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Cardiovascular Research 1998 38(3):695-702; doi:10.1016/S0008-6363(98)00034-0
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Copyright © 1998, European Society of Cardiology

Free radical involvement in doxorubicin-induced electrophysiological alterations in rat papillary muscle fibres

P Venditti, M Balestrieri, T De Leo and S Di Meo*

Dipartimento di Fisiologia Generale ed Ambientale di Napoli, V. Mezzocannone 8, I 80134 Napoli, Italy

* Corresponding author. Tel.: +39 (81) 552-7736; Fax: +39 (81) 552-6194.

Received 22 July 1997; accepted 22 January 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: This work was designed to determine whether the doxorubicin-induced changes in heart electrical activity are due to increased free radical production and membrane oxidative damage. Methods: Four groups of rats (60 days old) were used. One group was untreated and the others were treated with doxorubicin (DXR), DXR and vitamin E, and DXR and N-acetylcysteine (NAC), respectively. DXR was administered by single i.p. injection (20 mg/kg b.wt.). Vitamin E was administered by ten daily i.m. injections (100 mg/kg), while NAC (100 mg/kg) was injected i.p. 1 h before and 7 h after DXR. The effectiveness of the drug in inducing oxidative stress in different tissues and of the antioxidants in offering protection was established by determining antioxidant capacity, susceptibility to oxidative stress, and lipid peroxidation in heart, liver, and blood. The drug effect on heart electrical activity was determined by measuring the heart rate in vivo and action potential configuration in papillary muscle fibres in vitro. Heart lipid peroxidation and electrical activity were also examined in both vitamin E and NAC-treated rats. Results: DXR treatment decreased antioxidant capacity and increased lipid peroxidation and susceptibility to oxidative stress in heart and blood, but not in liver. DXR administration to rats treated with antioxidants did not produce significant changes in antioxidant capacity and susceptibility to oxidative stress even in heart and blood. Furthermore, lipid peroxidation in heart and liver from DXR- and vitamin E-treated rats, and in liver from DXR- and NAC-treated rats was lower than in untreated controls. DXR treatment also increased the duration of ventricular action potentials in untreated rats, but not in antioxidant-treated rats. The treatment of control animals with the antioxidants affected lipid peroxidation, but not cardiac electrical activity. Conclusions: The protection offered by antioxidants against electrophysiological alterations indicates a free radical involvement in such alterations. In contrast, although electrical modifications are associated with increased peroxidative processes and both are prevented by the antioxidants, it is not yet clear whether a causative relationship exists between them.

KEYWORDS Antioxidant; Doxorubicin; Vitamin E; Cardiac electrophysiology; Lipid peroxidation


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The clinical usefulness of doxorubicin (DXR) and other anthracyclines, which have been shown to be effective in treating various tumours, is limited by cardiotoxicity [1]. Congestive heart failure, cardiomyopathy and electrocardiogram (ECG) changes have been observed following doxorubicin administration to both experimental animals and patients [1]. Studies to determine the DXR-induced alterations in the electrical activity of individual cardiac cells related to ECG modifications have been controversial [2–6]. Furthermore, the cell membrane changes underlying the electrophysiological alterations and the mechanisms, by which ionic channels of cardiac cell membrane are modified, still need to be defined.

There are several hypotheses to explain doxorubicin cardiotoxicity, but no single hypothesis adequately integrates available information [7]. However, oxidative damage to membrane lipid and other cellular components is believed to be a major factor in the DXR toxicity [7]. DXR and its iron chelate undergo redox cycling, resulting in the generation of free radicals and reactive oxygen species (ROS). Lipid peroxidation and depletion of tissue non-protein sulphydryl compounds in response to DXR administration [8, 9]and reduction of both lipid peroxidation and cardiac toxicity of DXR by free radical scavengers [8]support an oxidative mechanism of toxicity. One finding apparently inconsistent with the free radical hypothesis is that the anthracycline-induced increase in oxygen consumption [10]is greater in liver than in heart, while only cardiac tissue develops clinically significant pathologic changes. It has been suggested that myocardial cells have a limited capacity to detoxify oxygen radicals [11, 12]and, hence, are particularly susceptible to injury from reactive oxygen species. However, this view is based on a partial evaluation of the tissue antioxidant defences.

We have recently used an enhanced luminescence technique to determine overall antioxidant capacity [13]and susceptibility of liver to oxidative stress [14]. The application of such methods to other tissues has shown that the heart has less antioxidant capacity significantly than the liver, whereas its susceptibility to in vitro oxidative stress is higher [15].

We designed the following study to establish the possible contribution of free radicals to the changes in heart electrophysiological properties induced by acute in vivo treatment with DXR. Because evidence of an interrelation between cell injury and free radical production can be supplied by the ability of antioxidants to reduce functional disturbances, the effect of DXR treatment on heart electrophysiology was also studied in both N-acetylcysteine (NAC) and vitamin E-treated rats. To assess DXR effectiveness in inducing oxidative alterations in tissues endowed with different susceptibility to the drug, lipid peroxidation, antioxidant capacity, and susceptibility to oxidative stress of liver, heart and blood were determined.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Animals
Male Wistar rats (60 days old) were used in the experiments. The animals purchased at weaning from Nossan (Correzzana, Italy), were housed one to two per cage at 24±1°C, with an artificial lighting cycle (light/dark 8:20 h). They were all provided with water ad libitum and a commercial rat chow diet (Nossan) containing 150 mg/kg vitamin E. Rats were randomly assigned to one of four groups: control untreated rats; DXR-treated rats, animals treated with single intraperitoneal injection of DXR (20 mg/kg b.wt.); DXR+VE-treated rats, animals treated for 10 days with daily intramuscular injections of vitamin E (100 mg/kg) and single intraperitoneal injection of DXR (20 mg/kg); and DXR+NAC-treated rats, animals treated with single intraperitoneal injection of DXR (20 mg/kg) and two intraperitoneal injections of NAC (100 mg/kg) 1 h before and 7 h after DXR. The above doses of DXR, vitamin E and NAC can increase the duration of action potential in papillary muscle [3], attenuate the thyroid hormone-induced modification of heart electrical activity [16], and decrease the DXR-induced appearance of microscopic myocardial lesions [8], respectively. Data were collected and analyzed on eight rats per group.

Subsequently, the effects of antioxidant treatment alone on lipid peroxidation and electrical activity of the heart were determined on four rats from three groups: control, vitamin E- and NAC-treated.

Animal care, housing and killing met the guidelines of the Italian Health Ministry.

2.2 Experimental procedure
Twenty-four hours after the DXR injection the rats were anaesthetized with Ethrane (Abbot, Aprilia, Italy) and subjected to electrocardiographic recording. The animals were then killed by decapitation, blood samples were collected, and livers and hearts were rapidly excised. The hearts were placed in cold oxygenated Krebs' solution containing (mmol/l): NaCl 135, KCl 5, MgCl2 1, CaCl2 2, NaHCO3 13, NaH2PO4 1, glucose 11, pH 7.4, and right ventricular papillary muscles were removed to be used in the electrophysiological determinations. The heart and livers were placed in cold 175 mmol/l KCl, 15 mmol/l Tris, pH 7.4. The heart great vessels and valves were trimmed away, the ventricles and atria were cut open and rinsed free of blood. The livers were freed of connective tissue. After the tissues were weighed, 20% (w/v) homogenates were prepared with a Potter–Elvehjem homogenizer set at a standard velocity (500 rpm) for 2 min in the above medium.

2.3 Level of tissue antioxidants and susceptibility to oxidative stress
Vitamin E levels in heart, liver and blood were measured using a high-performance liquid chromatography procedure [17].

Determinations of the response to oxidative stress and overall antioxidant capacity of tissue preparations were performed in microtitre plates using reagents and instrumentation of the Amerlite System (Johnson and Johnson, Cinesello Balsamo, Italy) as previously reported [13–15]. Briefly, the homogenates were diluted with equal volume of 15 mmol/l Tris containing 0.20% Lubrol PX (Sigma Chimica, Milano, Italy) at pH 8.5, so that their concentration was 10% (w/v). The blood was diluted ten times so that the final suspension contained 0.10% Lubrol in 15 mmol/l Tris, pH 8.5. Several dilutions of the homogenates and blood samples up to a concentration of 0.002% were prepared with 15 mmol/l Tris, pH 8.5. Enhanced chemiluminescence reactions were initiated by adding 250 µl of reaction mixture to 25 µl of the samples. The reaction mixture was obtained by dissolving a tablet containing substrate in excess (sodium perborate) and signal generating reagents (sodium benzoate, indophenol and luminol) (Amerlite Signal Reagent Tablets) in buffer at pH 8.6 (Amerlite Signal Reagent Buffer). The plates were incubated at 37°C for 30 s under shaking and then transferred to a luminescence analyzer (Amerlite Analiser), which supplied the emission values as percentages of the emission of a standard constituted by 25 µl of a solution of 22 ng/ml horseradish peroxidase. Such values were used to fit dose–response curves using Fig. P Program (Biosoft, Cambridge, UK). To determine the antioxidant capacity, 250 µl of the above reaction mixture was added to 10 µl of 110 ng/ml peroxidase plus 15 µl of either desferrioxamine, at concentrations ranging from 0.01 to 3 mmol/l, in 15 mmol/l Tris (pH 8.5) or buffer alone. Equal volumes of reaction mixture were also added to both 10 µl of 110 ng/ml peroxidase plus 15 µl of 10% homogenate (or blood) samples (samples) and 10 µl of 15 mmol/l Tris (pH 8.5) plus 15 µl of the same samples (blanks). The emission values obtained from the mixture of peroxidase and desferrioxamine were reported against the desferrioxamine concentration on logarithmic coordinates, supplying a standard curve. The differences between the emission values obtained from samples and blanks were referenced against the standard curve, and this allowed the tissue antioxidant capacity to be expressed as equivalent desferrioxamine concentration.

2.4 Measurement of lipid peroxidation
As a measure of lipid peroxidation the malondialdehyde (MDA) content in 10% homogenates in 0.175 mol/l KCl, 15 mmol/l Tris, pH 7.4, was determined with thiobarbituric acid reaction, according to the method of Buege and Aust [18].

2.5 Transmembrane potential determination
Right ventricular papillary muscles were mounted horizontally in a chamber between bipolar silver electrodes and superfused, at a rate of 11 ml/min, with Krebs' solution gassed with 95% O2–5% CO2 and warmed to 25±1°C. The preparations were allowed to equilibrate for 1 h under stimulation at 0.1 Hz before measurements were taken. During measurements, muscles were stimulated at 1 Hz. Transmembrane potentials were measured by cellular impalement using 3 M KCl-filled glass capillary microelectrodes and standard microelectrode technique [19]. The action potential signal was displayed and monitored on an oscilloscope (Tektronix 502A) throughout the experiment. Each action potential was recorded digitally on-line at 80 µs intervals on an IBM PC AT and stored on hard disc for subsequent processing. The transmembrane potentials were analyzed by a computer programme for the following characteristics: resting membrane potential, depolarization time, action potential amplitude, area above 20% depolarization, action potential duration (APD) from 10 to 90% of repolarization.

2.6 Statistical analysis
As impalements in approximately 20 cells from each preparation were performed, mean values of the electrical parameters were calculated, and the sample means were averaged together. Resulting values were used to supply traces of action potentials characteristic of each group. Some values were reported as means±s.e.m. in the tables and indicated by vertical bars in the figures. The data were analyzed with a one-way analysis of variance method. When a significant F ratio was found, Student–Newmann–Keuls multiple range test was used to assess differences among group means. Values were considered significantly different when P<0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 General characteristics
The body and heart weight were 243±12 and 0.63±0.04 g in untreated, 206±11 and 0.56±0.03 g in DXR-treated, 211±12 and 0.58±0.03 g in DXR+VE-treated rats, and 202±10 and 0.52±0.03 g in DXR+NAC-treated rats, respectively. The average concentrations for vitamin E in these hearts were 47.16±6.12, 42.75±5.48, 68.91±6.96 and 38.55±4.13 nmol/g, respectively. Therefore, the DXR-treatment did not significantly decrease heart content of the vitamin, while the vitamin treatment effectively elevated the content above control values.

3.2 Heart rate
DXR treatment gave rise to no significant increase in heart rate from 359±5 to 394±11 beats/min, which was reduced by vitamin E treatment to 364±8 beats/min and by NAC treatment to 371±11 beats/min (Fig. 1). Cardiac arrhythmias have not been shown in rats treated with DXR.


Figure 1
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  		Fig. 1 Effect of doxorubicin or doxorubicin and vitamin E or N-acetylcysteine administration on heart rate (HR). Untreated=control, untreated rats; DXR-treated=doxorubicin-treated rats; DXR+VE-treated=doxorubicin and vitamin E-treated rats; DXR+NAC-treated=doxorubicin and N-acetylcysteine-treated rats.

 
3.3 Lipid peroxidation and antioxidant capacity
Despite its limits, the MDA measurement by the thiobarbituric acid reaction is an useful index of lipid peroxidation. Therefore, data in Table 1 indicate that DXR treatment significantly increases peroxidative processes in heart and blood. The vitamin E administration protects the animals against the peroxidative processes due to both normal metabolism and doxorubicin action so that in liver and heart MDA concentration was lower than in control animals. NAC produced such an effect only in the liver. Significant decreases in antioxidant capacity were found in heart and in blood, but not in the liver of the doxorubicin-treated rats. In rats treated with antioxidants, the DXR administration did not produce significant changes of antioxidant levels even in heart and blood (Table 1).


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  		Table 1 Lipid peroxidation and antioxidant level in rat tissues

 
3.4 Susceptibility to in vitro oxidative stress
The luminescence response to changes of concentration of tissue preparations in untreated rats can be described by the equation: E=axC/exp(bxC). The a value depends on the concentration of substances inducing the luminescent reaction, such as the haemoproteins, while the b value, which corresponds to the inverse of the concentration at which the maximum emission is obtained, depends on the concentration of tissue antioxidants [14, 15]. The responses obtained for the tissues from DXR-treated rats exhibited analogous characteristics and were described by the same equation (Fig. 2). However, the emission values were higher than in control preparations, showing a higher susceptibility to oxidative stress. This indicates a greater release of hydroxyl radicals from haemoproteins, acting as peroxidases, and/or a reduced interception by scavenger systems [15]. The treatment with vitamin E effectively opposed the effects of DXR administration in all preparations. The examination of parameters characterizing light emission shows that DXR administration does not modify the a value in all tissues from untreated and antioxidant-treated rats. A similar pattern is shown for the b value in liver and blood preparations (Table 2). In contrast, the DXR administration significantly increases the values of b and Emax (an index of susceptibility to oxidative stress) in control rats, but not in those treated with antioxidants.


Figure 2
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  		Fig. 2 Light emission from tissues of control (solid line), doxorubicin-treated (dotted line), doxorubicin and vitamin E-treated (closely-dashed line), and doxorubicin and N-acetylcysteine-treated (dashed line) rats. The experimental data are obtained by modifying the concentration of homogenate or blood samples. Emission is given in relative units with 100 units equivalent to the emission of 44 ng/ml peroxidase. The curves are computed from experimental data using the equation: E=axC/exp(bxC).

 

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  		Table 2 Parameters characterizing light emission from rat tissues stressed with sodium perborate

 
3.5 Intracellular action potential characteristics
The effect on heart rate was consistent with those on action potential characteristics, clearly shown by the superimposed recordings of action potentials from papillary muscles in Fig. 3. These recordings demonstrate that the treatments are associated with marked alterations in action potential configuration, the most pronounced change being an increase in action potential duration in DXR-treated rats, that is reduced by vitamin E.


Figure 3
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  		Fig. 3 Effect of doxorubicin or doxorubicin and antioxidant administration on action potentials recorded from rat papillary muscle fibres. The working frequency was 1 Hz, the temperature 26°C. The arrows indicate the action potential duration at 90% repolarization. Untreated=control, untreated rats; DXR-treated=doxorubicin-treated rats; DXR+VE-treated=doxorubicin and vitamin E-treated rats; DXR+NAC-treated=doxorubicin and N-acetylcysteine-treated rats.

 
The quantitative values show that the resting potential and action potential amplitude are not affected by either DXR or vitamin E (Table 3). The depolarization time is significantly lengthened in DXR-, DXR+VE- and DXR+NAC-treated rats. The repolarization phase of the action potential is significantly slowed down in DXR-treated animals so that the action potential duration is markedly lengthened. Furthermore, the area of the action potential is increased. The antioxidant administration attenuates the DXR-induced modifications of the repolarization phase of the action potential and reinstates control values of APD (Table 3).


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  		Table 3 Membrane potential parameters in rat papillary muscle fibres

 
3.6 Antioxidant effects on control animals
Heart lipid peroxidation was significantly reduced by antioxidants. In fact, MDA was 42.72±2.21, 32.80±2.11 and 35.15±2.02 nmol/g wet weight in control, vitamin E- and NCA-treated rats. In contrast, heart electrical activity was not affected by antioxidants. In the above groups, HR was 342±6, 354±7 and 345±7 beats/min, while APD90 was 62.6±3.2, 64.0±3.6 and 67.70±3.83 ms, respectively.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
In agreement with previous studies using acute and chronic treatment with doxorubicin [2, 3], our results show that this anthracycline gives rise to a lengthening of APD in rat ventricular fibres. They also supply information on the mechanism by which the above alteration is induced. It is well known that anthracyclines significantly increase ROS production in cardiac mitochondria, cytosol, and sarcoplasmic reticulum [20]. This explains the pathological picture of anthracycline cardiac toxicity characterized by disruption of heart mitochondrial and sarcoplasmic reticular membranes [21]by drug-induced free radical formation in specific myocardial compartments [20]. The free radical hypothesis has been supported by the finding that free radical scavengers attenuate anthracycline-induced myocardial morphofunctional alterations [22]. On the other hand, the free radical hypothesis appears to be inconsistent with the lack of evidence of oxidative stress [23]or free radical production [24]at doses of doxorubicin that cause cardiac injury, and the frequent failure of antioxidants to attenuate doxorubicin-induced histological lesions [25, 26]and ameliorate systolic and diastolic dysfunction [27]. Thus, alternative hypotheses implicating prostaglandins [28], histamine [29], and C-13 anthracycline metabolites [30]as mediators of cardiac toxicity have been proposed. Although it is possible that the above mediators contribute in different measure to the various alterations which characterize the doxorubicin-induced cardiomyopathy, the disappearance of DXR-induced APD changes in animals treated with antioxidants suggests that ROS are alone responsible for DXR effects on cardiac electrophysiology.

This view is supported by the ROS ability to induce alterations in the cardiac electrophysiological properties. Several studies have shown similar changes in resting membrane potential and action potential amplitude [31–34], but different changes in APD: increases [33], decreases [34], or initial increases followed by marked reductions [31, 32]. Because this variability seems to depend on time and intensity of exposure to ROS, higher values giving rise to APD shortening, we suggested that APD shortens only when the ratio between ROS and antioxidant concentrations in preparations reaches a critical value [35].

In this frame, it is interesting that doxorubicin increases APD in rat [2, 3]and guinea pig ventricular fibres [6], and in dog Purkinje fibres [36]and decreases it in rabbit ventricular fibres [4, 5]. Lacking further data, it is not possible to establish whether the different DXR effects on APD are due to species-related differences in antioxidant capacity or in susceptibility of specific ionic channels to oxidative reactions.

Even though the intracellular antioxidant concentration is not responsible for the direction of the APD changes, it determines the wideness of such changes. In effect, it is well known that alterations in the intracellular concentration of antioxidant defense systems can modify tissue toxicity from drug-induced ROS. Thus, the idea is widely shared that differences in antioxidant capacity can explain why cardiac tissue develops significant pathologic changes after doxorubicin administration, whereas other tissues, such as the liver, are relatively resistant to damage, although the heart generates less free radicals [9]. It has been reported that the activity of antioxidant enzymes is higher in rat liver than in heart [11, 12], but comparative information on other defense systems is lacking. On the other hand, the measurement of the activities of the various scavenger systems does not allow the relative antioxidant status of the tissues to be determined. In a previous paper, using a enhanced luminescence technique we found that the order of antioxidant capacity was liver>blood>heart>muscle [15]. In contrast, the order of susceptibility to oxidative stress was blood>heart>muscle>liver. The discrepancies are due to differences in tissue content of substances, such as haemoproteins, able to catalyze the production of •OH radicals by hydrogen peroxide. We have used the above methods to determine antioxidant capacity and susceptibility to oxidative stress of heart, liver and blood, and have related them to peroxidative modifications induced by DXR administration to control, vitamin E- and NAC-treated rats. The results confirm the pattern previously found in untreated rats [15]and support the view that differential acute organ toxicity after doxorubicin is related to the antioxidant defenses in the tissues, even though liver ability to metabolize the drug can play a role.

Moreover, they also show that the DXR treatment significantly reduces antioxidant capacity in heart and blood which accounts for the increased susceptibility to oxidative stress of the above tissues. Thus, the DXR-induced increases in heart and blood lipid peroxidation agree with the high susceptibility to oxidative stress of such tissues in untreated animals. Interestingly, in DXR+VE-treated rats the MDA content is lower than in control animals. The idea that the vitamin E reduces both the baseline levels of lipid peroxidation and the DXR-induced increase is confirmed by the heart MDA levels found in rats treated with vitamin E alone.

Recent reports indicate that cardiac microsomes contain a similar mechanism as that inhibiting doxorubicin-dependent peroxidation in liver microsomes and requiring glutathione and both tocopherol and heat labile ‘free radical reductase’ activity in the membrane [37, 38]. The greater capacity of the heart microsomes to oppose peroxidative processes [38]is consistent with the higher tocopherol concentration found in the heart of control rats and only apparently disagrees with the higher increases of malondialdehyde shown in hearts of rats treated with doxorubicin. In fact, the effectiveness of vitamin E in reducing lipid oxidation in membranes is in part due to its regeneration by glutathione. Thus, severe oxidative challenge can reduce the poor antioxidant defenses of the heart to such low levels that the efficiency of the vitamin E system is impaired.

Evidence has been found that DXR-induced APD prolongation in guinea pig ventricular fibres is due to substantial suppression of delayed rectifier K+ current [6], but the mechanisms by which the membrane permeability is modified are not known. The protection selectively offered by vitamin E against membrane alterations suggests that electrophysiological changes observed in DXR-treated rats are induced through modifications of membrane components. Although the doxorubicin-initiated lipid peroxidation appears to be a contributing factor to the development of the cardiac toxicity [8], its involvement in the different cellular and subcellular effects of doxorubicin is doubtful. After cardiac preparation exposure to ROS-generating systems, both increased lipid peroxidation and altered action potential configuration [34]and membrane currents [39]have been found. However, our results show that antioxidant treatment reduces lipid peroxidation in both control and DXR-treated rats, but decreases APD only in DXR-treated rats. Moreover, mechanisms can play a critical role in free radical-induced APD alteration. Oxygen radical stress causes oxidation of membrane sulphydryl-containing proteins [40], which could lead to alterations in channel activity and changes in the membrane currents. Furthermore, other more indirect mechanisms may also be involved, since ion channels are sensitive to a variety of intracellular factors that might be influenced by oxidative stress. For example, the level of intracellular Ca2+, which affects non-selective cation conductance [41], is altered by oxidative stress [42]as well as DXR treatment [43]. On the other hand, recent evidence suggests that calcium accumulation may be a manifestation rather than a cause of anthracycline cardiomyopathy. In fact, free radical-induced membrane structural rearrangements, initiated as a result of both phospholipase activation and lipid peroxidation, which are inhibited by vitamin E, seem to induce Ca2+ overload [34, 44]. Moreover, the DXR-induced rise in intracellular Ca+2 is in part due to a sustained influx of the ion associated with APD prolongation [6]. Thus, although the present study indicates that free radical generation and membrane oxidative damage are responsible for DXR-induced electrophysiological alterations, underlying myocardial membrane changes remain to be understood.

Time for primary review 31 days


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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M. E. Buyukokuroglu, S. Taysi, M. Buyukavci, and E. Bakan
Prevention of acute adriamycin cardiotoxicity by dantrolene in rats
Human and Experimental Toxicology, May 1, 2004; 23(5): 251 - 256.
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