JNK pathway

Biochemical and Biophysical Research Communications 359 (2007) 759–764 www.elsevier.com/locate/ybbrc

Inhibition of Thioredoxin reductase induces apoptosis in neuronal cell lines: Role of glutathione and the MKK4/JNK pathway Jan Seyfried *, Ullrich Wu¨llner Department of Neurology, University of Bonn, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany Received 23 May 2007 Available online 4 June 2007

Abstract The Thioredoxin (Trx)/Thioredoxin reductase (TrxR)-system has emerged as a crucial component of many cellular functions particularly antioxidant defence. We investigated the effect of the selective TrxR inhibitor 1-chloro-2,4-dinitrobenzene (CDNB) on survival and redox status in neuronal cell lines. CDNB was found to cause apoptosis without depletion of glutathione or loss of mitochondrial complex I-activity. Cells treated with CDNB displayed an early increase of reactive oxygen species and rapid activation of stress inducible protein kinases c-Jun N-terminal kinase (JNK) and mitogen activated protein kinase kinase 4 (MKK4). Thus TrxR inhibition by CDNB results in generation of reactive oxygen species and subsequent activation of stress-inducible kinases without impairment of the cellular antioxidant status or mitochondrial function. Inhibition of the specific kinases involved in cell death triggered by Trx/TrxR dysfunction could represent a novel and selective therapeutic approach in neurodegenerative disorders. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Thioredoxin reductase; Glutathione; Oxidative stress; Neuronal cell lines; c-Jun N-terminal kinase pathway; Parkinson’s disease

Thioredoxin (Trx) is a ubiquitous 12 kDa Protein with multiple functions in mammalian cells. Secreted Trx acts as growth factor that stimulates proliferation of various cell types and tumour cell lines [1]. Trx also plays an important role in the antioxidant defence of cells [2]. All known functions of Trx involve formation of an intramolecular disulfide bond. The reduced species Trx(SH)2 is a powerful protein disulfide reductase. Oxidized TrxS2 is regenerated by Thioredoxin reductase (TrxR) in a NADPH-dependent reaction. Glutathione (GSH) is the most abundant cellular thiol and antioxidant. A deficiency in glutathione, particularly in mitochondria has been implicated in neurodegenerative conditions [3]. Because of its high reactivity the TrxR/ Trx system occupies a key position among the various antioxidant small molecules and enzyme systems.

* Corresponding author. Address: University of Manchester, School of Biological Sciences, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK. Fax: +44 (0) 161 275 5600. E-mail address: [email protected] (J. Seyfried).

0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.05.176

The importance of the Trx/TrxR-system for cellular oxidative balance is highlighted by the existence of mitochondrial isoforms of both proteins (Trx2 and TrxR2) [4,5]. Mitochondria are the main source of oxidative stress within a respiring cell. Silencing of Trx2 gene expression in mice results in embryonic lethality at the stage of mitochondrial maturation in development [6]. Repression of Trx2 expression in cultured cells causes overproduction of ROS and apoptosis [7]. Studies of Trx mRNA and protein expression detected high Trx levels in brain areas with high energy demand [8] and high levels of Trx2 in dopaminergic neurons of the substantia nigra. Dopaminergic neurons are highly vulnerable to oxidative stress because animals treated systemically with non-toxic doses of mitochondrial toxins develop experimental Parkinsonism [9]. Decreased levels of thioredoxin have been found in brain tissue of Alzheimer’s disease patients and thioredoxin reductase protects against amyloid b-peptide toxicity in cultured neurons [10]. The notion that Trx functions as a redox sensor was supported by the discovery that TrxSH2 is an inhibitory binding protein of apoptosis signal-

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regulating kinase 1 (ASK1) [11]. When oxidised, Trx dissociates from ASK1 which then activates its downstream kinases MKK4 and JNK/p38. 1-Chloro-2,4-dinitrobenzene (CDNB) is an electrophilic compound that reacts with thiol-groups by alkylation. CDNB depletes GSH in cultured cells and is used as a substrate in enzyme assays of glutathione-S-transferases. Arner et al. [12] reported that CDNB selectively inhibits TrxR at concentrations much lower than necessary for derivatization of other thiols. Because only reduced TrxR is inhibited by CDNB it is thought that CDNB alkylates a highly reactive thiol-group of the enzyme. The CDNBinactivated TrxR enzyme does not lose its NADPH oxidase activity, which is increased. Therefore inactivation of TrxR could at the same time compromise cellular antioxidant defence and increase endogenous ROS levels. The aim of the present study was to investigate cellular glutathione status and the role of mitochondria in cell death induced by TrxR inhibition in neuronal cells. Materials and methods Cell culture. PC12 cells (European collection of cell cultures or RIKEN cell bank, Japan) were maintained in RPMI 1640 medium supplemented with 5% heat inactivated foetal calf serum (FCS), 10% heat inactivated horse serum, 100 U/ml penicillin and 100 lg/ml streptomycin in a constant 5% CO2 atmosphere at 37 °C. Rat mesencephalic dopaminergic CSM 14.1 cells and rat C 6 glioma cells (American type cell culture collection, ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% heat inactivated FCS, 100 U/ml penicillin and 100 lg/ml streptomycin in 5% CO2 atmosphere at 33 °C (CSM 14.1 cells) and 37 °C (C 6 cells), respectively. Viability assay. Cellular viability was measured by the reduction of the water-soluble dye Alamar Blue (BioSOURCE, Fleurus, Belgium) after incubation at 37 °C for 2 h according to the instructions of the manufacturer. Fluorescence was monitored on a Spectramax Gemini plate fluorescence reader (Molecular Devices). Untreated cells were used for background readings. Enzyme activity assays. TrxR activity was assayed as described [13] measured as the Thioredoxin-dependent formation of free thiols in insulin. Treated cells were collected by trypsination, lysed by repeated freezing/ thawing and cleared by centrifugation. Protein content of the supernatant was determined with the Bradford reagent (Bio-Rad) using bovine serum albumin (BSA) as standard. Activity of mitochondrial complex I was assayed as described [14]. Glutathione assay. Glutathione was assayed by the enzymatic recycling assay according to Tietze [15] modified for use in microtiter plates. Briefly, cells were washed in cold PBS and lysed in 1% sulfosalicylic acid (SSA). Total glutathione (GSX) content was determined after addition of 5,5 0 dithiobis-2-nitrobenzoacid (DTNB, 150 lM), NADPH (200 lM), and glutathione reductase by monitoring absorbance 405 nm over 10 min. To determine oxidized glutathione (GSSG) levels, reduced glutathione (GSH) was derivatized with 2.5% 2-vinylpyridine for 60 min prior to addition of DTNB. Reduced glutathione (GSH) was calculated according to GSX = GSH + 2*GSSG. Protein content of the acid insoluble pellet was determined using the Lowry method [16] using BSA as standard. Western blotting. Cells or cellular fractions were lysed in buffer (120 mM NaCl, 5 mM EDTA, 0.5% Nonidet P 40, 50 mM Tris–HCl, pH 8, PMSF 100 lg/ml, aprotinine 2 lg/ml, leupeptin 10 lg/ml). Solubilised proteins were heat (95 °C) denatured in Laemmli buffer, separated by 12% SDS–PAGE and electroblotted on nitrocellulose membranes. Incubation with primary antibody was carried out overnight at 4 °C in PBS/5% nonfat dry milk, followed by three washes in PBS/0.5% Tween-20. For detection peroxidase-coupled anti-mouse IgG (Amersham) was used at

1:1000–1:5000 dilution (1 h at room temperature) and signals were visualized using the enhanced ECL detection kit (Amersham, Braunschweig, Germany). Subcellular fractionation. For subcellular localization of TrxR, fractions of cell homogenates were prepared by a combination of differential centrifugation and percoll gradient centrifugation steps as described [17]. Electron microscopy. Cells were fixed with 0.2 M sodiumcacodylate buffer (pH 7.2) containing 2.5% glutaraldehyde and 0.8% paraformaldehyde, osmificated and embedded into epon by standard procedures. Ultrathin sections were contrasted with uranyl acetate and lead citrate and studied with an EM 900 (Zeiss). Measurement of reactive oxygen species. PC 12 cells were grown in 10 cm dishes until 80% confluence. After treatment, medium was changed to serum-free medium containing 10 lM 5-(and-6)-chloromethyl-carboxy2 0 7 0 -dichlorodihydrofluoresceindiacetate acetylester (Molecular Probes) and cells were loaded for 2 h. Cells were analyzed on a FACScan (Becton– Dickinson, Heidelberg, Germany) equipped with a 15 mW, 488 nm aircooled argon-ion laser at a 600 nm wavelength emission filter. Data acquisition and analysis was performed using the Cell Quest (BectonDickinson) software. In vitro kinase activity assays. Cells were lysed in Triton lysis buffer (TLB) [18]. Protein content was determined with the Bradford reagent (Bio-Rad) using BSA as standard. Cellular lysates (300 lg total protein) were incubated (2 h at 4 °C) with 5 lg GST-c-jun fusion protein (prepared as described [18] and 30 ll of GSH-agarose beads (50% slurry, Pharmacia) for assay of JNK or with 4 ll of anti-MKK4 antibody and 30 ll of Protein A-sepharose (Sigma) for assay of MKK4. The beads/sepharose pellets were washed twice with TLB and three times with kinase buffer (KB) [18] and resuspended in 15 ll KB, 1 ll ATP (1 mM), 0.1 lCi ATc32P and 1 lg GST-JNK (prepared as described, [18]) in case of MKK4 assay, and incubated for 30 min at 30 °C. Samples were separated by SDS–PAGE (12%, 1.5 mm) and gels were stained with Comassie brilliant blue, dried and exposed to autoradiography film (Kodak Biomax) at 80 °C for 4– 24 h. Phosphorylation of substrate protein bands was quantified by PhosphoImager (Fuji FLA 3000) analysis. Statistics. Values are presented as means ± standard deviation (SD) or standard error of the mean (SEM) of at least three independent experiments. Statistical evaluation of raw data from several groups was performed using one-way analysis of variance (ANOVA) followed (if significant: p < 0.05) by Turkey’s post hoc test. Materials. Cell culture media, sera and antibiotics were obtained from Invitrogen (Heidelberg, Germany). The following antibodies were used: Upstate # 07-078 (Biomol, Hamburg, Germany) for the detection of TrxR1 and Upstate # 07-079 (Biomol, Hamburg, Germany) for the detection of TrxR2. For kinase activity assay of MKK4 antibody #964 from Santa Cruz (Santa Cruz, USA) was used. All chemicals were of analytical grade and purchased from Sigma (Deisenhofen, Germany) or Merck (Darmstadt, Germany).

Results Expression and subcellular localization of thioredoxin reductase in PC 12 cells To confirm expression of thioredoxin reductase in PC 12 cells, western blots of fractionated cell lysates were performed with antibodies directed against the two TrxR-isoforms (Fig. 1). The antibody specific for cytosolic TrxR1 detected a single band of expected size in the cytosolic fraction of PC 12 cells and a weaker band in the fraction containing nuclei and cell fragments. TrxR2 was detected in the mitochondrial fraction of PC 12. Consistent with the manufacturer’s information, the antibody directed against TrxR2 crossreacts with the larger cytosolic isoform.

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Fig. 1. (A) Western blot of fractionated homogenates of PC 12 cells. 10 lg protein per lane was separated, electroblotted and probed with antibodies for TrxR1 and TrxR2, respectively. Abbreviation of fractions: N, nuclear and cell debris; C, cytosolic; E, endosomal; M, mitochondrial. (B) Measurement of Thioredoxin Reductase activity in PC 12 cells. Cells were grown to 80–90% confluency, treated as indicated and TrxR activity assayed as described in Materials and methods. Data are presented as means ± SEM, n = 3, ***p < 0.001 vs. vehicle treated controls.

10 μM

Measurement of TrxR activity in PC 12 cells treated with micromolar concentrations of CDNB confirmed that TrxR is inhibited in a concentration-dependent manner (Fig. 1). Treatment with ethacrynic acid (EA) did not result in inhibition of TrxR. Toxicity of CDNB and ethacrynic acid The effect of CDNB on viability of PC 12 cells was examined after incubation with different concentrations for 24 h. As a comparison we used ethacrynic acid (EA), an electrophile that is conjugated to glutathione by cellular glutathione-S-tranferases [19]. In PC 12 cells (Fig. 2) and in CSM14 and C6 cells (Supplementary Fig. 1) CDNB was markedly more toxic (EC 50 in PC12 cells: 3.9 lM, in CSM 14 cells: 5.3 lM) than EA (EC 50 in PC12 cells: 244 lM, in CSM 14 cells: 232 lM). In PC 12 cells no difference in toxicity of CDNB was seen in either native cells or cultures differentiated for two weeks (data not shown). PC 12 cells treated with 10 lM CDNB showed distinct apoptotic morphology (Fig. 2 and Supplementary Fig. 3) with chromatin condensation, cell shrinkage and vacuolization whereas 1 lM CDNB elicited early stages of apoptotic morphology. Glutathione levels under CDNB treatment To examine the relevance of cellular glutathione in CDNB-induced cell death, glutathione levels were mea-

Fig. 2. (A) Viability of PC 12 cells treated for 24 h. Cells were seeded in 96-well plates at 100,000/ml, CDNB or EA was added after 24 h and cultures were stained with Alamar blue after further 24 h. Data are presented as means ± SD, n = 10. (B) Electron microscopy of PC 12 cells treated 24 h with 1 or 10 lM CDNB. Cells were grown until 50% confluency when CDNB for 24 h and processed for ultrastrucutral examination as described in Materials and methods. The bar represents 1 lm.

sured glutathione in PC 12 cells treated with CDNB and EA (Fig. 3). Low concentrations of ethacrynic acid (
J. Seyfried, U. Wu¨llner / Biochemical and Biophysical Research Communications 359 (2007) 759–764

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Fig. 4. JNK- and MKK4-kinase activity in CDNB-treated PC 12 cells. Cells were grown until approximately 50% confluence and treated with CDNB or vehicle (veh) for 8 h (A). Cells were treated with 10 lM CDNB for the indicated time periods (B). At the end of each treatment period cells were processed as described in Materials and methods.

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Fig. 3. (A) Glutathione levels of PC 12 cells after 24 h treatment with CDNB, EA, and BSO. Cells were grown until 50% of confluence and treated as indicated. Cells were harvested and glutathione was assayed as described in Materials and methods. Absolute reduced glutathione (GSH) content measured in untreated cells was: 42.8 ± 2.2 nmol/mg. Data are presented as means ± SEM, n = 3, ***p < 0.001 vs. vehicle treated controls. (B) Measurement of reactive oxygen species by flow cytometry of CM-H2DCFDA-stained PC 12 cells as described in Materials and methods. Cells were incubated with for 2 h with the indicated concentrations. 20,000 cells per condition were analyzed. Data are presented as percentage of cells displaying high FL1-fluorescence intensity (>10e3 arbitrary units). Data are presented as means ± SD, n = 3, ***p < 0.01 vs. vehicle treated controls.

sulfoximine (BSO), an inhibitor of the rate limiting enzyme of glutathione synthesis, c-glutamylcysteinyl-synthetase was used as a positive control. The same pattern was found in CSM 14 and C 6 cells: CDNB depleted glutathione only at concentrations higher than those necessary for induction of cell death (Supplementary Fig. 2). Co-treatment of PC 12 cells with sublethal (
Generation of reactive oxygen species by CDNB treatment Levels of oxidative stress in CDNB treated cells were examined by fluorescence measurement of the oxidation of a dichlorodihydrofluorescein-derivate. In PC 12 cells, 1 lM CDNB decreased the proportion of fluorescent cells, whereas 5 and 10 lM CDNB elicited a massive increase after only 2 h of treatment (Fig. 3). Ethacrynic acid was used as a positive control and generated a massive increase

of reactive oxygen species at 200 lM as described previously [20]. Activation of stress regulated kinases by CDNB To test whether CDNB-treatment activates the ASK/ MKK4/JNK-pathway of stress induced kinases, we performed kinase activity assay. Activation of JNK and its upstream kinase MKK4 in PC 12 cells was detected after 8 h treatment with 10 lM but not 1 lM CNDB (Fig. 4). Analysis of the time course showed that JNK- and MKK4-activity started to increase after 45 min of treatment with 10 lM CDNB (Fig. 4). These results confirm that activation of this stress induced kinase module is an early event in apoptosis caused by TrxR-inhibition. Discussion This study examines the role of cellular redox homeostasis in neuronal cell death induced by CDNB. Glutathione is the main cellular antioxidant and the redox status of many cellular proteins is thought to be maintained by glutathione. In three neuronal cell lines CDNB was markedly more toxic than the glutathione-depleting agent ethacrynic acid. Toxic EC 50 concentrations of CDNB were at least an order of magnitude lower than those for EA. The morphology of cell death induced by CDNB was confirmed to be apoptotic, in accordance with previous studies [21]. At non-toxic concentrations CDNB caused an increase of cellular glutathione. This is most likely due to electrophile/antioxidant response elements regulating the expression of c-glutamyl cysteinyl-synthetase (cGCS) [22]. Even at concentrations higher than the toxic EC 50, CDNB failed to deplete cellular glutathione. We conclude that apoptosis caused by inhibition of thioredoxin reductase occurs without perturbation of intracellular redox balance. The additional challenge of co-treatment with the

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cGCS-inhibitor BSO acerbated CDNB-induced cell death, though only when concentrations lower than EC 50 of CDNB were used. Oxidative stress can trigger apoptosis via oxidation of Trx, which then dissociates from and thereby activates ASK-1 [11]. A significant increase of ROS was measured after 2 h treatment of PC 12 cells with CDNB thus preceding the onset of cell death. The most likely source of this early increase in cellular ROS is the increased NADPH-oxidase activity of CDNB-inhibited TrxR [12]. Alternatively, inhibition of TrxR2 could cause damage to mitochondrial key proteins and increased leakage of electrons from the mitochondrial respiratory chain and generation of ROS. Deficiency of complex I is an early event of stress-induced mitochondrial degeneration in vitro and in vivo [23]. We found complex I activity was not decreased in CDNB-treated cells prior to the onset of cell death (31.3 ± 3.3 nmol NADH/min/mg after 24 h at 5 lM CDNB, untreated cells: 27.0 ± 1.6 nmol NADH/min/mg). This result argues against a mitochondrial origin of the reactive oxygen species detected in early stages of CDNBtreatment. A three tier-cascade comprising the protein kinases ASK1, SEK1/MKK4 (stress activated protein kinase 1/mitogen activated kinase kinase 4) and JNK (c-jun N-terminal protein kinase) induces cell death in response to oxidative stress. MKK4 and JNK are indeed activated by CDNB-treatment of PC 12 cells and the activation of these kinases is not accompanied by depletion of cellular glutathione. Inhibition of TrxR appears to be sufficient for activation of the ASK1 pathway. From the fact that toxic concentrations of CDNB inhibit thioredoxin reductase in agreement with previous studies [12] we conclude that inhibition of TrxR is a crucial step in CDNB-toxicity. The comparison with EA demonstrates that this toxicity is not caused by unspecific electrophilic reactions of CDNB. The concept that Trx/TrxR play a crucial role for survival of neuronal cells has been supported by the expression pattern of Trx in the brain [8], decreased levels of Trx activity in post mortem brain tissue of patients suffering from Alzheimer’s disease [10] and the protection of transgenic mice overexpressing Trx in models of ischemia [24]. An acquired or genetic dysfunction of Trx or TrxR could predispose of neurons for degeneration. Deficiency in antioxidants in general and glutathione in particular has been implicated in the pathogenesis of neurodegenerative diseases. Activation of the ASK1/MKK4/JNK cascade of stress induced kinases has been found in models of neuronal degeneration [25]. The evidence presented here suggests that the activation of the ASK1 stress kinase module following inhibition of TrxR in neuronal cells is triggered by an early increase of oxidative stress. Cell death is initiated without changes in the overall cellular redox balance and without early oxidative damage at the level of mitochondria. Pallis et al. [26] draw similar conclusions from experiments in a leukaemia cell line. This model provides an explanation why dietary supplementation with

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antioxidants failed to ameliorate the course of Parkinson’s disease [27] despite widespread evidence that oxidative stress occurs in early stages of Parkinson’s disease. Acknowledgments We are greatly indebted to Dr. C. Tournier (University of Manchester) for providing reagents for the kinase- and TrxR-activity assays. We thank Dr I. Boehm (Department of Dermatology, University of Bonn, Germany) for generous help with the flow cytometry analysis. We are indebted to K. Tolksdorf (Department of Neurology, University of Bonn) for carrying out the electron microscopy. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2007. 05.176. References [1] G. Powis, D. Mustacich, A. Coon, The role of the redox protein thioredoxin in cell growth and cancer, Free Radic. Biol. Med. 29 (2000) 312–322. [2] A. Mitsui, J. Hamuro, H. Nakamura, N. Kondo, Y. Hirabayashi, S. Ishizaki-Koizumi, T. Hirakawa, T. Inoue, J. Yodoi, Overexpression of human thioredoxin in transgenic mice controls oxidative stress and life span, Antioxid. Redox. Signal 4 (2002) 693–696. [3] J.B. Schulz, J. Lindenau, J. Seyfried, J. Dichgans, Glutathione, oxidative stress and neurodegeneration, Eur. J. Biochem. 267 (2000) 4904–4911. [4] G. Spyrou, E. Enmark, A. Miranda-Vizuete, J. Gustafsson, Cloning and expression of a novel mammalian thioredoxin, J. Biol. Chem. 272 (1997) 2936–2941. [5] A. Miranda-Vizuete, A.E. Damdimopoulos, J.R. Pedrajas, J.A. Gustafsson, G. Spyrou, Human mitochondrial thioredoxin reductase cDNA cloning, expression and genomic organization, Eur. J. Biochem. 261 (1999) 405–412. [6] L. Nonn, R.R. Williams, R.P. Erickson, G. Powis, The absence of mitochondrial thioredoxin 2 causes massive apoptosis, exencephaly, and early embryonic lethality in homozygous mice, Mol. Cell Biol. 23 (2003) 916–922. [7] T. Tanaka, F. Hosoi, Y. Yamaguchi-Iwai, H. Nakamura, H. Masutani, S. Ueda, A. Nishiyama, S. Takeda, H. Wada, G. Spyrou, J. Yodoi, Thioredoxin-2 (TRX-2) is an essential gene regulating mitochondria-dependent apoptosis, EMBO J. 21 (2002) 1695–1703. [8] A. Lippoldt, C.A. Padilla, H. Gerst, B. Andbjer, E. Richter, A. Holmgren, K. Fuxe, Localization of thioredoxin in the rat brain and functional implications, J. Neurosci. 15 (1995) 6747–6756. [9] R. Betarbet, T.B. Sherer, G. MacKenzie, M. Garcia-Osuna, A.V. Panov, J.T. Greenamyre, Chronic systemic pesticide exposure reproduces features of Parkinson’s disease, Nat. Neurosci. 3 (2000) 1301–1306. [10] M.A. Lovell, C. Xie, S.P. Gabbita, W.R. Markesbery, Decreased thioredoxin and increased thioredoxin reductase levels in Alzheimer’s disease brain, Free Radic. Biol. Med. 28 (2000) 418–427. [11] M. Saitoh, H. Nishitoh, M. Fujii, K. Takeda, K. Tobiume, Y. Sawada, M. Kawabata, K. Miyazono, H. Ichijo, Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1, EMBO J. 17 (1998) 2596–2606. [12] E.S. Arner, M. Bjornstedt, A. Holmgren, 1-Chloro-2,4-dinitrobenzene is an irreversible inhibitor of human thioredoxin reductase. Loss of thioredoxin disulfide reductase activity is accompanied by a large

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