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Introduction
Doxorubicin (DOX) is one of the anthracycline antibio-tics widely used in cancer chemotherapy, yet it is limited by severe
and cumulative cardiotoxicities despite its
effectiveness[1,2]. The mortality of cardiomyopathy caused by DOX is as high as
40% in patients without treatment[3], and the incidences of congestive heart failure secondary to DOX-induced
cardiomyopathy are 4%_36% in patients receiving cumulative dosages of 500_600 mg per square meter of body surface
area[4]. The mechanism of DOX cardiotoxicity is still not well understood. It is widely accepted that oxidative stress and the production
of free radicals are involved in DOX
cardiotoxicity[4], in which iron was suggested to play an important role. DOX combines
with Fe3+ in vivo. This
DOX-Fe3+ complex can reduce oxygen to hydroxyl radical and other reactive oxygen species
(ROS)[5], which initiate severe damage to cardiac tissues. Efforts have been made to prevent the DOX cardiotoxicity with a number of
antioxidants and iron chelators such as
melatonin[6] and ICL670A
(deferasirox)[7]. So far there is only one agent, dexrazoxane
(Zinecard), approved by the Food and Drug Administration (FDA) to prevent cardiomyopathy induced by DOX. However,
dexrazoxane may potentiate the myelosuppression caused by DOX, and as a parenteral iron chelator, dexrazo-xane must be
reconstituted with a sodium lactate injection and given slowly intravenously. Therefore, it is necessary and urgent to seek
alternatives for clinical treatment besides dexrazoxane.
Deferiprone (L1, CP20) is the first oral iron chelator, licensed in the European Union for treating iron overload in thalassaemia
patients unable to use deferoxamine[8]. Deferi-prone forms stable complexes with iron at a molar ratio of
3:1 (chelate:Fe3+)[9]. Several clinical trials have found that thalassaemia patients treated with deferiprone had higher ejection
fractions than those treated with deferoxamine, and deferiprone was more effective than the latter in the removal of
myocardial iron[10]. A previously published study of cultured hepatocytes showed that by scavenging superoxide radicals, both
deferiprone and Fe3+-deferiprone complexes were able to protect the cells against hypoxia-reoxygenation injury
effectively[11]. Further studies of cultured heart cells showed that deferiprone eliminated DOX-induced cardiac
damage[12,13]. Together, these studies support the cardiac protective activity of deferiprone. The mechanism was possibly related to the iron
chelating of deferiprone. As a small molecule, deferiprone is able to enter cardiac myocytes
rapidly[13] and is capable of efficiently preventing free radical-based cardiotoxicity by quickly displacing iron from the complex with
DOX[13]. Moreover, deferiprone and its complex with
Fe3+ have antioxidant properties. It has been suggested that forming a redox complex with
Fe3+ was the initial step for deferiprone to reduce oxidative damage: the complex could directly scavenge superoxide radicals
with a free electron transference between
them[11]. With the advantages of cheap synthesis and oral
activity[14,15], deferiprone is promising to be an alternative besides dexrazoxane by improving compliance and easing financial burden.
The purpose of this study is to investigate the effects of deferiprone on DOX cardiotoxicity at tissue level with
spontaneously-beating isolated paired atria from rats. Changes of atrial contractility
(dF/dt) and mitochondrial structure
induced by DOX were observed with or without the pretreatment of deferiprone. The experimental model we chose was due
to 2 reasons. First, it was necessary to ensure that deferiprone could protect cardiac function from DOX before further
studies in vivo since there are few reports about the effects of deferiprone on cardiac function. In addition, deferi-prone has
a far shorter elimination time (elimination
t1/2: 47 to 134
min[16]) than that of DOX (elimination
t1/2: 20 to 48 h) in humans, which could interfere with the effects of
deferiprone in vivo. Thus, we chose the model of isolated rat atria to avoid the possible interaction, which could also be
useful for a rapid evaluation of DOX cardiotoxicity. Second, the effective dosages or dosage ratio of deferiprone to DOX is
still unknown. As a chelator, deferiprone is
administered at the dosage of 75 mg/kg per day in patients with high iron levels; this dosage might be too high or
unsafe for DOX-treated patients with normal iron levels. In the present study, different concentrations of deferiprone were
set in order to find the effective concentrations for later studies
in vivo.
Materials and methods
Materials Deferiprone was purchased from Acros
Organics (New Jersey, USA). DOX was purchased from Sigma Chemical Co (St Louis, Missouri, USA ). The chemicals were
dissolved in distilled water and were diluted in tissue bath solution to achieve the final concentrations.
Animals Forty eight adult male Sprague-Dawley rats at 10 weeks of age, weighing 350_450 g (Grade II), were
obtained from the Department of Laboratory Animal Sciences of Fudan University (Shanghai, China). The rats were housed
in a standard environment at a constant temperature of 22 °C under a 12-h light/dark cycle with free access to food and
drinking water.
Preparation of spontaneously-beating isolated paired atria
The rats were killed after anesthesia by urethane
(1 g/kg) administered intraperitoneally. Both the left and right atria were dissected from the ventricles, cleaned of fatty
tissues and mounted vertically at pH 7.4 at 30 °C in tissue bath filled with 6 mL Krebs-Henseleit buffer (in mmol/L: NaCl, 118;
KCl, 4.7; KH2PO4, 1.2;
MgSO4, 1.2; CaCl2, 1.25;
NaHCO3, 25; glucose, 11) and aerated with 5%
CO2 in oxygen. The paired atria were allowed to beat spontaneously under a resting tension of 1 g. An equilibrium period of 30 min was given before the
application of drugs. Tension changes in the tissues were recorded every 10 min via a force-displacement transducer
attached to SMUP-E Model Biosignal Processing System. Contractility
(dF/dt) was assessed by recording the maximum rate
of isometric force development. Control values (=100%) refer to the
dF/dt before the addition of
drugs[17].
Experimental protocols
Spontaneously-beating isolated atria were divided randomly into 4 groups after the equilibrium
period, and the experiment lasted 70 min.
DOX-treated atria were preincubated for 10 min without any drugs before incubation with DOX for 60 min. We tested the
IC50 concentration of DOX (0.035 mmol/L) in a previous
study[18], and then adjusted it to 0.03 mmol/L as the preliminary study
showed a negative inotropic effect of 50% after 60 min. Deferiprone-pretreated atria were incubated with both deferiprone
and DOX for 60 min; the addition of deferiprone was 10 min before that of DOX. The concentrations of deferiprone were 10
or 40 times higher than that of DOX (deferiprone:1.2 mmol/L+DOX:0.03 mmol/L or deferiprone:0.3 mmol/L+DOX:0.03 mmol/L).
In a preliminary study, several concentrations of deferiprone had been tested with 0.03 mmol/L DOX. The results had not
shown significant cardiac protection against DOX-induced damage on atria pretreated with 0.3 mmol/L deferiprone. Thus,
0.3 mmol/L was selected to be the low concentration of deferiprone. The control atria were incubated without any drugs during
the whole process.
After the experiment, the cardiac tissues around the sinuatrial nodes were taken and fixed in 2.5% glutaraldehyde; the
remains were homogenized (10%, w/v) at 4 °C in homogenization buffer consisting of 0.01 mol/L sucrose, 0.01 mol/L Tris-HCl,
0.1 mmol/L EDTA-2Na and 8% solution of NaCl, pH 7.4. Homogenates were distributed and stored at -70 °C until different
assessments were conducted.
Histological studies Tissue samples were post-fixed with osmium tetroxide and embedded in araldite (epoxyresin).
Semifine sections (0.5 µm) were stained with toluidine blue and observed by light microscopy; ultra-thin sections (80 nm)
contrasted with uranyl acetate and lead citrate were
examined by a Philips CM120 transmission electron microscope (Eindhoven, the Netherlands).
Determination of Cu, Zn-SOD and MDA levels
Cu, Zn-SOD activity was measured with a spectrophotometer at 550
nm. Treatment with 2% sodium dodecyl sulfate (SDS) at 37 °C for 30 min eliminated Mn-SOD, but not Cu, Zn-SOD activity. After
removing the excess sodium dodecyl sulfate by precipitation with 2% KCl, the supernate was assayed using the xanthine
oxidase-cytochrome c method. One unit of SOD was defined as the amount of enzyme required to inhibit 50% of the rate of
cytochrome c reduction at 25 °C, and data were expressed as units per
microgramme[19].
MDA concentrations were determined after incubation at 95 °C and pH 3.4 with thiobarbituric acid as an indicator of lipid
peroxidation. The pink colour produced by these reactions was measured at 532 nm in order to measure MDA
concentrations[20].
Determination of SDH
activity The activity of SDH was estimated by quantifying the deoxidization at the speed of 2,
6-dichlorophenol indophenol (DCPIP), which absorbs strongly at 600 nm when oxidized, but becomes colorless in
its reduced form. The reaction mixture contained 0.12
mmol/L DCPIP, 10 mmol/L succinate and 20 µL of homogenate in a final volume of
2 mL (pH 7.8). The reaction was started by the addition of succinate and was monitored for 3 min. One activity unit of SDH
was defined as 0.01 decreases in absorbance at 600 nm per mg protein per
min[21].
Protein determination Protein determinations were performed by the Bradford method based on the specific binding of
Coomassie blue G-250 dye to proteins. The binding caused a shift in the absorption maximum of the dye from 465 to 595 nm,
and it was the increase in absorption at 595 nm that was
monitored[22]. The Bradford reagent contained
0.01% w/v Coomassie Blue G-250. The protein concentrations were determined from a standard BSA curve prepared in
parallel.
All of the colorimetry assays were performed with a UV-2401PC spectrophotometer (Shimadzu, Kyoto, Japan).
Statistical analysis
All results were expressed as mean± SD and compared statistically by Student's
t-test. The numbers of animals (n) used per group are stated in Figures 1 and 2
and Table 1. The differences between means were considered
significant if P<0.05.
Results
Effects of deferiprone on the contractility
(dF/dt) of DOX-treated atria To determine whether deferiprone can protect
cardiac function from DOX, we measured the contractility
(dF/dt) of isolated atria with or without the presence of deferiprone
(Figure 1). At the end of the equilibration period, the absolute value for
dF/dt of the control atria was 19.3±
5.3 g/s; DOX-treated atria was 21.6±6.1 g/s, and the deferiprone-pretreated atria (1.2 mmol/L or 0.3 mmol/L) was 20.4±5.9 g/s
or 19.2±5.4 g/s. Incubation with 0.03 mmol/L DOX resulted in a gradual reduction of contractility of DOX-treated atria. Forty
min after the addition of DOX, the contractility became significantly lower than that of the control atria
(P<0.01), and decreased by 49.34%±4.79% (10.9±4.2
g/s) at the end of incubation. Neither the 1.2 mmol/L nor 0.3 mmol/L concentrations of deferiprone had significant effects on
basal contractility 10 min after pretreatment, and the contractility of both deferiprone-pretreated atria did not decrease until
50 min after the addition of DOX. Furthermore, pretreatment of deferiprone did moderately attenuate DOX-induced
negative inotropic effect. Compared with the DOX-treated atria, the contractility of the 1.2 mmol/L or 0.3
mmol/L concentrations of deferiprone-pretreated atria was significantly increased 40 min after the addition of DOX
(P<0.01), and was 25.70% or 20.44% higher than that of DOX-treated atria at the end of the incubation, respectively
(P<0.01).
Effects of deferiprone on the level of MDA and Cu, Zn-SOD activity of DOX-treated atria
Cu, Zn-SOD activity in the DOX-treated atria decreased by 21.42%
(P<0.01) and MDA levels increased by 44.24%
(P<0.05) compared with the control atria (Table 1). However, there was no statistically significant difference between the control and the
deferiprone-pretreated atria. Moreover, Cu, Zn-SOD activity in the 1.2 mmol/L or 0.3 mmol/L deferiprone-pretreated atria were
notably increased by 12.97% (P<0.01) or 12.11%
(P<0.05) respectively. MDA levels were reduced by 29.12%
(P<0.01) or 39.82% (P<0.01) compared with the DOX-treated atria.
Effects of deferiprone on SDH activity of DOX-treated atria
The results (Table 1) show that SDH activity in the
DOX-treated atria decreased by 31.38% compared with the control atria,
(P<0.05). However, in the 1.2 mmol/L or 0.3 mmol/L
deferiprone-pretreated atria, SDH activity increased by 25.15% or 34.76%
(P<0.05) compared with the DOX-treated atria.
Effects of deferiprone on ultrastructure of DOX-treated atria
Electron microscopic studies revealed that mitochondria
swelling, disruption of mitochondrial crista and decreased electron density of the matrices were found in the cardiac myocytes
of the DOX-treated atria. However, these lesions were ameliorated in the cardiac myocytes of the1.2 mmol/L
deferiprone-pretreated atria (Figure 2).
Discussion
The present study differs from other published studies in that it provides evidence that deferiprone can protect against
damage to cardiac contractility induced by DOX. The results of our study also suggest that deferiprone should be tested
in vivo as a novel therapy for DOX-induced cardiotoxicity.
The effect of deferiprone on DOX cardiotoxicity was evaluated via spontaneously-beating isolated paired atria from rat.
Recording changes of contractility of isolated atria can quantitate the effect of deferiprone on cardiac function. DOX was
administered at the concentration of 0.03 mmol/L for 60 min in tissue bath solution. Atria contractility decreased gradually
and nearly 50% of the contractility was lost 40 min after the addition of DOX, which was similar to the negative inotropic effect
on the DOX-treated mouse atrium[18]. The contractility of both 1.2 mmol/L and 0.3
mmol/L deferiprone-pretreated atria decreased 50 min after the DOX addition, but they were 25.70% or 20.44% higher than that of the DOX-treated atria at the end of the
incubation. The results indicate that pretreatment with deferiprone remarkably preserved the contractility of atria, although
it could not entirely reverse the negative inotropic effects induced by DOX. A previously published study reported that
deferiprone prevented cultured cardiac myocytes from DOX-induced damage monitored with the release of lactate
dehydrogenase[13]. Our study further suggests that deferiprone can also protect cardiac contractile function from damage induced by
DOX.
After incubation, we investigated the effects of deferi-prone on the mitochondria and SDH activity of the isolated atria
treated with DOX. Cardiac myocytes contain the highest volume density of mitochondria in the body due to the
extraordinary demand for continuous synthesis of
ATP[23]. Unfortunately, mitochondria are also the target for
DOX[24] and its complex with
iron[12]. It was found that DOX had high affinity for cardiolipin, the phospholipid in the inner mitochondrial
membrane[24]. This affinity results in the accumulation of DOX in cardiac myocytes with higher concentrations than that in other
cells[25], and causes oxidative damage on mitochondrial membranes, the respiratory chain, and
DNA[26_28]. On the other hand, the mitochondrial respiratory chain is the major source of superoxide, thus, mitochondria are more susceptible to oxidative
damage than the rest of the cells[29]. SDH is a component of complex II of the respiratory chain and is the only tricarboxylic
acid cycle enzyme embedded to the inner mitochondrial membrane that is essential for full respiratory
activity. As a mitochondrial marker enzyme, the activity
of SDH decreases when its vicinal thiols (SH group) are
oxidized[30]. Previous studies have shown that the SH group is very sensitive and SDH is inactivated by oxidized
DOX[31].
In our study, significant mitochondrial structural lesions and the decrease of SDH activity were observed in DOX-treated
atria, which indicates the damage caused by DOX on the cardiac mitochondria. Conversely, the pretreatment of deferiprone
resulted in fewer ultrastructure lesions and maintained SDH activity. These results suggest that deferiprone is able to protect
both mitochondrial structure and function from DOX-induced damage and supports previous
research[12] in which deferiprone had eliminated DOX-induced inhibition of mitochondrial function with a partial reduction of
14C-palmitate utilization in cardiac myocytes.
However, other studies have reported that DOX has little effect on SDH
activity[32,33]. The discrepancy
may be attributed to the fact that in the current study, an acute DOX treatment lasting 60 min was performed
in vitro, while in previous studies, DOX were repeatedly administered
in vivo over several months or years. Nevertheless, these
contradicting findings suggest that the inactivation of SDH might be an index of acute mitochondrial lesions rather than chronic ones.
We also investigated the antioxidant effects of deferiprone on the cardiac tissues of DOX-treated atria. Iron-based
oxida-tive stress has been implicated to be responsible for DOX-induced
cardiotoxicity[34]. Fe3+ reacts with DOX in a redox reaction
after which the iron atom accepts an electron and a
Fe2+ DOX-free radical complex is produced to stimulate ROS, including
extremely damaging hydroxyl
radicals[5,35]. In addition, the lack of natural antioxidants enhances the susceptibility of cardiac
tissues to oxidative damage, which is suggested to be the inherent causes of DOX
cardiotoxicity[36]. Assessments of SOD and
the lipid peroxidation end product MDA have been always used as markers for oxidative damage. Cu, Zn-SOD is the major
intracellular form of SOD, which is located in the cytoplasma and
nucleus[37]. In this study, DOX induced a decrease in Cu,
Zn-SOD activity and an increase in MDA levels in DOX-treated atria, which indicates that the oxidative damage caused by
DOX are consistent with findings of previous
studies[33,38]. Conversely, MDA levels were lower and Cu, Zn-SOD activity was
maintained in the atria pretreated with deferiprone at concentrations 10 and 40 times higher than that of DOX. This result
demonstrates the antioxidant property of deferiprone against DOX, and it is this antioxidant property that makes deferiprone
surpass other iron chelators such as deferoxamine and ICL670A in cardio protection. Neither deferoxamine nor its complex
with Fe3+ could scavenge superoxide radicals or
reveal cytoprotective activity[11].
ICL670A was found to not protect cardiac myocytes against
DOX[7]. The antioxidant property of deferiprone would be in part due to its direct
superoxide radical scavenging[11] and iron-chelating
activity[5]. It was demonstrated that deferiprone not only could quickly remove
Fe3+ from its complex with DOX extracellularly, but also
penetrated myocytes rapidly and displaced iron from its complex with the intracellularly-loaded fluorescent dye
calcein[13]. Those findings suggest that like dexrazoxane, deferiprone is able to prevent the formation of
Fe3+-DOX complex in the cardiac myocyte and as a result, prevent site-specific iron-based oxygen radical
damage[13].
Both pretreatments of 1.2 mmol/L and 0.3 mmol/L deferiprone were shown to protect against DOX-induced cardiotoxicity
without statistical differences between them. The results suggest that 10:1 is the approximate effective concentration rate of
deferiprone to DOX in the current study, which must be further verified
in vivo. However, in the preliminary study,
pretreatment with 0.3 mmol/L deferi-prone had not shown significant protection on the contractility after 60 min co-incubation with
0.03 mmol/L DOX. One of the possible reasons for this was that fewer rats were tested in preliminary study.
In conclusion, our findings indicate that deferiprone is able to improve cardiac contractility efficiently and protect
mitochondrial structure and function from DOX-induced damage. The effectiveness of deferiprone could be due to defense
capability against oxidative damage induced by DOX.
Acknowledgements
The authors thank Prof Jun PAN for her critical review and Mr Yin-xiang CAO and Mr Yuan QIAN for their excellent
technical support on the software and adjustment of the biosignal processing system.
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