Peroxynitrite treatment reduces adenosine uptake via the equilibrative nucleoside transporter in rat astrocytes

Neuroscience Letters 498 (2011) 52–56

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Peroxynitrite treatment reduces adenosine uptake via the equilibrative nucleoside transporter in rat astrocytes Akiko Tanaka, Kentaro Nishida, Hiroto Okuda, Takeshi Nishiura, Youichirou Higashi 1 , Sadaki Fujimoto, Kazuki Nagasawa ∗ Department of Environmental Biochemistry, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan

a r t i c l e

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Article history: Received 23 March 2011 Received in revised form 11 April 2011 Accepted 23 April 2011 Keywords: Adenosine uptake Astrocyte Nucleoside transporter Oxidative stress SIN-1

a b s t r a c t In the oxidative stress-loaded brain, extracellular adenosine levels are elevated and thereby neuronal damage is attenuated, but mechanisms underlying alteration of the extracellular kinetics of adenosine remain unclear. Here we investigated whether oxidative stress might alter functional expression of nucleoside transporters (NTs), a predominant regulatory system for nucleoside kinetics, in cultured rat astrocytes. Treatment of astrocytes with 0.5 mM SIN-1 for 3 h caused apparent cellular accumulation of nitrotyrosine, but had no effect on their viability, indicating load of oxidative stress to astrocytes without any change in their viability. Under the condition, [3 H]adenosine uptake was significantly less than that by control cells. This decreased uptake was due to decrease in adenosine uptake mediated by an equilibrative NT (ENT) 1 which was inhibited by low concentrations (≤0.1 ␮M) of nitrobenzylthioinosine (NBMPR), but not by sodium-dependent or high concentrations (≥1 ␮M) of NBMPR-inhibitable nucleoside transporters. The expression level of ENT1 was not altered, while the Michaelis constant, but not the maximum rate, of adenosine uptake was increased. These findings suggest that under oxidative stressloaded conditions, decreased adenosine clearance via astrocytic ENT1 might involve, at least in part, in an elevated extracellular adenosine level in the brain. © 2011 Elsevier Ireland Ltd. All rights reserved.

Endogenous nucleosides are important modulators of cell survival and activity in the brain. Adenosine has significant neuroprotective effect against ischemia/hypoxia, glutamate toxicity, etc., mainly by stimulating adenosine A1 and A3 receptors [12]. Furthermore, adenosine is involved in the coupling of cellular metabolism to nucleotide fueling, and adenine nucleotides are also used as energy to maintain their electrical and synaptic activity in neurons [9]. On the other hand, neurons and astrocytes communicate to each other by releasing nucleotides, cytokines, etc., especially ATP [27,29,32]. Since the brain lacks a pathway for the de novo biosynthesis of nucleotides, the supply of nucleosides to neurons and astrocytes is physiologically essential to maintain their functions. Under oxidative stress-loaded conditions such as ischemia– reperfusion, an excess amount of ATP is released from astrocytes, activated microglia and leaks from dead cells [16], and thus the extracellular ATP concentration is elevated to a level, which leads to neuronal death via activation of purine receptors such as P2X7 ones [7]. Frenguelli et al. found the increased lev-

∗ Corresponding author. Tel.: +81 75 595 4648; fax: +81 75 595 4756. E-mail address: [email protected] (K. Nagasawa). 1 Present address: Department of Neurosurgery, Kochi Medical School Kochi University, Nankoku, Kochi 783-8505, Japan. 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.04.060

els of adenosine as a metabolite of ATP in brain slices loaded with oxidative stress [11]. However, whether or not the adenosine clearance alters under oxidative stress-loaded conditions are unrevealed yet. As for regulation of in the intra- and extra-cellular adenosine levels in brain neuronal cells, nucleoside transporters (NTs), which comprise isoforms, play central roles [1,5,8,13,25,31]. We previously reported that equilibrative NTs (ENTs), ENT1 (Slc29a1) and ENT2 (Slc29a2), are major molecules that clear extracellular adenosine, and astrocytes are more prominent as to this clearance than neurons [18,20]. In a study of Kobayashi et al. [15], exposure of cultured neuronal PC12 cell to oxidative stress decreased expression of ENT1 mRNA, resulting in an increase in the extracellular adenosine level. In cultured cardiomyocyte HL-1 cells, load of oxidative stress to them had no effect on ENT1 mRNA expression, while the expression of phosphorylated protein kinase C␧ (pPKC␧), by which ENT1 activity is regulated, was decreased by the treatment and thus adenosine transport activity via ENT1 was impaired [6]. These findings clearly imply that oxidative stress induces alteration of the functional expression of ENT1. In this study, we examined the characteristics of adenosine transport and functional expression of ENTs in oxidative stressloaded cultured rat astrocytes. As an oxidative stressor, we used SIN-1 (3-morpholinosydnonimine), which generates peroxynitrite. Under ischemia–reperfusion conditions, peroxynitrite is known

A. Tanaka et al. / Neuroscience Letters 498 (2011) 52–56

to be released from activated microglia and deteriorate neuronal death [7]. Adenosine was purchased from Wako Pure Chemical Ind. (Osaka, Japan), and nitrobenzylthioinosine (NBMPR) and uridine were from Sigma Chemical Co. (St. Louis, MO, USA). [3 H]Adenosine ([8-3 H]adenosine, 40 Ci/mmol) was obtained from American Radiolabeled Chemicals Inc. (St. Louis, MO, USA). All other reagents were of commercial or analytical grade requiring no further purification. NBMPR is a representative inhibitor of Na+ -independent ENTs, and the sensitivity of ENT1 and ENT2 to NBMPR is well-known to be different, the former being blocked by 0.1 or less ␮M of it, but the latter by only 1 or more ␮M [1,33]. Primary astrocyte cultures were prepared from the cortices of 1to 2-day-old newborn Wistar rats (Japan SLC, Hamamatsu, Japan), as described previously [18,20]. The experiments were approved by the Experimental Animal Research Committee of Kyoto Pharmaceutical University and were performed according to the Guidelines for Animal Experimentation of Kyoto Pharmaceutical University. Briefly, the cortices were isolated and dissociated in papain/DNase mixture, prior to plating on cell culture plates or dishes in Eagle’s minimal essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS) and 2 mM l-glutamine. At confluence (days 14–16), cultures were treated with 20 ␮M cytosine arabinoside for 48 h to prevent microglial proliferation, followed by replacement with fresh EMEM supplemented with 3% FBS and 2 mM l-glutamine. Astrocyte cultures were used at days 20–40 in vitro. The cultures comprised >95% astrocytes and <5% microglia, as confirmed by immunocytochemistry for GFAP (glial fibrillary acidic protein) and Iba-1 [20]. SIN-1 treatment was performed using a balanced salt solution (BSS; 3.1 mM KCl, 134 mM NaCl, 1.2 mM CaCl2 , 1.2 mM MgSO4 , 0.25 mM KH2 PO4 , 15.7 mM NaHCO3 and 2 mM glucose, pH 7.2) as the incubation buffer in a 5% CO2 incubator. After the culture medium had been replaced with BSS, astrocytes were incubated with the designated concentrations of SIN-1 for 3 h, and then washed twice with an appropriate buffer and used for experiments. In the cell viability assay, astrocytes were incubated in BSS with SIN-1 for 0, 3, 6, 12 or 24 h, and then they were subjected to the viability assay described below. Astrocyte death was quantified by measuring intracellular lactate dehydrogenase (LDH) activity as described previously [19]. The uptake experiments involving [3 H]adenosine were performed by the inhibitor stop method described previously [20], using HEPES-HBSS (136.9 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl2 , 0.4 mM MgSO4 , 0.5 mM MgCl2 , 0.3 mM Na2 HPO4 , 0.4 mM KH2 PO4 , 4.2 mM NaHCO3 , 5.56 mM d-glucose and 25 mM HEPES, pH 7.2) as the transport buffer, and in the case of Na+ -free conditions, cholinereplaced HEPES-HBSS, in which NaCl, Na2 HPO4 and NaHCO3 were replaced by choline chloride, K2 HPO4 and KHCO3 , respectively, was used. The reason for the use of HEPES-HBSS as the transport buffer, but not BSS, was to maintain the buffer pH during the experimental period, and we confirmed that there was no difference in the timedependent uptake of [3 H]adenosine between HEPES-HBSS (used in a water bath in room air) and BSS (used in a 5% CO2 incubator). Protein concentrations were measured by the method of Bradford [3] using Coomassie brilliant blue reagent (nacalai tesque, Kyoto, Japan) with bovine serum albumin (Sigma) as the standard. Immunostaining was performed as reported previously [19]. The following primary antibodies were used: mouse antinitrotyrosine antibodies (1:1000; Alfa Diagnostic, San Antonio, TX, USA), rabbit anti-SLC29A1 (ENT1) antibodies (1:100; Proteintech, Chicago, IL, USA), and mouse anti-GFAP antibodies (1:500; Chemicon, Billerica, MA, USA). Alexa Fluor 594- or Alexa Fluor 488labelled IgG (1:1000; Molecular Probes, Eugene, OR, USA) were used for visualization with a confocal laser microscope (LSM510META; Carl Ziess, Jena, Germany).

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Expression of ENT1, PKC␧, pPKC␧ and ␤-actin was evaluated by western blotting as described previously [19,21] with the following antibodies: rabbit anti-SLC29A1 antibodies (1:1000), mouse antiPKC␧ antibodies (1:1000; BD Biosciences, San Jose, CA, USA), goat anti-pPKC␧ antibodies (Ser729) (1:3000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, # sc-12355), or mouse anti-␤-actin antibodies (1:10,000; Sigma). An anti-rabbit, anti-mouse or anti-goat IgG horseradish peroxidase (HPR)-conjugated antibody was used as a secondary antibody at 1:10,000 dilution, and the signals of ENT1, PKC␧, pPKC␧ and ␤-actin were detected using Western Lightning Chemiluminescence Reagent (Perkin Elmer Life Sciences, Boston, MA, USA). The optical density of each protein band was measured with the NIH Image J software and normalized as to the corresponding ␤-actin one. The data are expressed as means ± SE. Comparisons between two or more groups were performed by means of analysis of variance (ANOVA, followed by Fischer’s PLSD) or a paired t-test, differences with a p-value of 0.05 or less being considered statistically significant. First, we examined the effect of SIN-1 treatment under various conditions on astrocyte viability. Treatment of astrocytes with SIN1 at the concentrations of 0.1 and 0.5 mM, but not 1 mM, for 3 h did not have any effect on their viability, while further extension of the treatment period up to 24 h resulted in an apparent decrease in the cell viability (Fig. 1A). Next, whether or not astrocytes were subjected to oxidative stress by exposure to SIN-1 was investigated. Apparent immunoreactivity to nitrotyrosine, a marker of oxidative stress, was detected in astrocytes treated with 0.5 mM SIN-1 for 3 h (Fig. 1B), and astrocytes under the condition also exhibited a representative reactive form such as sharpened astrocytic foot processes (data not shown). Thus, astrocytes treated with 0.5 mM SIN-1 for 3 h were subjected to oxidative stress without any change in cell viability, and we performed transport experiments just after this treatment. Fig. 2 shows the time courses of adenosine uptake by astrocytes treated with or without SIN-1. Although the uptake of [3 H]adenosine time-dependently increased up to 15 min, the uptake by SIN-1-treated astrocytes was significantly less than that by the control ones (Fig. 2A). The uptake of [3 H]adenosine in the presence of extracellular Na+ was comparable to that in its absence. Under the both conditions, the [3 H]adenosine uptake decreased with the SIN-1 treatment, and the decreased levels were almost the same for both conditions (Fig. 2B), implying no involvement of a Na+ -dependent concentrative NTs (CNTs) in the decreased uptake of adenosine by SIN-1-treated astrocytes. In the control cells, [3 H]adenosine uptake was decreased by the addition of 0.01 and 0.1 ␮M of NBMPR, and the great inhibition was seen in the range of 100 ␮M of it. In the case of SIN-1-treated astrocytes, 0.01 and 0.1 ␮M NBMPR had no effect on the uptake of [3 H]adenosine, while 100 ␮M of it significantly decreased the uptake, suggesting the ENT1, but not ENT2, was affected by the SIN-1 treatment (Fig. 2C). Expression of ENT1 was evaluated in the control and SIN-1treated astrocytes. As shown in Fig. 3, there was no apparent difference in the expression amount (Fig. 3A and B) and profile (Fig. 3C) of ENT1 between the two groups. In addition, we could not detect any alteration in the expression levels of PKC␧ and pPKC␧ in the SIN-1-treated astrocytes (Fig. 3A and B). For further characterization, we examined the concentrationdependence of [3 H]adenosine uptake. The [3 H]adenosine uptake showed saturation at concentrations over 150 ␮M in the control and SIN-1-treated groups, but the uptake in the latter was apparently less than that in the former (Fig. 4A). Comparing the Akaike’s information criteria obtained by analysis of an EadieHofstee plot (Fig. 4B), these plots in the both control and SIN-1

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Fig. 1. Effects of SIN-1 treatment on cell viability (A) and nitrotyrosine accumulation (B) in cultured rat astrocytes. (A) Cells were incubated in BSS with 0 mM (closed circles), 0.1 mM (open circles), 0.5 mM (open triangles) or 1 mM (open squares) SIN-1 for 0, 3, 6, 12 or 24 h at 37 ◦ C. After extensive washing, cell viability was assessed by means of the LDH assay. Each point represents the mean ± SE (N = 3). *p < 0.05, significantly different from control. (B) After cells had been incubated in BSS with 0.5 mM SIN-1 for 3 h at 37 ◦ C, nitrotyrosine accumulation was assessed by immunocytochemical analysis. Representative photomicrographs are shown. Bar = 20 ␮m.

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impaired clearance of extracellular adenosine via ENT1 is considered to explain, at least in part, the increase in the extracellular adenosine level under oxidative stress-loaded conditions. The administration of adenosine to oxidative stress-loaded astrocytes was reported to have cellular protective effect via activation of adenosine A1 and A3 receptors [2]. Pascual et al. reported that ENT1 was involved in the regulation of synaptic transmission [24], and Rose et al. indicated that ENT1 is a key molecule for the cardioprotective effect of adenosine [26]. Since we have demonstrated that astrocytes are prominent cells in adenosine clearance from extracellular space [18,20], it is suggested that ENT1 expressed by astrocytes may play an important role in the rescue of neuronal cells from oxidative stress. With the SIN-1 treatment, the affinity of adenosine to a transporter, probably ENT1, was decreased, while there was no change in the uptake capacity (Vmax ), resulting in decrease in the uptake clearance (Vmax /Km ). Almost the same expression level and pattern of ENT1 in the control and SIN-1 groups reasonably explains the

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groups were judged to be well fitted by two components, saturable and non-saturable ones. The Michaelis constant (Km ) for adenosine uptake via the saturable component by SIN-1treated astrocytes (37.7 ± 8.38 ␮M) was found to be significantly (p < 0.05) greater than that by untreated ones (12.1 ± 1.06 ␮M), while there was no change in the maximum uptake rate (Vmax ) (5.81 ± 2.18 and 4.13 ± 0.278 nmol/mg protein per 5 min, respectively), resulting in decrease in the uptake clearance, i.e., the Vmax /Km value (0.156 ± 0.0390 and 0.343 ± 0.0130 mL/mg protein per 5 min, respectively). In this study, we found that oxidative stress caused by the SIN-1 treatment decreased the ENT1-mediated uptake of adenosine due to decrease in its affinity to the transporter. In addition, we confirmed that an adenosine deaminase inhibitor, erythro-9(2-hydroxy-3-nonyl) adenine (EHNA) [14,23], had no effect on adenosine uptake by astrocytes under control and SIN-1-treated conditions (data not shown). Thus, in addition to increased release of adenosine as a metabolite of ATP and NAD+ [4,10,22], the

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Fig. 2. Characteristics of [3 H]adenosine uptake by cultured rat astrocytes. (A) Cells were incubated in BSS with (open circles) or without (closed circles) 0.5 mM SIN-1 for 3 h at 37 ◦ C. After extensive washing and preincubation of the cells in fresh HEPES-HBSS for 10 min, they were incubated with 2.5 nM (0.03 ␮Ci/well) [3 H]adenosine for 2, 5, 10 or 15 min at 37 ◦ C. Each point represents the mean ± SE (N = 3). *p < 0.05, significantly different from the value for the control group. (B) Cells were incubated in BSS with (triangles) or without (circles) 0.5 mM SIN-1 for 3 h at 37 ◦ C. After extensive washing and preincubation in fresh HEPES-HBSS (closed symbols) or choline-replaced HEPES-HBSS (opened symbols) for 10 min, they were incubated with 5 nM (0.06 ␮Ci/well) [3 H]adenosine for 2, 5, 10 or 15 min at 37 ◦ C. Each point represents the mean ± SE (N = 4). (C) Cells were incubated in BSS with (open bar) or without (closed bar) 0.5 mM SIN-1 for 3 h at 37 ◦ C. After extensive washing and preincubation in fresh HEPES-HBSS for 10 min, they were incubated with 5 nM (0.06 ␮Ci/well) [3 H]adenosine for 5 min in the presence or absence of the indicated concentrations of NBMPR at 37 ◦ C. Each bar represents the mean ± SE (N = 6). *,# p < 0.05, significantly different from the value for the 0 ␮M NBMPR group without and with SIN-1, respectively. †,§ p < 0.001, significantly different from the value for the 0.1 ␮M NBMPR group without and with SIN-1, respectively.

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Fig. 3. Expression of ENT1, PKC␧ and pPKC␧ by SIN-1-treated cultured rat astrocytes. After cells had been incubated in BSS with 0.5 mM SIN-1 for 3 h at 37 ◦ C, cell and protein samples were obtained, and subjected to western blot (A and B) or immunocytochemical (C) analyses. (B) Quantification results for each protein shown in panel (A). Each bar represents the mean ± SE (N = 3). Representative photomicrographs are shown in panel (C) (N = 3).

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Fig. 4. Concentration-dependence of [3 H]adenosine uptake by SIN-1-treated cultured rat astrocytes. Cells were incubated in BSS with (open circles) or without (closed circles) 0.5 mM SIN-1 for 3 h at 37 ◦ C. After extensive washing and preincubation in fresh HEPES-HBSS for 10 min, they were incubated with the indicated concentrations (0.5–200 ␮M) of [3 H]adenosine for 5 min at 37 ◦ C. (A and B) Uptake-concentration profile and the Eadie–Hofstee plot, respectively. Each point represents the mean ± SE (N = 3).

finding of the lack of a change in the uptake capacity. A postulated mechanism underlying the decrease in the affinity of adenosine is a structural change, modification of the transporter protein and so on. Peroxynitrite inhibits ion pumps such as Ca2+ pump and Na+ /K+ -ATPase by their nitration and inactivation, and then induces myocardial dysfunction [17,28,30]. We detected increase in tyrosine nitration on SIN-1 treatment of astrocytes, and so there is a possibility that ENT1 might be nitrated by peroxynitrite, resulting

in its dysfunction, but more detail investigation is needed to clarify this. In summary, we demonstrated that adenosine uptake via ENT1 expressed by astrocytes was impaired by oxidative stress induced by SIN-1, which results from decrease in function, but not expression, of ENT1. This might explain, at least in part, elevation of the brain extracellular adenosine level under oxidative stress-loaded conditions.

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Acknowledgements A part of this study was financially supported by a Grant-in-Aid for Scientific Research (C) (21590107) from the Ministry of Education, Science and Culture of Japan, and ‘Academic Frontier’ Project for Private Universities from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government. References [1] S.A. Baldwin, S.Y. Yao, R.J. Hyde, A.M. Ng, S. Foppolo, K. Barnes, M.W. Ritzel, C.E. Cass, J.D. Young, Functional characterization of novel human and mouse equilibrative nucleoside transporters (hENT3 and mENT3) located in intracellular membranes, J. Biol. Chem. 280 (2005) 15880–15887. [2] O. Bjorklund, M. Shang, I. Tonazzini, E. Dare, B.B. Fredholm, Adenosine A1 and A3 receptors protect astrocytes from hypoxic damage, Eur. J. Pharmacol. 596 (2008) 6–13. [3] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [4] S. Bruzzone, L. Guida, E. Zocchi, L. Franco, A. De Flora, Connexin 43 hemi channels mediate Ca2+ -regulated transmembrane NAD+ fluxes in intact cells, FASEB J. 15 (2001) 10–12. [5] C.E. Cass, J.D. Young, S.A. Baldwin, Recent advances in the molecular biology of nucleoside transporters of mammalian cells, Biochem. Cell Biol. 76 (1998) 761–770. [6] N. Chaudary, Z. Naydenova, I. Shuralyova, I.R. Coe, Hypoxia regulates the adenosine transporter, mENT1, in the murine cardiomyocyte cell line, HL-1, Cardiovasc. Res. 61 (2004) 780–788. [7] H.B. Choi, J.K. Ryu, S.U. Kim, J.G. McLarnon, Modulation of the purinergic P2X7 receptor attenuates lipopolysaccharide-mediated microglial activation and neuronal damage in inflamed brain, J. Neurosci. 27 (2007) 4957–4968. [8] C.R. Crawford, D.H. Patel, C. Naeve, J.A. Belt, Cloning of the human equilibrative, nitrobenzylmercaptopurine riboside (NBMPR)-insensitive nucleoside transporter ei by functional expression in a transport-deficient cell line, J. Biol. Chem. 273 (1998) 5288–5293. [9] J.W. Deitmer, Strategies for metabolic exchange between glial cells and neurons, Respir. Physiol. 129 (2001) 71–81. [10] T.V. Dunwiddie, L. Diao, W.R. Proctor, Adenine nucleotides undergo rapid, quantitative conversion to adenosine in the extracellular space in rat hippocampus, J. Neurosci. 17 (1997) 7673–7682. [11] B.G. Frenguelli, E. Llaudet, N. Dale, High-resolution real-time recording with microelectrode biosensors reveals novel aspects of adenosine release during hypoxia in rat hippocampal slices, J. Neurochem. 86 (2003) 1506–1515. [12] C.V. Gomes, M.P. Kaster, A.R. Tome, P.M. Agostinho, R.A. Cunha, Adenosine receptors and brain diseases: neuroprotection and neurodegeneration, Biochim. Biophys. Acta 808 (2011) 1380–1399. [13] M. Griffiths, S.Y. Yao, F. Abidi, S.E. Phillips, C.E. Cass, J.D. Young, S.A. Baldwin, Molecular cloning and characterization of a nitrobenzylthioinosine-insensitive (ei) equilibrative nucleoside transporter from human placenta, Biochem. J. 328 (Pt 3) (1997) 739–743. [14] A.J. Hirsh, J.R. Stonebraker, C.A. van Heusden, E.R. Lazarowski, R.C. Boucher, M. Picher, Adenosine deaminase 1 and concentrative nucleoside transporters 2 and 3 regulate adenosine on the apical surface of human airway epithelia: implications for inflammatory lung diseases, Biochemistry 46 (2007) 10373–10383. [15] S. Kobayashi, H. Zimmermann, D.E. Millhorn, Chronic hypoxia enhances adenosine release in rat PC12 cells by altering adenosine metabolism and membrane transport, J. Neurochem. 74 (2000) 621–632.

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