Toxicology in Vitro 24 (2010) 15841591

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Toxicology in Vitro
journal homepage: www.elsevier.com/locate/toxinvit

Enhanced toxicity and ROS generation by doxorubicin in primary cultures

of cardiomyocytes from neonatal metallothionein-I/II null mice
Ze Fu a, Jiabin Guo a, Li Jing a, Ruisheng Li b, Tingfen Zhang a, Shuangqing Peng a,*
a
Evaluation and Research Centre for Toxicology, Institute of Disease Control and Prevention, Academy of Military Medical Sciences,
20 Dongdajie Street, Fengtai District, Beijing 100071, PR China
b
Laboratory Animal Center, 302 Military Hospital of China, Beijing 100039, PR China

a r t i c l e

i n f o

Article history:
Received 29 March 2010
Accepted 17 June 2010
Available online 22 June 2010
Keywords:
Metallothionein
Doxorubicin
Cardiomyocytes
Reactive oxygen species

a b s t r a c t
The clinical use of doxorubicin (Dox), a potent anticancer drug, is limited by its concurrent dose-dependent cardiotoxicity. We previously found that metallothionein-I/II (MT-I/II) null mice are more vulnerable
to Dox-induced cardiomyopathy, but it is unknown whether depletion of MT would sensitize cardiomyocytes to Dox toxicity in vitro since the protective effect of MT still remains controversial. In the present
study, a primary culture system of cardiomyocytes from neonatal MT-I/II null (MT/) and corresponding
wild type (MT+/+) mice was established to unequivocally determine the effect of MT deciency on Doxinduced toxicity. MT concentrations in the MT/ cardiomyocytes were about 2.5-fold lower than those
in MT+/+ cardiomyocytes. MT/ cardiomyocytes were more sensitive to Dox-induced cytotoxicity than
MT+/+ cardiomyocytes as measured by morphological alterations, lactate dehydrogenase leakage, cell viability, and apoptosis. Dox time- and concentration-dependently increased reactive oxygen species (ROS)
formation in MT+/+ cardiomyocytes, and this effect was exaggerated in MT/ cardiomyocytes. Antioxidant N-acetylcysteine (NAC) and glutathione (GSH) signicantly rescued MT+/+ but not MT/cardiomyocytes from Dox-induced cell death and ROS generation. These ndings suggest that basal MT provide
protection against Dox-induced toxicity in cardiomyocytes, particularly highlight the important role of
MT as a cellular antioxidant on scavenging ROS.
2010 Elsevier Ltd. All rights reserved.

1. Introduction
Doxorubicin (Dox) is a highly effective anticancer drug that is
frequently employed to treat haematological and solid tumors
including leukemia, breast cancer, and soft tissue sarcomas (Umlauf and Horky, 2002; Takemura and Fujiwara, 2007). However,
the therapeutic effectiveness of Dox is compromised by its dosedependent and cumulative cardiotoxic side effects (Singal et al.,
2000; Buyukokuroglu et al., 2004; Minotti et al., 2004). In acute
cases, patients treated by Dox present with hypotension, tachycardia and arrhythmia. Notably, patients given cumulative dose above
600 mg/m2, may cause severe chronic toxicity as manifested by
congestive heart failure (Singal and Iliskovic, 1998). It has been
widely accepted that the mechanisms of Dox-induced cardiotoxicity are mainly attributed to the formation of reactive oxygen species (ROS) and promotion of myocardial oxidative stress, which
cause DNA and protein damage, and lipid peroxidation, thus eventually lead to cell necrosis as well as apoptosis (Gewirtz, 1999). The
critical role of ROS in Dox-induced cardiotoxicity is further supported by the reports that treatment of animals with a variety of
antioxidants such as probucol, dexrazoxane, and melatonin
* Corresponding author. Tel./fax: +86 10 66949631.
E-mail address: pengsq@hotmail.com (S. Peng).
0887-2333/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tiv.2010.06.009

protects heart against the toxicity of Dox (Li et al., 2000; Xu

et al., 2001; Lebrecht et al., 2007; Simunek et al., 2009).
Metallothionein (MT) is a ubiquitous, low-molecular-weight,
and thiol-rich protein which is highly inducible in response to oxidative stress (Thirumoorthy et al., 2007). In mammals there are four
isoforms (MT-IIV), of which MT-I and MT-II (MT-I/II) are the best
characterized MT protein in the heart. Many studies suggest that
MT plays an important role in the homeostasis of essential metals
such as zinc and copper, detoxication of toxic metals such as cadmium, and protection against oxidative stress (Romero-Isart and
Vasak, 2002; Klaassen et al., 2009; Ngu and Stillman, 2009). In particular, increasing evidence indicates that MT can function as antioxidant on scavenging free radicals though the precise biological
functions of MT still remain indistinct (Sato and Kondoh, 2002;
Futakawa et al., 2006). Preinduction of MT in vivo has been shown
to ameliorate oxidative stress-associated injuries by starvation,
ischemia/reperfusion, toxic chemical exposure, and ionizing radiation (Kang et al., 2003; Kang, 2007; Shibuya et al., 2008).
By using a cardiac-specic metallothionein-overexpressing
transgenic mouse model, studies from Dr. Kangs Laboratory have
demonstrated that MT overexpression inhibits Dox-induced cardiac toxicity (Kang et al., 1997; Sun et al., 2001; Feng et al., 2006).
Consistently, we previously reported that MT-I/II deciency exacerbates Dox-induced cardiomyopathy (Guo et al., 2006; Shuai et al.,

Z. Fu et al. / Toxicology in Vitro 24 (2010) 15841591

2007), indicating the protective effect of basal MT. However, what is

the least level to which cardiac MT has to be elevated for the protection to Dox toxicity is still controversial. Dr. DiSilvestro et al. (1996)
reported that transgenic mice overexpressing metallothionein are
not resistant to Dox-induced toxicity. More recently, a study demonstrated that MT null mice do not exhibit signicantly worse longterm behavioral outcomes following neonatal hypoxiaischemia
(McAuliffe et al., 2008). These controversial results indicate that
further studies are necessary to examine the effect and its mechanism of MT on Dox-induced cardiotoxicity.
Because of the complicated environment in in vivo test where
systemically metabolic, hormonal, and neuronal inuences as well
as compensatory effects can not be excluded, it is unknown
whether depletion of MT would sensitize cardiomyocytes to Dox
toxicity. Therefore, the linkage between the effect of MT deciency
on Dox-induced toxicity and ROS generation in cardiomyocytes has
not been delineated. To this end, the present study was undertaken
to establish a primary cardiomyocyte culture system from MT-I/II
null (MT/) and corresponding wild type (MT+/+) neonatal mice,
and to test whether MT deciency enhances Dox toxicity and ROS
generation in cardiomyocytes. Moreover, N-acetylcysteine (NAC)
and glutathione (GSH), two typical antioxidants, were used to evaluate the role of MT in the cardiac antioxidant defense system.
2. Materials and methods
2.1. Materials
Dox hydrochloride (Adriamycin), bromodeoxyuridine (BrdU), 3[4,5-dimethylthiazol-2-yl]-2, 5-diphenylterazolium bromide (MTT),
20 ,70 -dichlorouorescineiacetate (DCFH-DA), N-acetylcysteine (NAC)
and glutathione (GSH) were purchased from Sigma Aldrich. Fetal
bovine serum (FBS) was obtained from Hyclone. Dulbeccos modied eagle medium (DMEM), and trypsin (1:250) were purchased
from Gibco. Hoechst 33258 staining kit was obtained from Beyotime Institute of Biotechnology. LDH assay kit was obtained from
Biosino Bio-Technology and Science Inc. Bicinchoninic acid (BCA)
protein assay regents were obtained from Applygen Technologies
Inc. All other reagents were at least analytic grade.

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were discarded, whereas the cells from subsequent digestion were

added to an equal volume of Dulbeccos Modied Eagle Medium
(DMEM) supplemented with 5% fetal bovine serum (FBS) until all
cardiac cells were isolated. The resulting mixture was centrifuged,
and the cells were resuspended in the FBS-DMEM (DMEM supplemented with 15% FBS (v/v), 100 U/ml penicillin, 100 U/ml streptomycin, and 2 mM L-glutamine). To exclude non-muscle cells, the
isolated cells were rst plated in tissue culture dishes at 37 C for
2 h in an incubator with 5% CO2 and 95% atmospheric air. The
non-adherent myocytes were then collected and plated at a density
of 1.0  105 cells/ml in 35-mm culture dishes, 25-cm2 culture asks
or 96-well culture plates, and incubated under the same conditions
as above. 48 h after plating, bromodeoxyuridine (BrdU) was added
to the medium to inhibit broblast growth. Cultures used in experiments contained >95% cardiac myocytes as determined by immunocytochemical staining for a-sarcomeric actin.
2.4. Drug treatments
Dox was dissolved in deionized water (2 mM stock solution) and
further dilutions were made with culture medium. At the time of
Dox exposure, the FBS-DMEM was removed and replaced with fresh
serum-free DMEM containing different concentrations of Dox as
indicated. In some experiments, cultures were pre-treated with
antioxidant NAC (5 mM) or GSH (1 mM) for 30 min, and exposure
to the antioxidant was continued during subsequent Dox treatment.
2.5. Determination of cellular MT concentrations
MT concentrations in the cardiomyocytes were determined by
the cadmiumhemoglobin afnity assay (Eaton and Cherian,
1991), using rat red blood cell hemolysate to remove nonmetallothionein-bound cadmium. On the sixth day of culturing, cells were
harvested and centrifuged at 800g for 10 min. Cell pellet was resuspended with TrisHCl (10 mM), and was then pulse-sonicated on
ice for 15 s repeated three times with a 30 s interval. After centrifugation at 12,000g for 10 min, 100 ll of supernatant was collected
for MT analysis, and another 100 ll of supernatant was sampled
for total protein concentration determination using the BCA protein assay reagents.

2.2. Animal care

2.6. Morphological observations

C57BL/OLA129 mice which are decient in MT-I and MT-II

genes (MT/) and homozygous wild type (MT+/+) mice were originally obtained from the Murdoch Institute of the Royal Childrens
Hospital (Parkville, Australia), and breeding colonies were established. Animals were kept in a ventilated animal room maintained
at 22 C with a standard 12/12 light/dark cycle. Food and tap water
were provided ad libitum. Animals were housed, and all experiments were carried out with protocols approved by the Institutional Animal Care and Use Committee according to the Guide
for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

To observe cell morphological alterations, 6-day-old MT+/+ and

MT/ cardiomyocytes were treated with 0, 0.1, 1, and 10 lM
Dox. After 24 h Dox treatments, morphological observation of the
cultured cardiomyocytes was performed by an inverted microscope (Olympus, Japan).

2.3. Neonatal mouse primary cardiomyocyte culture

Cardiac ventricular myocytes were prepared from 1 to 3-day-old
neonatal MT-I/II knockout (MT/) or wild type (MT+/+) mice and
cultured as described previously (Wang et al., 1999). In brief, mice
were euthanized by cervical dislocation. Hearts were removed
aseptically, retaining the ventricles only, and kept in ice-cold DHanks balanced salt solution. The ventricles were washed three
times with the same solution and minced into small fragments.
Cardiac cells were dissociated with trypsin (0.05%, w/v) and obtained by centrifugation. The cells released after the rst digestion

2.7. Determination of LDH release

On the sixth day of culturing, cardiomyocytes were treated with
0, 0.1, 1, 10, and 100 lM Dox for 24 h. At the end of the treatments,
the supernatants of culture media were collected. Lactate
dehydrogenase (LDH) activity was measured by means of a colorimetric assay using a commercial LDH assay kit according to the
manufacturers instructions.
2.8. Determination of cell viability
Cell viability was quantied by measurement of the mitochondrial reduction of MTT to produce a dark-blue formazan product as
described previously (Liu et al., 2007). Cells were plated in 96-well
tissue culture plates (2.5  104 cells/well) and cultured for 4 days
before exposed to Dox. MTT was dissolved in phosphate-buffered
saline and added to each well at the nal concentration of

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Z. Fu et al. / Toxicology in Vitro 24 (2010) 15841591

0.25 mg/ml after completion of Dox treatment. Cells were then

incubated at 37 C for 4 h to allow MTT reduction. Subsequently,
the medium was removed and replaced with 100 ll dimethylsulfoxide to dissolve the formazan blue crystals. Absorbance at 570 nm
was measured by a microplate reader. Data were normalized to
measurements from control cultures which were considered as
100% cell survival.

observed between MT+/+ and MT/ cardiomyocytes as evaluated

by the time for cell attachment (within 24 h for both of the two
types of cells), spontaneous beating rate (98 and 107 times/min,
respectively, for MT+/+ and MT/ cardiomyocytes on the sixth
day after culturing), and cell morphology. MT concentrations in
MT+/+ cardiomyocytes were about 2.5-fold higher than those in
MT/ cardiomyocytes (1.08 and 0.42 lg/mg protein, respectively)
on the sixth day after culturing.

2.9. Determination of apoptotic cells

3.2. MT deciency exacerbates Dox-induced cytotoxicity
Apoptotic cells were determined by Hoechst 33258 staining,
which allows determination and quantication of cells with fragmented and condensed chromatin. Hoechst 33258 is a blue uorescent dye that can easily permeate cell membrane. The cells
have condensed chromatin would be stained with brighter uorescence than normal healthy cells. On the sixth day of culturing, cells
were treated with 0, 0.1, 1, and 10 lM Dox for 24 h. After Dox
exposure, cells were washed twice with cold PBS and xed in 4%
formaldehyde at 4 C overnight. Afterward, the xed cells were
washed and labeled with Hoechst 33258 (5 lg/ml) at room temperature in the dark for 10 min. Subsequently, the cells were
washed three times with cold PBS and one drop of mounting solution was added. Cell nuclei were observed and imaged by an inverted uorescence microscope (DMI 3000 B, Leica Microsystems
Ltd.) with excitation at 350 nm and emission at 460 nm. The number of apoptotic nuclei was determined on at least six randomly selected areas from three coverslips of every experimental group.
Data were expressed as a percentage of apoptotic cells relative to
the total number of cells.
2.10. Analysis of intracellular ROS generation
Cellular ROS accumulation in cardiomyocytes was measured by
a H2-DCFDA staining method as described previously (Li et al.,
2010). This assay is based on the principle that the non-polar,
non-ionic H2-DCFDA crosses cell membranes and is enzymatically
hydrolyzed by intracellular esterases to non-uorescent H2-DCF. In
the presence of ROS, H2-DCF is rapidly oxidized to become highly
uorescent DCF. For the concentrationresponse experiments,
MT/ and MT+/+ cardiomyocytes were treated with 0, 0.1, 1, and
10 lM Dox for 3 h. For the time-course experiments, the cultures
were treated with Dox at 1 lM for 0, 1, 3, and 6 h. Immediately
after Dox treatment, the cultures were washed three times with
cold PBS and then incubated for 40 min at 37 C with 4 lM H2DCFDA dissolved in culture medium. The cultures were then xed
with a xative containing 2% glutaraldehyde and 2% formaldehyde
dissolved in PBS and visualized by a inverted uorescence microscope (DMI 3000 B, Leica Microsystems Ltd.) with excitation settings of 488 nm. The uorescence intensities of DCF in cells were
analyzed by Image-Pro Plus 6.0 software.
2.11. Statistical analysis
All data were expressed as mean SD. Statistical analysis of the
data was determined by one-way ANOVA for multiple group comparisons. Differences between MT/ mice cardiomyocytes and
MT+/+ controls were analyzed by t-test. A level of p < 0.05 was considered signicantly different.

The cytotoxicity of Dox in the primary cultures of MT+/+ and

MT/ cardiomyocytes was evaluated by morphological alterations, LDH leakage, cell viability, and apoptosis. Dox-induced concentration-dependent morphological changes in both MT+/+ and
MT/ cardiomyocytes, included intermediate pseudopodia, vacuoles, and granules with disruption of monolayer (Fig. 1). However,
the damage to the MT/ cardiomyocytes was more severe than
the damage to the MT+/+ cardiomyocytes as obvious differences
in the morphological changes induced by Dox was observed.
LDH leakage, as a marker of necrotic cellular death, was measured in the cell culture medium supernatant. There were no signicant differences between Dox untreated MT+/+ and MT/
cardiomyocytes with the LDH levels of 93.0 and 91.4 U/L, respectively. Dox induced a typical concentration-dependent increase in
the LDH release in both MT+/+ and MT/ cardiomyocytes. 24 h
treatment with 0.1 lM Dox caused a slight but signicant increase
of the LDH release in MT/ cardiomyocytes, but only caused a
minimal LDH leakage in MT+/+ cardiomyocytes. When cells were
challenged by Dox at P1 lM, both MT+/+ and MT/ cardiomyocytes showed signicant LDH release. Again, MT/ cardiomyocytes were more susceptible to Dox-induced LDH leakage
compared to MT+/+ cardiomyocytes (Fig. 2). The results obtained
from cell viability assay were consistent with the ndings from
LDH assay. Dox concentration-dependent decreased cell viability
in both MT+/+ and MT/ cardiomyocytes, while signicant lower
cell viabilities were found in MT/ cardiomyocytes compared to
MT+/+ cardiomyocytes treated with the same concentration of
Dox (Fig. 3).
Apoptosis is a critical cellular process involved in Dox-induced
cytotoxicity. Dox has been shown to induce apoptosis both
in vitro and in vivo (Kalyanaraman et al., 2002). As shown in
Fig. 4, nuclei of both Dox untreated MT+/+ and MT/ cardiomyocytes had regular contours, and were round and large in size
(Fig. 4A). The cells treated with 1 lM Dox displayed either shrunken and irregularly shaped or degraded with aggregation and fragmentation of chromatin (arrows indicated the apoptotic cells),
which were much more obvious in MT/ cardiomyocytes. The
number of apoptotic cells was determined and expressed as percentage of apoptotic index (Fig. 4B). There were no signicant differences between the untreated MT+/+ and MT/ cardiomyocytes
with percentage apoptotic cell of 5.1% and 4.7%, respectively. After
24 h treatment with various concentrations of Dox, concentrationdependent increases of apoptotic cells in both MT/ and MT+/+
cardiomyocytes were observed. At the concentrations of 0.1, 1,
and 10 lM Dox, a signicant increase of apoptotic cell to 14.1%,
23.1%, and 42.2% was observed in MT/ cardiomyocytes, which
was to 7.5%, 16.6%, and 34.1% in MT+/+ cells.
3.3. Enhanced ROS generation in MT/ cardiomyocytes

3. Results
3.1. Primary cultures of MT+/+ and MT/ cardiomyocytes
MT+/+ and MT/ neonatal mouse cardiomyocytes were isolated
and cultured separately. No obvious different characteristics were

Increase of ROS generation has been found to be the early event

in Dox-induced cell death (Bernuzzi et al., 2009). The present study
therefore exposed neonatal primary mouse cardiomyocytes to Dox
for a relative short-term. To examine the effects of DOX on the
intracellular ROS formation, H2-DCFDA was used as a uorescent

Z. Fu et al. / Toxicology in Vitro 24 (2010) 15841591

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Fig. 1. Phase-contrast photomicrograph of primary neonatal MT+/+ and MT/ mouse cardiomyocytes treated with or without Dox for 24 h. No obvious difference was found
between (A) untreated MT+/+ and (E) MT/ cardiomyocytes. (B) MT+/+ cardiomyocytes exposed to 0.1 lM Dox exhibiting no obvious changes, while (F) MT/ cardiomyocytes
exposed to 0.1 lM Dox exhibiting minimal pseudopodia, intermediate vacuolization. (C) MT+/+ cardiomyocytes exposed to 1 lM Dox exhibiting minimal pseudopodia and
intermediate vacuolization, while (G) MT/ cardiomyocytes exposed to 1 lM Dox exhibiting obvious pseudopodia and intermediate vacuolization. (D) MT+/+ cardiomyocytes
exposed to 10 lM Dox exhibiting pseudopodia and intermediate vacuolization, while (H) MT/cardiomyocytes exposed to 10 lM Dox exhibiting obvious granulation around
the nuclei with signicant disruption of the monolayer (original magnication, 400).

Fig. 2. LDH leakage in MT+/+ and MT/ cardiomyocytes after Dox treatment. Cells
were treated with varying concentrations of Dox for 24 h. Values represent
mean S.D. (n = 4) from quadruplicate samples for each treatment at varying
concentration levels. #Signicantly different from Dox untreated MT+/+ cardiomyocytes. oSignicantly different from Dox untreated MT/ cardiomyocytes. *Significantly different from MT+/+ cardiomyocytes for the same treatments (p < 0.05).

Fig. 3. Determination of cell viability in MT+/+ and MT/ cardiomyocytes. Cells

were treated with various concentrations of Dox for 24 h, cell viability was then
determined by MTT method. Data are expressed as mean S.D. (n = 4). #Signicantly different from Dox untreated MT+/+ cardiomyocytes. oSignicantly different
from Dox untreated MT/ cardiomyocytes. *Signicantly different from MT+/+
cardiomyocytes for the same treatments (p < 0.05).

probe. There was no signicant difference in the intracellular ROS

formation between Dox untreated MT+/+ and MT/ cardiomyo-

cytes. Dox induced a concentration and time-dependent elevation

of the ROS level in both two types of cells. However, this effect was

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Z. Fu et al. / Toxicology in Vitro 24 (2010) 15841591

Fig. 4. Effect of Dox on apoptosis in MT+/+ and MT/ cardiomyocytes. (A) Representative uorescence images of Hoechst 33258-stained MT+/+ and MT/ cardiomyocytes
treated with or without 1 lM Dox for 24 h. Nuclei have fragmented and condensed chromatin with brighter staining were identied as apoptotic cells (arrow). Scale bar,
100 lm. (B) Percentage of apoptotic cells in MT+/+ and MT/ cardiomyocytes. Data are expressed as mean S.D. (n = 3). #Signicantly different from Dox untreated MT+/+
cardiomyocytes. oSignicantly different from Dox untreated MT/ cardiomyocytes. *Signicantly different from MT+/+ cardiomyocytes for the same treatments (p < 0.05).

much more remarkable in MT/ cardiomyocytes, indicating that

Dox induces more ROS accumulation in cardiomyocytes when
MT is decient (Fig. 5).
3.4. Antioxidant NAC and GSH can not compensate MT deciency in
protection against Dox-induced cell death and ROS generation
In order to evaluate the role of MT in the cellular antioxidant
defense system under oxidative stress, both MT+/+ and MT/
cardiomyocytes were pre-treated with typical antioxidant NAC or
GSH, and exposure to the antioxidant was continued during the
subsequent Dox treatment. Both MT+/+ and MT/ cardiomyocytes
treated with NAC or GSH alone did not show obvious effects on cell
viability and ROS production (Data not shown). Either NAC or GSH
almost fully inhibited Dox-induced decrease of cell viability in
MT+/+ cardiomyocytes with the levels of 107.2% and 108.2%,
respectively (Fig. 6A). However, both of the two antioxidants only
partially inhibited Dox-induced cell death in MT/ cardiomyo-

cytes (86% and 95%, respectively, for NAC and GSH pre-treated
cells). Consistently, both NAC and GSH almost fully inhibited
Dox-induced increase of ROS generation in MT+/+ but not MT/
cardiomyocytes. As compared to cells in control, only a slight increase of ROS level was observed in MT+/+ cells treated with Dox
plus NAC or GSH by 10% and 20%, respectively. However, ROS levels
in MT/ cells treated with Dox plus NAC or GSH were signicantly
increased by 30% and 40%, respectively (Fig. 6B). These results indicated that NAC and GSH can not compensate MT deciency in protection against Dox-induced cell death and ROS generation.
4. Discussion
Dox has been widely used in cancer chemotherapy for more
than three decades. However, the clinical application of Dox was
restricted because it causes dose-dependent cardiomyopathy. Severe cardiac damage and mortality were detected in patients as
well as animal models treated with Dox, indicating that Dox was

Z. Fu et al. / Toxicology in Vitro 24 (2010) 15841591

Fig. 5. ROS generation in MT+/+and MT/ cardiomyocytes measured by H2-DCFDA

staining. (A) Both MT+/+ and MT/ cardiomyocytes were exposed to 1 lM Dox for 1,
3, and 6 h. (B) Cells were exposed to different concentrations of Dox for 3 h. Values
represent mean S.D. (n = 3). #Signicantly different from Dox untreated MT+/+
cardiomyocytes. oSignicantly different from Dox untreated MT/ cardiomyocytes.
*Signicantly different from MT+/+ cardiomyocytes for the same treatments
(p < 0.05).

selectively more toxic to cardiac muscle cells. In the present study,

we used primary cultures of cardiomyocytes from neonatal MT-I/II
null mice (MT/) and the corresponding wild type mice (MT+/+) to
investigate whether depletion of MT would sensitize cardiomyocytes to Dox-induced toxicity. We found that MT/ cardiomyocytes were more susceptible than MT+/+ cardiomyocytes to Doxinduced toxicity. Dox time- and concentration-dependently increased the levels of ROS in MT+/+ cardiomyocytes, and this effect
was exaggerated in MT/ cardiomyocytes. In addition, antioxidant
GSH and NAC signicantly rescued MT+/+ but not MT/cardiomyocytes from Dox-induced cell death and ROS generation. These ndings clearly demonstrate that basal MT provides protection against
Dox-induced toxicity in cardiomyocytes, which possibly associates
with inhibition of ROS generation.
Studies examining the effects of doxorubicin in cardiac myocytes in vitro have demonstrated that Dox directly induces cellular
injury in several forms, including structural disruption, enzymatic
alteration, and cell death. It has further been reported that Dox induces apoptosis in cardiomyocytes in experimental animals as
well as patients, and it is suggested that apoptosis plays a crucial

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Fig. 6. Effects of NAC and GSH on Dox-induced cell death and ROS generation in
MT+/+ and MT/ cardiomyocytes. Cells were pre-treated with NAC (5 mM) or GSH
(1 mM) for 30 min before exposed to 1 lM Dox. (A) Cell death and (B) ROS
generation were assayed after 24 and 3 h Dox treatment, respectively. Data are
0
mean S.D. (n = 4). a,a Signicantly different from Dox untreated MT+/+ and MT/
0
cardiomyocytes, respectively. b,b Signicantly different from MT+/+ and MT/
cardiomyocytes treated with 1 lM Dox alone, respectively. cSignicantly different
from MT+/+ cardiomyocytes for the same treatments (p < 0.05).

role in the progression of myocardial dysfunction induced by Dox

(Kotamraju et al., 2000). Recently, the primary cultures of cardiomyocytes isolated from MT transgenic mice have been used as
useful tools to exam the events of myocardial injury. It has been
shown that MT-overexpression cardiomyocytes are more resistant
to hydrogen peroxide, streptozotocin, and Dox-induced cell death.
In the present study, we established a primary culture system of
cardiomyocytes from MT/ and MT+/+ mice to directly investigate
the effects of MT deciency on Dox-induced cardiac myocytes injury. Our results clearly demonstrated that MT deciency signicantly increases the sensibility of cardiomyocytes to Doxinduced cytotoxicity as evidenced by enhanced cell morphological
alteration, increase of LDH release, decreased cell viability, and increased apoptotic cells in MT-I/II depleted cardiomyocytes. Thus,
these ndings strongly support the protective effects of basal
MT against Dox toxicity in primary cultured cardiomyocytes. A
study from Dr. Klaassens laboratory demonstrated that transgenic
mice overexpressing MT are not resistant to Dox cardiotoxicity, in
which MT from transgenic mice was elevated less than 3-fold
higher than wild type mice. However, studies from Dr. Kangs Laboratory have shown that 10-fold elevation of cardiac MT provided
a signicant protection against Dox toxicity. Taken together, it is

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Z. Fu et al. / Toxicology in Vitro 24 (2010) 15841591

suggested that a certain level of MT provides protection against

Dox-induced toxicity, and particularly highlight the basal MT in
cardiomyocytes is an imperative factor in affecting DOX toxicity.
Several hypotheses have been proposed to explain Dox-induced
cardiotoxicity, such as free-radicals generation, calcium overloading, and mitochondrial dysfunction (Olson and Mushlin, 1990;
Kimura et al., 2000). It is widely accepted that cardiac toxicity of
Dox is mainly mediated by ROS produced during its intracellular
metabolism (Wallace, 2003; Tokarska-Schlattner et al., 2006). The
metabolic characteristics of Dox in rodents are highly similar with
that in human (Licata et al., 2000). Dox has a high afnity for myocardium tissue, which leads to Dox accumulation in cardiomyocytes (Quiles et al., 2002). The quinine moiety of Dox undergoes
a one-electron reduction that is catalyzed by NAD(P)H reductases
to yield a semiquinone free-radical intermediate, which regenerates its parent quinone by reacting with O2 to form O
2 and H2O2,
members of the broad family of ROS (Menna et al., 2008). These
ROS can then be transformed into the more potent hydroxyl radical
HO which is capable of damaging DNA and proteins, and initiating
membrane lipid peroxidation, eventually leading to cell death and
cardiotoxicity. In the present study, our result shown that Dox
time- and concentration-dependently increased ROS in cardiomyocytes, and this effect was signicantly aggravated in the absence of
MT, indicating that MT exerts antioxidative and free-radical scavenging properties in cardiomyocytes in response to Dox-induced
increase of ROS generation.
The mechanisms by which MT scavenges ROS are poorly understood. MT is a thiol-rich, zinc-binding protein that is found in all
eukaryotes and in some prokaryotes. It has been shown that all
20 of the thiolate groups are involved in the radicals quenching
process and MT is more potent than GSH in scavenging hydroxyl
radical. A study using HL-60 cells has demonstrated that the thiolate groups in MT can react with hydrogen peroxide directly (Quesada et al., 1996). In particular, the thiolate groups in MT were the
preferential attacking targets of hydrogen peroxide compared with
the other sulfhydryl residues from glutathione and protein fractions. Alternatively, the zinc-binding property of MT and the release of zinc under oxidative stress may contribute to the action
of MT in scavenging ROS (Muller et al., 1994; Maret, 2000). MT is
the only known protein that is implicated in zinc distribution.
Increasing evidence indicates that zinc also exerts antioxidant
properties by inhibiting hydroxyl radical formation through antagonism of redox-transition metals. In the present study, cardiomyocytes from MT/ mice were presumed to have less zinc release
due to MT-I/II deletion. Indeed, it has been reported that tissues
from MT/ mice contain less zinc compare to MT+/+ mice (Zhou
et al., 2002). Therefore, it is likely that zinc plays some role in
MTs protection against Dox-induced toxicity.
Cellular antioxidant defense system is an important factor in
determining the fate of cells in response to oxidative injury. Of
all antioxidants found in cardiomyocytes, GSH is one of the most
important antioxidants against ROS. In vitro studies have shown
that GSH and NAC, a precursor of GSH, can directly scavenge free
radicals and reduce oxidant-induced cell damage and cell death
(Kern and Kehrer, 2005). The use of biothiols, such as GSH, N-acetylcysteine (NAC), to mitigate acute oxidative stress induced by
anticancer drugs has long been proposed (Park et al., 2003; Shi
et al., 2009). In the present study, the application of NAC and
GSH almost completely inhibited Dox-induced increases of ROS
production in MT+/+ cardiomyocytes, which may account for the
eventual full protection against Dox-induced cell death. However,
both NAC and GSH can only partially protect against Dox-induced
ROS production and cell death in MT/ cardiomyocytes where
MT-I/II is absent, indicating that these antioxidants cannot compensate the deciency of MT in response to Dox-induced oxidative

stress. This nding particularly highlights the important role of MT

as a cellular antioxidant in cardiomyocytes.
In summary, the present study clearly demonstrates that Doxinduced toxicity and ROS production are enhanced in the primary
cultures of cardiomyocytes derived from neonatal MT-I/II null
mice, and particularly highlights the important role of MT as a cellular antioxidant on scavenging ROS. Dox is a widely used and
important chemotherapy agent though its application is limited
by the incidence of cumulative cardiomyopathy. In the past decades, signicant efforts have been made to discover and develop
agents that can effectively protect against the cardiac toxicity of
Dox. Therefore, exploring the protective effects of MT may provide
a possible therapeutic strategy in prevention and/or treatment of
Dox-induced cardiomyopathy.
5. Disclosure/conict of interest
The authors state no duality of interest.
Acknowledgements
This project was supported by the National Natural Science
Foundation of China (30873130), and National Key Project on Drug
Development from the Ministry of Science and Technology of
China (2009ZX09501-034).
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