cTnT
R141W
WT CYP2E1 cTnT
R141W
WT CYP2E1 cTnT
R141W
WT CYP2E1 cTnT
R141W
WT CYP2E1 cTnT
R141W
WT CYP2E1 cTnT
R141W
WT CYP2E1 cTnT
R141W
Fig. 6. Determination of the levels of cytochrome c release,
cleaved caspases 3 and 9, as well as apoptotic cells in heart tis-
sues. Protein levels of cytochrome c (cyt-c) in mitochondrial (mito)
or cytosolic (cyto) fractions (A), cleaved caspase 9 (C) and cleaved
caspase 3 (E) extracted from heart tissues of WT mice, and
CYP2E1 and cTnT
R141W

Æmg
)1
protein) =
D
546
⁄ 9.53 ⁄ 0.2⁄ 60 ⁄ 7.1 · 10
3
.
Echocardiography
Mice were lightly anesthetized by intraperitoneal injection of
tribromo-ethanol at a dose of 180 mLÆkg
)1
body weight. M-
mode echocardiography of the left ventricle was recorded at
the tip of the mitral valve apparatus with a 30 MHz trans-
ducer (Vevo770; VisualSonics, Toronto, Canada) [22].
Histological analysis
Cardiac tissue from mice at the age of 5 months was fixed
in 4% formaldehyde and mounted in paraffin blocks. Sec-
tions were stained with hemotoxylin and eosin or Masson’s
trichrome.
RT-PCR
Total RNA was isolated from heart tissue from each trans-
genic mouse at the age of 5 months using TRIzol Reagent (In-
vitrogen, Carlsbad, CA, USA). First-strand cDNA was
synthesized from 2 lg of total RNA using random hexamer
primers in accordance with the Superscript b reverse trans-
criptase manufacturer’s protocol (Invitrogen). Detection of
mRNA for Col3a1 was carried out by the RT-PCR, using
GAPDH for normalization. Primers were: 5¢-GGCAGTGA

enized rapidly in nine volumes of buffer (0.15 molÆL
)1
KCl,
1.0 mmolÆL
)1
EDTA) to obtain 1 : 10 (w ⁄ v) homogenates.
Homogenates were centrifuged at 13 000 g (4 °C) for
30 min to collect the supernatant for assay. Levels of MDA
were evaluated by the thiobarbituric acid reactive sub-
stances method [41]. Levels of H
2
O
2
were measured using
an assay kit (DE3700; R&D Systems, Minneapolis, MN,
USA). Levels of GSH were measured using the GSH-400
colorimetric assay kit (Promega, Madison, WI, USA).
Levels of T-AOC were measured using assay kit ab65329
(Abcam). The protein concentration in heart homogenates
was determined by the Bradford method using BSA as a
standard [42].
TUNEL assay
The in situ TUNEL assay was performed in sections of
heart tissues using the In Site Cell Death Detection Kit
(Roche Diagnostics GmbH, Mannheim, Germany) in
accordance with the manufacturer’s instructions. Eight
images per heart (three hearts per genotype group) were
acquired and positive cells were counted individually. The
results were expressed as the percentage of apoptotic cells
among the total cell population.

radish peroxidase conjugated to goat anti-rabbit IgG as the
second antibody (Santa Cruz Biotechnology; dilution
1 : 20 000) using a chemiluminescent detection system
(Western Blotting Luminal Reagent, Santa Cruz Biotech-
nology). Variations in sample loading were normalized rela-
tive to the GAPDH signal. Bands were quantified by the
densitometry function of quantity one software (Bio-Rad,
Hercules, CA, USA).
Statistical analysis
All measurement data are expressed as the mean ± SEM.
Statistical significance of differences among groups was
analyzed by one-way analysis of variance. P < 0.05 was
considered statistically significant.
Acknowledgements
The present work was supported in part by the
Ministry of Health Foundation (200802036) and the
National Science and Technology Major Projects
(2009ZX09501-026). The authors declare that there are
no conflicts of interest.
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CYP2E1 causes apoptosis of cardiomyocytes in vivo W. Zhang et al.
1492 FEBS Journal 278 (2011) 1484–1492 ª 2011 The Authors Journal compilation ª 2011 FEBS