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(Circulation. 2001;103:290.)
© 2001 American Heart Association, Inc.

Basic Science Reports

Morphine Mimics Preconditioning via Free Radical Signals and Mitochondrial K_ATP Channels in Myocytes

Bradley C. McPherson, BA; Zhenhai Yao, MD, PhD

From the Department of Anesthesia and Critical Care, the University of Chicago, Chicago, Ill.

	Abstract

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Background—We tried to determine whether morphine mimics preconditioning (PC) to reduce cell death in cultured cardiomyocytes and whether opioid ₁ receptors, free radicals, and K_ATP channels mediate this effect.

Methods and Results—Chick embryonic ventricular myocytes were studied in a flow-through chamber while flow rate, pH, and O₂ and CO₂ tension were controlled. Cardiomyocyte viability was quantified with propidium iodide (5 µmol/L), and production of free radicals was measured with 2',7'-dichlorofluorescin diacetate. PC with 10 minutes of simulated ischemia before 10 minutes of reoxygenation or morphine (1 µmol/L) or BW373U86 (10 pmol/L) infusion for 10 minutes followed by a 10-minute drug-free period before 1 hour of ischemia and 3 hours of reoxygenation reduced cell death to the same extent (*P<0.05) (PC, 20±1%, n=7*; morphine, 32±4%, n=8*; BW373U86, 21±6%; controls, 52±5%, n=8). Like PC, morphine and BW373U86 increased free radical production 2-fold before ischemia (0.35±0.10, n=6*; 0.41±0.08, n=4* versus controls, 0.15±0.05, n=8, arbitrary units). Protection and increased free radical signals during morphine infusion were abolished with either the thiol reductant 2-mercaptopropionyl glycine (400 µmol/L), an antioxidant; naloxone (10 µmol/L), a nonselective morphine receptor antagonist; BNTX (0.1 µmol/L), a selective opioid ₁ receptor antagonist; or 5-hydroxydecanoate (100 µmol/L), a selective mitochondrial K_ATP channel antagonist.

Conclusions—These results suggest that direct stimulation of cardiocyte opioid ₁ receptors leads to activation of mitochondrial K_ATP channels. The resultant increase of intracellular free radical signals may be an important component of the signaling pathways by which morphine mimics preconditioning in cardiomyocytes.

Key Words: free radicals • ion channels • receptors • preconditioning

	Introduction

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Stimulation of endogenous opioid receptors has been shown to increase survival in mice after hypoxia.^{1 2} Activation of opioid receptors in anesthetized rats mimicked ischemic preconditioning to reduce myocardial infarct size.³ In rabbits, the nonselective opioid receptor antagonist naloxone abolished the protective effect of preconditioning.⁴ Whether opioid receptor agonists reduce the size of a myocardial infarct via a direct effect on cardiomyocytes or via indirect systemic effects is not clear. A recent study provided the first evidence that the opioid morphine can precondition cardiomyocytes directly.⁵ We tried to determine whether morphine mimics ischemic preconditioning to reduce cell death in isolated cardiomyocytes and to discover the intracellular signaling pathways involved in preconditioning.

Assuming that free radicals participate in initiation of hypoxic preconditioning in cardiocytes,^{6 7 8 9} we also tried to determine the role of free radical signals in morphine-induced preconditioning and the importance of opioid receptors in producing intracellular free radical signals. It has been suggested that K_ATP channel activation mediates morphine-induced preconditioning.⁵ Whether activation of mitochondrial K_ATP channels results in an increase of free radical signals or vice versa is not clear. We tested the hypothesis that stimulation of opioid receptors, which results in opening of mitochondrial K_ATP channels, also increases free radical signals during morphine-induced preconditioning in cultured cardiomyocytes. For this purpose, we used the opioid receptor antagonist naloxone and the mitochondrial selective K_ATP channel blocker 5-hydroxydecanoate (5-HD).^{5 9 10 11}

	Methods

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Cardiomyocyte Preparation
Ten-day-old embryonic chick ventricular myocytes were prepared according to a method described by Barry et al¹² and modified by Vanden Hoek et al.⁸ Briefly, hearts were harvested and placed in Hanks’ balanced salt solution lacking magnesium and calcium (Life Technologies, Inc). Ventricles were minced and myocytes were dissociated by use of 4 to 6 repeats of trypsin (0.025%, Life Technologies, Inc) degradation at 37°C with gentle agitation. Then isolated cells were transferred to a solution with trypsin inhibitor for 8 minutes, filtered through a 100-µm mesh, centrifuged for 5 minutes at 1200 rpm at 4°C, and finally resuspended in a nutritive medium described previously by Chandel et al¹³ and Duranteau et al.⁷ Resuspended cells were placed in a Petri dish in a humidified incubator (5% CO₂, 95% air at 37°C) for 45 minutes to promote early adherence of fibroblasts. Nonadherent cells were counted with a hemocytometer, and viability was measured with trypan blue (0.4%). Approximately 1x10⁶ cells in nutritive medium were pipetted onto coverslips (25 mm) and incubated for 3 to 4 days, after which synchronous contractions of the monolayer were noted. Experiments were performed on spontaneously contracting cells at day 3 or 4 after isolation.

Perfusion System
Glass coverslips containing spontaneously beating chick myocytes were placed in a stainless steel flow-through chamber (1 mL volume, Penn Century Co). Thin gaskets were used to minimize O₂ exchange between the parts of the chamber. The chamber was mounted on a temperature-controlled platform (37°C) on an inverted microscope. A water-jacketed glass equilibration column mounted above the microscope stage was used to equilibrate the perfusate to known O₂ tensions (PO₂: 140 mm Hg for normoxic and 3 mm Hg for simulated ischemic solution). The standard perfusion medium consisted of a buffered salt solution (BSS, in mmol/L: NaCl 117, KCl 4.0, NaHCO₃ 18, MgSO₄ 0.8, NaH₂PO₄ 1.0, CaCl₂ 1.21, and glucose 5.6), which was equilibrated for 1 hour before the experiment by bubbling with a gas mixture of 21% oxygen, 5% carbon dioxide, and 74% nitrogen. A simulated ischemia solution composed of BSS containing no glucose with 2-deoxyglucose (20 mmol/L) added to inhibit glycolysis was bubbled with a gas mixture of 20% carbon dioxide and 80% nitrogen for 1 hour before the experiments. Chick cardiomyocytes have a high glycolytic capability. Our previous results and those of others revealed that the combined insults were essential to produce adequate ischemia and reperfusion injury in the control series.^{7 8 9 14} Stainless steel or polymer tubing with low O₂ solubility connected the equilibration column to the flow-through chamber to minimize ambient O₂ transfer into the perfusate. In previous studies, the low PO₂ in the chamber was confirmed under conditions identical to those in experiments that used an optical phosphorescence quenching method (Oxyspot, Medical Systems Inc).^{15 16}

Determination of Cell Viability
An inverted microscope equipped for epifluorescent illumination included a xenon light source (75 W), a 12-bit digital cooled CCD camera (Princeton Instruments), a shutter and filter wheel (Sutter), and appropriate excitation and emission filter cubes. The microscope also was equipped with Hoffman-modified phase illumination to accentuate surface topology, facilitating the measurement of contractile motion (see below). Fluorescent cell images were obtained with an x10 objective (Nikon Plan Fluor). There were 600 cardiomyocytes under the selected field for each experiment. Multiple fields were examined and compared before each study; the field with normal synchronous contraction was chosen and monitored throughout experiments. Data were acquired and analyzed with Metamorph software (Universal Imaging). Cell viability was quantified with the nuclear stain propidium iodide (PI, 5 µmol/L, Molecular Probes), an exclusion fluorescent dye that binds to chromatin on loss of membrane integrity.¹⁷ PI is not toxic to cells over the course of 8 hours, permitting its addition to the perfusate throughout the experiments. At the completion of each experiment, digitonin (300 µmol/L) was added to the perfusate for 1 hour. Digitonin disrupts the membrane integrity of all cells, allowing PI to enter and maximum PI value to be obtained. Percent loss of viability (cell death) was then expressed relative to the maximum value after 1 hour of digitonin exposure (100%).

Measurement of Free Radicals
Free radical generation in cells was assessed with the probe 2',7'-dichlorofluorescin (DCFH). The membrane-permeable diacetate form of DCFH, DCFH-DA, was added to the perfusate at a final concentration of 5 µmol/L. Within the cell, esterases cleave the acetate groups on DCFH-DA, thus trapping DCFH intracellularly.¹⁸ Free radicals in the cells lead to oxidation of DCFH, yielding the fluorescent product DCF.¹⁹ DCFH in cardiomyocytes is readily oxidized by H₂O₂ or hydroxyl radical but is relatively insensitive to superoxide.^{7 8 14} Change in DCF fluorescence intensity is a useful marker of free radical generation in cells.^{7 9 14 15 16 20 21} Fluorescence was measured with an excitation wavelength of 480 nm, dichroic 505-nm long pass, and emitter bandpass of 535 nm (Chroma Technology) with neutral density filters to attenuate the excitation light intensity. Fluorescence intensity was assessed in clusters of several cells identified as regions of interest. The background was identified as an area without cells or with minimal cellular fluorescence. Intensity values are reported as the percentage of initial values after subtraction of the background value.

Because DCFH is much less sensitive to superoxide, the production of superoxide was measured with dihydroethidium (DHE).

Chemicals
Morphine sulfate was purchased from Elkins Sinn, Inc. 2-Mercaptopropionyl (2-MPG), BW373U86, and 5-hydroxydecanoate (5-HD) were purchased from Sigma Chemical Co. Naloxone was purchased from Research Biochemical International. Morphine sulfate, 2-MPG, naloxone, or 5-HD was dissolved in BSS buffer before administration. PI, DHE, and DCFH-DA were purchased from Molecular Probes.

Experimental Design
Twelve groups of cardiomyocytes (control, preconditioning, morphine, BW373U86, naloxone, naloxone+morphine, 7-benzylidenenaltrexone [BNTX, 0.1 µmol/L], BNTX+morphine, 2-MPG, 2-MPG+morphine, 5-HD, and morphine+5-HD) were studied. Cells in the nonpreconditioned groups were subjected to 60 minutes of ischemia before 3 hours of reoxygenation. Preconditioned cells underwent the same protocol preceded by 10 minutes of ischemia and 10 minutes of reoxygenation. Instead of preconditioning, saline (control series) or morphine (1 µmol/L) was added to the perfusate for 10 minutes in nonpreconditioned cells. The other cells were treated with naloxone (10 µmol/L), 2-MPG (400 µmol/L), or 5-HD (100 µmol/L) in perfusate during the 1-hour period of baseline before 60 minutes of ischemia.

In 11 additional series of studies (treatment with saline, morphine, BW373U86, 2-MPG, 2-MPG+morphine, naloxone, naloxone+ morphine, BNTX, BNTX+morphine, 5-HD, and 5-HD+morphine), we examined the role of opioid receptors and mitochondrial K_ATP channels in regulating free radical signals in mediating the beneficial effect of morphine.

The doses of various antagonists were chosen on the basis of preliminary studies⁹ that showed that these drugs alone had no significant effects on baseline free radical generation compared with controls. Antagonists used in this study were infused during the first 60-minute period before the prolonged simulated ischemic period.

Statistical Analysis
Data are expressed as mean±SEM. Differences between groups for cell death and free radical production were compared by a 2-factor ANOVA and Fisher’s least significant difference test. Differences between groups were considered significant at values of P<0.05.

	Results

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Cell Death
The percentage of cell death (% PI uptake) was monitored intermittently throughout each experiment, and the data are summarized in Figure 1. After 3 hours of reoxygenation, cell death averaged 52.3±5.0% (n=8) in the control series. Cell death was markedly reduced by preconditioning (18.9±1.4%, n=7, P<0.05) or morphine (31.2±3.5%, n=8, P<0.05). Pretreatment with either naloxone, 2-MPG, or 5-HD abolished the protective effects of morphine (48.0±7.3%, n=6; 42.7±6.2%, n=6; 45.3±6.6%, n=6). Infusion of naloxone, 2-MPG, or 5-HD alone did not increase cell death compared with ischemic controls (44.3±8.3%, n=4; 49.2±5.5%, n=4; 54.9±5.7%, n=4).

View larger version (34K): [in this window] [in a new window]   Figure 1. Effects of ischemic preconditioning (PRE) and morphine on cell death after ischemia-reperfusion as assessed by PI uptake. Cardiocytes equilibrated at normoxic conditions for 40 minutes (baseline) were subjected to 10 minutes of ischemia (PRE) or of morphine (1 µmol/L) infusion, followed by 10 minutes of normoxia or a drug-free period, respectively. Control cells were equilibrated at normoxic conditions for 60 minutes (CONT). All cells were subjected to 60 minutes of ischemia and 3 hours of reoxygenation. PRE and morphine (Mor) significantly reduced cell death compared with nonpreconditioned controls. Treatment for 60 minutes with either naloxone (10 µmol/L) (Nalo+Mor), 2-MPG (400 µmol/L) (2-MPG+Mor), or 5-HD (100 µmol/L) (5-HD+Mor) before 60 minutes of ischemia completely abolished protective effects of morphine. *P<0.05.

The protection afforded by morphine was abolished by pretreatment with BNTX (0.1 µmol/L), a selective opioid ₁ receptor antagonist that had no effects on cell death. Reduction of cell death by administration of the selective opioid ₁ receptor agonist BW373U86 (10 pmol/L) was similar to that with morphine or preconditioning (Figure 2).

View larger version (23K): [in this window] [in a new window]   Figure 2. Effects of opioid 1 receptors on morphine-reduced cell death after ischemia-reperfusion as assessed by PI uptake. Cardiocytes equilibrated at normoxic conditions for 40 minutes (baseline) were subjected to 10 minutes of morphine (1 µmol/L) or BW373U86 (10 pmol/L) infusion, followed by 10 minutes of a drug-free period. Control cells were equilibrated at normoxic conditions for 60 minutes (CONT). All cells were subjected to 60 minutes of ischemia and 3 hours of reoxygenation. Morphine (Mor) or BW373U86 significantly reduced cell death compared with controls. Treatment for 60 minutes with BNTX (0.1 µmol/L) before 60 minutes of ischemia abolished protective effects of morphine. *P<0.05.

Intracellular Free Radical Measurement
Figure 3 documents 1 typical experiment from each series. In the control cells, the intensity of the DCF fluorescence increased slightly over 1 hour. Infusion of morphine (1 µmol/L) for 10 minutes followed by 10 minutes of a drug-free period markedly increased production of reactive oxygen species. The increase in DCF fluorescence during morphine infusion was abolished by pretreatment with 2-MPG (Figure 3A), naloxone (Figure 3B), or 5-HD (Figure 3C), which had no effects on baseline free radical production.

View larger version (24K): [in this window] [in a new window]   Figure 3. Representative experiment from all series. In control cells, intensity of DCF fluorescence increased slightly over 1 hour (CONT). Infusion of 1 µmol/L of morphine for 10 minutes followed by 10 minutes of a drug-free period increased DCF fluorescence markedly (Mor). Increase of fluorescence intensity was abolished with pretreatment of naloxone (Nalo+Mor), 2-MPG (2-MPG+Mor), or 5-HD (5-HD+Mor).

Figure 4 summarizes results of the peak DCF fluorescence during 10 minutes of morphine infusion and the following 10 minutes of a drug-free period. Treatment with morphine increased intensity of DCF fluorescence 35% above baseline (35±10%, n=6, P<0.05); the increase was only 15% in controls (15±5%, n=8). DCF fluorescence induced with morphine was completely abolished by pretreatment with naloxone (11±4%, n=5), 2-MPG (14±5%, n=6), or 5-HD (10±6%, n=6) compared with that in controls (15±5%, n=8). Finally, treatment with naloxone, 2-MPG, or 5-HD alone for 1 hour did not affect DCF fluorescence signals.

View larger version (11K): [in this window] [in a new window]   Figure 4. Peak free radical production was assessed by increased intensity of DCF fluorescence during 10 minutes of morphine infusion. Morphine markedly increased free radical burst (35% above baseline). In controls, free radicals were only 15% higher than at baseline over 1 hour. Morphine-induced free radical burst was completely abolished by pretreatment with naloxone (Nalo+Mor), 2-MPG (2-MPG+Mor), or 5-HD (5-HD+Mor). *P<0.05.

In addition, we observed that BW373U86, a selective opioid ₁ receptor agonist, markedly increased DCF fluorescence intensity and that the increase of DCF fluorescence intensity with morphine was abolished by pretreatment with BNTX, a selective antagonist for opioid ₁ receptors (Figure 5). Finally, morphine and BW373U86 had no effects on DHE oxidation (Eth-DNA fluorescence intensity) either in the presence or in the absence of the superoxide dismutase inhibitor DDC (Figure 5B).

View larger version (14K): [in this window] [in a new window]   Figure 5. A, Peak free radical production was assessed by increased intensity of DCF fluorescence during 10 minutes of drug infusion. Morphine (1 µmol/L) or opioid 1 receptor agonist BW373U86 (10 pmol/L) markedly increased free radical burst (35% and 41% above baseline, respectively). In controls, free radicals were only 15% higher than at baseline over 1 hour. Morphine-induced free radical burst was abolished by pretreatment with BNTX (BNTX+Mor). BNTX (0.1 µmol/L) had no effect on free radical production by itself. B, Peak value of superoxide production assessed by increased intensity of Eth-DNA fluorescence (DHE oxidation) during 10 minutes of drug infusion. Morphine and BW373U86 had no effects on DHE oxidation in either absence or presence of superoxide dismutase inhibitor DDC. *P<0.05.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Morphine reduced cell death as much as did preconditioning in embryonic chick cardiomyocytes. The cardioprotective effect of morphine correlates with its ability to increase intracellular free radical signals before the start of ischemia. Our results suggest that morphine mimics preconditioning via direct stimulation of opioid ₁ receptors and activation of mitochondrial K_ATP channels and increase in free radical signals.

Ischemic preconditioning markedly reduced cell death in an in vitro model of myocyte ischemia and reperfusion.^{5 9 10 14 22} Numerous efforts have been made to identify and develop an agent capable of mimicking the powerful endogenous myocardial protection of ischemic preconditioning.²³ Morphine mimicked ischemic preconditioning in anesthetized rats and rabbits,^{3 4} probably via an opioid receptor–related mechanism.³ However, whether morphine protects via direct effects on myocardial opioid receptors or by affecting the opioid receptors of other systems is less clear. Our results and those of others reveal that morphine protects against ischemia-reperfusion injury in cardiomyocytes. Functional opioid receptors exist in ventricular myocytes.^{5 21} Using isolated cultured myocytes, we found that morphine or BW373U86, a selective ₁ opioid receptor agonist,^{24 25} was able to mimic ischemic preconditioning. Liang and Gross found that morphine preconditioned cardiomyocytes via direct stimulation of opioid receptors.⁵ Morphine-induced preconditioning in cardiomyocytes was abolished by either the nonselective opioid receptor antagonist naloxone or the selective ₁ antagonist BNTX.²⁶ The results of others using the same cardiocyte preparation showed that the protection of morphine was blocked by naloxone or BNTX.⁵ Taken together, cardioprotection with morphine appears to be opioid ₁ receptor–mediated. The subtypes of opioid receptors involved have not been established, although morphine has a high affinity for the µ-opioid receptor.^{5 21}

The mechanism by which direct stimulation of cardiomyocyte opioid receptors mimics preconditioning is unknown. In our study, as with preconditioning, free radical production increased 2-fold. This effect correlated with a reduction in cell death. Both effects were abolished with the antioxidant 2-MPG. It has been suggested that biological oxidants regulate intracellular signal transduction.^{6 27} Free radicals are intracellular signaling components in hypoxia-, ischemia-, and acetylcholine-mediated preconditioning in cardiocytes.^{6 7 9} In addition, we found that the increased free radical signals were abolished by naloxone or BNTX, a selective opioid ₁ receptor antagonist. Furthermore, stimulation of opioid ₁ receptors with BW373U86 markedly increased oxygen free radical production. Our results and those of others^{5 6 21 27} indicate that an increase in free radical signals is opioid ₁ receptor–mediated and plays an important role in the protective effects of morphine.

Because DCFH is not sensitive to superoxide, which may be associated with early programmed cell death (apoptosis),²⁸ we used an additional molecular probe, DHE, to determine the effects of morphine and opioid ₁ receptor stimulation on superoxide production. Morphine or BW373U86 had no effects on DHE oxidation, an index of superoxide generation, in the absence or presence of the superoxide dismutase inhibitor DDC. These findings suggest that morphine-induced oxygen radicals are mainly hydroxyl peroxide, unlikely to be superoxide.

The increase in free radical signals with morphine was abolished by 5-HD, which suggests that activation of mitochondrial K_ATP channels increases free radical signals. Activation of mitochondrial K_ATP channels was important in the acetylcholine-induced increase of free radical signals in isolated cardiomyocytes.⁹ Although the mechanism by which stimulation of opioid receptors increases free radical signals is not fully understood, the present data indicate that K_ATP channel activation is 1 important intermediate step after opioid receptor stimulation. We believe that stimulation of opioid receptors increases intracellular free radical signals by opening mitochondrial K_ATP channels. This pathway is important in morphine-produced preconditioning.

Free radical signals can activate potassium channels.²⁹ It has been shown that K_ATP channel activation mediates the cardioprotection of morphine and that the benefit of morphine is abolished by 5-HD or glibenclamide.⁵ We also found that the protection provided by morphine was abolished by 5-HD. Mitochondrial K_ATP channel activation contributes to the increased signal of free radicals. An increase in free radical signals further amplifies activation of the channel.

Free radicals with morphine also activate protein kinase C,³⁰ which is a component of the intracellular signaling pathway in opioid-mediated preconditioning in intact rabbit hearts.³¹ Activation of this enzyme is important in opioid-mediated preconditioning in cultured rat ventricular myocytes²¹ and in the chick embryonic myocytes used for our study.¹⁰ Activation of protein kinase C increases the activity of K_ATP channels in ventricular myocytes.³⁰ K_ATP channel activation also mediates the cardioprotection provided by morphine.^{3 5}

Morphine and opioid receptor stimulation activate nitric oxide synthase and release nitric oxide.^{32 33} Nitric oxide has been proposed as 1 pathway of preconditioning.^{20 34} Because DCFH oxidation is sensitive to hydroxyl peroxide and less sensitive to nitric oxide and superoxide, the present results suggest that the increased oxygen radicals are hydroxyl peroxide species. It is unlikely that the free radical signals produced by morphine and the ₁ opioid receptor agonist BW373U86 are peroxynitrate or nitric oxide. Obviously, more experiments are needed to confirm this possibility.

We recognize the limitations of the preparation used in our study. Our previous results and those of others have demonstrated that chick cardiocytes require the combined insults of anoxia, no glucose, and glycolytic inhibition to produce adequate ischemia-reperfusion injury in a control series, which may be caused by a higher endogenous glycolytic capability in this species.^{7 8 9 14} The ischemia-reperfusion models of other species do not require such severe insult. Like mammalian cardiocytes, however, chick cardiocytes have opioid receptors, adenosine receptors, K_ATP channels, protein kinase C, and reactive oxygen species to protect against ischemia-reperfusion injury.^{3 4 5 7 9 10 14 35} Nevertheless, application of our findings in this model to the mammalian system may not be warranted. Finally, naloxone did not block ischemic preconditioning in this preparation. Ischemic preconditioning may initiate several parallel intracellular signal transduction pathways, including stimulation of adenosine receptors, muscarinic receptors, nitric oxide synthases, protein kinase C, K_ATP channels, etc. Blockade of opioid receptors, although sufficient to abolish the morphine protection, was insufficient to block preconditioning.

In conclusion, our study demonstrates that morphine mimics ischemic preconditioning to reduce cell death in cardiomyocytes. Opioid ₁ receptor stimulation increases intracellular free radical signals through activation of mitochondrial K_ATP channels, which are important intracellular signaling components of morphine-induced preconditioning.

	Acknowledgments

 
This study was supported by National Heart, Lung, and Blood Institute USPHS grant HL-03881-02.

	Footnotes

 
Reprint requests to Zhenhai Yao, MD, PhD, Department of Anesthesia and Critical Care, The University of Chicago, 5841 S Maryland Ave, MC 4028, Chicago, IL 60637.

Received June 2, 2000; revision received July 18, 2000; accepted July 22, 2000.

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