Vulnerability to glucose deprivation injury correlates with glutathione levels in astrocytes

Brain Research 748 Ž1997. 151–156

Research report

Vulnerability to glucose deprivation injury correlates with glutathione levels in astrocytes Marios C. Papadopoulos a , Iphigenia L. Koumenis a , Laura L. Dugan b , Rona G. Giffard a

a, )

Department of Anesthesia, Room S-272, Stanford UniÕersity School of Medicine, Stanford UniÕersity Medical Center, Stanford, CA 94305-5117, USA b Departments of Neurology and Medicine, Washington UniÕersity, St. Louis, MO 63110, USA Accepted 22 October 1996

Abstract Astrocyte death from glucose deprivation appears to be mediated by free radicals. Reduced glutathione ŽGSH. was used as a measure of antioxidant defenses in primary cultures of cortical astrocytes. Glucose deprivation caused progressive, near complete loss of reduced glutathione ŽGSH.. Astrocytes were protected by increasing endogenous GSH levels. Depletion of GSH to 21.4 " 3.3% of controls by the glutathione synthetase inhibitor buthionine sulfoximine resulted in more rapid injury by glucose deprivation, yet depletion of glutathione alone did not kill astrocytes. Both enhanced lipid peroxidation and membrane rigidification were caused by glucose deprivation, both indicators of oxidative damage. Membrane peroxidation was detected as a 24 " 2% decrease in cis-parinaric acid fluorescence, membrane rigidification as a 6.3 " 0.8% increase in fluorescence anisotropy using diphenylhexatriene. Glucose deprivation under normoxic conditions may occur clinically in patients such as diabetics. In addition, oxidative damage in the setting of energy depletion occurs with other insults, including ischemic brain injury. Glucose deprivation may thus be a clinically relevant model of hypoglycemic astrocyte injury, and may be useful to investigate the effects of glutathione and redox modulation on second messenger systems and gene regulation. Keywords: Free radical; Glutathione; Anisotropy; Mouse; Astrocyte; Lipid peroxidation; Starvation; Primary culture

1. Introduction Hypoglycemia is a clinical problem which can result in brain damage w1x. During hypoglycemia, neurons may die from an extracellular overflow of excitatory amino acids w1,21x, but the mechanisms by which hypoglycemia leads to glial cell injury are not completely understood. To determine whether there is a direct link between glucose deprivation ŽGD. and oxidative stress, we investigated changes in glutathione ŽGSH. levels, membrane fluidity and lipid peroxidation in primary cultures of astrocytes deprived of glucose. The tripeptide GSH plays a critical role in combating

Abbreviations: BSO, L-buthionine-Ž S,R .-sulfoximine; BSS 5.5 , balanced salt solution containing 5.5 mM glucose; BSS 0 , balanced salt solution lacking glucose; cis-PnA, cis-parinaric acid; DMTU, 1,3-dimethyl-2-thiourea; GD, glucose deprivation; GM, growth medium; GSH, reduced glutathione; LDH, lactate dehydrogenase; PBS, phosphatebuffered saline ) Corresponding author. Fax: q1 Ž415. 725-8052; e-mail: [email protected]

oxidative stress. Inhibition of GSH synthesis in rats is known to cause a striking enlargement of mitochondria in the brain w12x and severe brain damage accompanies inherited deficiencies of GSH synthesis in man w19x. GSH acts directly as a free radical scavenger and is important in recycling other antioxidants w18,26,27x. The maintenance of GSH levels is an energy requiring process. Suzuki and Kurata w31x showed that GSH regeneration in erythrocytes depended on the level of ATP. Energy-protein malnutrition is also associated with GSH depletion w4x. Loss of GSH may allow the oxidative species normally produced by mitochondria to damage cellular constituents. Oxidative species may react with polyunsaturated fatty acids in membranes to form lipid peroxides, which in turn lead to rigidification of membranes by crosslinking w6x. Rigidification of cell membranes has been reported after exposing myocardial w2x and erythrocyte membranes w33x to oxidative stress. Fluorescence anisotropy is a reliable and sensitive technique to measure membrane fluidity w3x, so it was used here to look for evidence of oxidative membrane changes. To confirm that membrane rigidification is a result of

0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 1 2 9 3 - 0

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lipid peroxidation, we used the probe cis-parinaric acid Žcis-PnA.. Cis-PnA contains four conjugated double bonds, which render it naturally fluorescent and which are readily attacked in lipid peroxidation reactions. Cis-PnA incorporates into membranes and its loss of fluorescence over time has been used to monitor lipid peroxidation in live cells w11,32x. Loss of cis-PnA fluorescence correlates well with the generation of thiobarbituric acid reactive substances w11x. Astrocytes are a central component of the brain’s antioxidant defense. Histochemical w28x and biochemical w24x studies show localization of GSH in glia with little GSH present in neurons. Astrocytes also export GSH to the brain extracellular fluid which may be crucial in protecting neurons from oxidative injury w34x. Primary astrocyte cultures are useful experimental models because they possess many of the morphological, immunohistochemical and electrophysiological properties of astrocytes in situ w14x, and were therefore used for these studies.

2. Materials and methods 2.1. Materials Endotoxin-free water, glutamine and MEM Ža330-1430. were obtained from Gibco ŽGrand Island, NY.. Fetal bovine serum and horse serum came from Hyclone Laboratories ŽLogan, UT.. Monochlorobimane and diphenyl-hexatriene were from Molecular Probes ŽEugene, OR.; bicinchoninic acid protein determination reagents came from Pierce ŽRockford, IL.; epidermal growth factor, Hoechst dye 33258 and all other chemicals for tissue culture were purchased from Sigma ŽSt. Louis, MO.; Falcon plasticware came from Becton Dickinson ŽLincoln, NJ.. Mice were from Simonsen ŽGilroy, CA.. 2.2. Methods 2.2.1. Astrocyte cultures Nearly pure astrocyte cultures were prepared from postnatal Žday 1–3. Swiss Webster mice as previously described w7x. All procedures were carried out according to a protocol approved by the Stanford University Animal Care and Use Committee, and were in keeping with the NIH Guide. Briefly, dissociated neocortical cells were plated in Falcon Primaria 24-well plates at a density of 1–2 hemispheres per multiwell, in Eagle’s Minimal Essential Medium supplemented with 10% equine serum, 10% fetal bovine serum, 21 mM Žfinal concentration. glucose and 10 ngrml epidermal growth factor. The cultures were maintained in a 378C humidified incubator with a 5% CO 2 in room air atmosphere. Once confluent, further cell replication was inhibited with 10 m M cytosine arabinoside. Astrocyte cultures were fed weekly with growth medium ŽGM., which is identical to the plating medium but lacks

fetal serum and epidermal growth factor. Mature cultures contained astrocytes by GFAP staining Ž) 98%., no detectable oligodendrocytes, and less than 2% microglia by staining with Bandereria simplicifolia lectin 1. All experiments were performed on cultures between 21 and 30 days in vitro. 2.2.2. Glucose depriÕation Cultures were deprived of glucose by changing the culture medium to a glucose-free balanced salt solution ŽBSS 0 . at pH 7.4, containing Žin mM. NaCl 116, CaCl 2 1.8, MgSO4 0.8, KCl 5.4, NaH 2 PO4 1, NaHCO 3 14.7, N-Ž2-hydroxyethyl .piperazine-N X -ethanesulfonic acid ŽHEPES. 10; and phenol red 10 mgrl. Control cultures were washed into the same balanced salt solution containing 5.5 mM glucose ŽBSS 5.5 .. Other control cultures were washed into fresh growth medium. The medium was replaced in each well twice in succession and a third time after a 10-min interval, resulting in a greater than 2000-fold dilution of the glucose concentration. 2.2.3. Assessment of injury Astrocyte injury was estimated morphologically by phase-contrast light microscopy and quantitated by measuring the lactate dehydrogenase ŽLDH. released from lysed cells into the bathing medium w15x. Total LDH release corresponding to complete astrocyte death was determined at the end of each experiment following freezing at y708C and rapid thawing. 2.2.4. GSH measurements GSH was measured using a modification of the technique described by Fernandez-Checa and Kaplowitz w8x. ´ Astrocyte cultures were washed twice with phosphatebuffered saline ŽPBS., incubated at 378C for 10 min with 50 m M monochlorobimane, then washed and lysed in 0.2% Triton X-100 in PBS. The insoluble debris was sedimented at 13 600 = g for 5 min. The concentration of protein in the supernatant was determined using the bicinchoninic acid method and the fluorescence of monochlorobimane in the supernatant was measured at 400 nm excitation, 480 nm emission, in a Perkin Elmer LS50B spectrofluorimeter. The concentration of GSH was calculated from standard curves and expressed as nmolrmg protein. The relationship between GSH and fluorescence at 480 nm was linear in the range found in the cells. 2.2.5. Anisotropy measurements Probe internalization is a potential problem when measuring anisotropy on intact cells w22x. To avoid this we measured anisotropy on liposomes prepared from lipid extracts. Total lipid extracts were prepared from astrocytes using 0.5 mlrwell hexanerisopropanol Ž2:3 vrv.. The lipids were completely dried under nitrogen, 1,6-diphenyl1,3,5-hexatriene was added to a final concentration of 1 m M in 3 ml 20 mM Tris-HCl, pH 7.4. Liposomes were

M.C. Papadopoulos et al.r Brain Research 748 (1997) 151–156

made using sonication for 10 s in room air. In control studies, sonication under nitrogen gave identical results. Anisotropy Ž R . was measured with a Perkin Elmer LS50B fluorescence spectrometer at 37.58C using excitation and emission wavelengths of 350 nm and 452 nm respectively. R was calculated using the formula R s Ž Ivv y G = Ivh .rŽ Ivv q 2Ž G = Ivh .., where I is intensity with the polarizers specified excitation first then emission and G s I hv rIhh . For example, Ivh is fluorescence intensity measured with the excitation polarizer passing vertically polarized light and the emission polarizer passing horizontally polarized light. 2.2.6. Cis-PnA assay of lipid peroxidation in intact astrocytes The cis-PnA assay was performed on live astrocytes as previously described w11,32x with minor modifications. Briefly, cis-PnA was dissolved in ethanol and was added to the culture medium for a final concentration of 10 m M Žfinal ethanol concentration, 0.5%.. The plates were covered with aluminum foil and kept at 378C for 7 h. The medium was then removed and the cells were gently resuspended in PBS. Essentially all the cells were viable after resuspension as assessed by the ability to exclude Trypan blue or propidium iodide. The fluorescence intensity was measured at 378C using an excitation wavelength of 312 nm and an emission wavelength of 414 nm. All manipulations were performed in the dark because of the photosensitivity of cis-PnA. Cis-PnA was stored under nitrogen at y208C to avoid oxidation and was tested for fluorescence at the above wavelengths before each use. 2.2.7. Statistical analysis Data are expressed as the mean " S.E.M. Statistical significance Ž P - 0.05. was determined by one-way ANOVA and the Bonferroni test or the Student t-test using SigmaStat ŽJandel..

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Fig. 1. GD leads to near complete loss of GSH. Astrocyte cultures were exposed to BSS 5.5 ŽBSSqG. or BSS 0 ŽBSSyG. and GSH levels were measured after 0, 6, 12, 15 and 24 h of exposure. Control cultures washed with GM had the same GSH levels at the beginning and end of the experiment. Incubation in BSS 5.5 led to a slight decrease of GSH levels, while GD led to a severe progressive loss of GSH. Each point represents the mean of measurements from four cultures"S.E.M. The BSS 0 curve was significantly different Ž P - 0.05. from the other two curves Ž ) .. Similar results were obtained in three additional experiments using astrocyte cultures from different dissections and GD for 8 h.

tration. to the bathing medium for 6 h prior to the start of the insult. Alternatively, the concentration of GSH was reduced to 21.4 " 3.3% of control by treatment with 100 m M L-buthionine-Ž S,R .-sulfoximine ŽBSO. for 12 h prior to the start of the insult. The added GSH or BSO was washed out when the cells were transferred to glucose free medium. Cultures treated with BSO to reduce GSH levels and not subjected to glucose deprivation did not die over 24 h. Fig. 2 shows that elevating GSH made astrocytes more resistant to GD, whereas reducing GSH rendered astrocytes more susceptible. Since GSH levels are important in this injury paradigm and ATP is necessary in the synthesis of GSH, we investi-

3. Results We first quantitated the effect of increasing durations of glucose deprivation ŽGD. on the level of GSH in astrocyte cultures, because of the central role played by GSH in the cell’s antioxidant defense. Control astrocytes in growth medium had 19.7 " 0.7 nmol GSHrmg protein Ž n s 11 different dissections.. Astrocytes exposed to balanced salt solution lacking other substrate but containing 5.5 mM glucose ŽBSS 5.5 . showed a slight decrease in GSH ŽFig. 1.. In contrast, cultures that also lacked glucose ŽBSS 0 . showed a progressive, nearly complete loss of GSH by 24 h. To find out whether the level of GSH alters the vulnerability of astrocytes to GD, we altered the level of GSH in the cultures prior to exposing them to GD of increasing durations. We elevated the concentration of GSH to 150.4 " 5.6% of control by adding 10 mM GSH Žfinal concen-

Fig. 2. The effects of altering GSH levels or adding a free radical scavenger on GD injury. Astrocyte cultures were deprived of glucose and LDH levels in the medium were measured after 6, 12, 18 and 24 h. Curve BSS 0 qGSH represents cultures exposed to 10 mM GSH for 6 h prior to the start of the experiment to increase GSH. This was protective. Curve BSS 0 qBSO represents cultures exposed to 100 m M BSO for 12 h prior to the start of the experiment to reduce GSH. This increased injury. Addition of 10 mM DMTU ŽBSS 0 qDMTU. during the experiment protected the cultures from GD injury. Astrocyte cultures had the following GSH levels measured at the start of the experiment compared with untreated astrocytes: 150.4"5.6% ŽBSS 0 qGSH. and 21.4"3.3% ŽBSS 0 qBSO.. Values shown are means"S.E.M. for ns 20 ŽBSS 0 ., ns 24 ŽBSS 0 qGSH., ns10 ŽBSS 0 qBSO. and ns14 ŽBSS 0 qDMTU.. Significant difference Ž P - 0.05. from BSS 0 is shown by ) .

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Table 1 Effects of inhibitors of macromolecular synthesis and other drugs on astrocyte survival after 18 hours of glucose deprivation

Table 2 Effects of macromolecular synthesis inhibitors and dimethylthiourea on GSH levels in astrocytes after 10 h of glucose deprivation

Condition

n

% LDH release

Condition

n

% GSH

BSS 0 BSS 0 qL-NAME Ž100 m M. BSS 0 qETYA Ž50 m M. BSS 0 qallopurinol Ž100 m M. BSS 0 qcycloheximide Ž35.5 m M. BSS 0 qactinomycin D Ž1 m M.

44 13 17 15 15 20

47.6"3.0 46.2"4.4 61.2"3.0 41.9"5.4 14.1"3.0 28.4"4.2

BSS5.5 BSS 0 BSS 0 qcycloheximide Ž35.5 m M. BSS 0 qactinomycin D Ž1 m M. BSS 0 qDMTU Ž10 mM.

13 14 14 11 11

100.0"3.0 54.8"1.8 70.4"4.6 78.0"4.0 62.8"3.1

a a

Astrocyte cultures were exposed to balanced salt solution lacking glucose ŽBSS 0 . for 18 h with the drugs present at the indicated concentrations then LDH activity in the medium was measured. Control cells kept in BSS5.5 in the presence or absence of each drug showed no drop in viability. L-NAME is N-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor; ETYA stands for 5,8,11,14-eicosatetraenoic acid, an arachidonate metabolism inhibitor. Values shown are mean"S.E.M. a Significant difference Ž P - 0.05. from BSS 0 .

gated whether protein and RNA synthesis inhibitors would ameliorate GD injury. Inhibition of protein synthesis would be expected to preserve ATP levels and amino acid precursors needed to synthesize GSH. Table 1 shows that cycloheximide Ž35.5 m M, 10 m grml. and actinomycin D Ž1 m M., inhibitors of translation and transcription, both protected astrocytes from GD. Consistent with a role for oxidative stress causing injury, addition of 10 mM 1,3-dimethyl-2-thiourea ŽDMTU., a free radical scavenger, was protective to about the same extent as raising GSH levels by 50% ŽFig. 2.. The free radical scavenger trolox, a vitamin E analog, also reduced injury by about 50% Ždata not shown.. To explore the source of the oxidative species that may contribute to GD injury, we exposed astrocyte cultures to GD in the presence of inhibitors of several free radical generating pathways which may become activated under stress. Table 1 shows that the nitric oxide synthetase

Fig. 3. GD produces membrane rigidification. Astrocyte cultures were washed with GM, BSS5.5 , BSS 0 , or with BSS 0 containing 10 mM dimethylthiourea ŽBSS 0 qDMTU.. After 8 h cells were extracted and liposomes made. The fluorescence anisotropy of liposomes from cultures deprived of glucose rose by 6.2"0.8% compared to BSS5.5 treated cultures, and by 6.9"0.8% compared to cultures washed with GM. The addition of DMTU significantly ameliorated the rise in anisotropy seen in glucose deprived astrocytes. Values shown are means"S.E.M. for ns12 ŽGM., ns 27 ŽBSS 5.5 ., ns 28 ŽBSS 0 . and ns16 ŽBSS 0 qDMTU.. The BSS 0 group differed Ž ) P - 0.05. from all other groups, which were not different from each other.

a

a a

GSH was measured after exposure to balanced salt solution containing ŽBSS 5.5 . or lacking ŽBSS 0 . glucose at 5.5 mM, with the drugs present at the concentrations shown. Values shown are means"S.E.M., normalized to the amount of GSH present in cells incubated in BSS 5.5 . a Significant difference Ž P - 0.05. from BSS 0 .

inhibitor N-nitro-L-arginine methyl ester Ž100 m M., the inhibitor of arachidonic acid metabolism 5,8,11,14 eicosatraenoic acid Ž50 m M. and the xanthine oxidase inhibitor allopurinol Ž100 m M. all failed to protect astrocytes from GD injury. If oxidative stress is contributing to the injury sustained during GD, the effects of oxidative damage, such as crosslinking of membrane lipids, should be detectable. Since crosslinking leads to rigidification of the membrane, membrane fluidity was determined by measuring anisotropy with the fluorescent probe 1,6-diphenyl-1,3,5hexatriene. Fig. 3 shows a significant rise in anisotropy, indicating membrane rigidification, after 8 h of GD. The rise in anisotropy caused by GD was partially prevented by addition of 10 mM DMTU. To directly demonstrate that GD leads to lipid peroxidation we loaded astrocytes with cis-PnA. Astrocyte cultures Ž n s 12. exposed to BSS 0 for 7 h had 76.0 " 2.2% Ž P 0.05. the cis-PnA fluorescence of cultures Ž n s 12. exposed to BSS 5.5 . Cis-PnA fluorescence measurements in astrocytes were unaffected by changing the temperature by 28C, a decrease which leads to a rise in anisotropy of 5–7% Ždata not shown.. This suggests that the loss of cis-PnA fluorescence observed with GD is not a consequence of the reduction in membrane fluidity but only reflects lipid peroxidation. To investigate whether the depletion of GSH that occurs with GD leads to oxidative injury or whether the converse is true, that is GD leads to oxidative stress which in turn depletes GSH, we exposed astrocytes to 10 h of GD in the presence of 10 mM DMTU and measured the level of GSH. Table 2 shows that DMTU failed to inhibit the fall in GSH to a statistically significant extent.

4. Discussion We suggest that oxidative stress occurs during GD of astrocytes in part, because the cells are unable to maintain their GSH levels which leaves them vulnerable to oxidative damage. It is known that synthesis of GSH from

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glutamate, cysteine and glycine requires ATP; recycling of GSH from glutathione disulfide requires NADPH w19,26x and synthesis of the amino acid precursor glutamate requires a carbon from glucose w23x. It is likely that GD inhibits the synthesis of GSH by reducing availability of precursors and ATP, and inhibits the reduction of GSSG to GSH by lowering NADPH. Although DMTU protected astrocytes from GD injury and reduced membrane rigidification, it failed to maintain GSH levels. The levels of GSH measured here in mouse cortical astrocytes are in good agreement with previous data w24,34x. Yudkoff and coworkers w34x reported that the amount of radiolabeled glutamate in GSH reached half the specific activity of the precursor pool in 180 min in the presence of glucose. This is a somewhat faster turnover rate than suggested in Fig. 1, probably because the change to BSS 0 does not lead to immediate complete inhibition of GSH synthesis, rather the extent of inhibition builds up with time as depletion of precursors and ATP gradually occur. Fig. 1 provides the first direct demonstration that GD causes a progressive fall in GSH in astrocytes. The protective effects of DMTU, trolox, or elevated GSH levels against GD suggest that the oxidative stress that occurs during GD leads to astrocyte death. Protection by trolox suggests that DMTU and elevation of GSH levels may enhance astrocyte survival by an antioxidant action rather than solely by protecting a sulfhydryl group. In the case of DMTU protection, the cells are significantly protected without preservation of GSH, suggestive of direct anti-oxidant activity. This is consistent with a report of protection of neurons from GD by trolox w5x. Increasing GSH levels may also protect by supplying extra amino acids which may provide indirect metabolic benefits. The membrane rigidification observed here with GD is of sufficient magnitude to alter membrane function and the function of membrane associated enzymes Žfor review see Stubbs and Smith w29x.. The loss in cis-PnA fluorescence brought about by GD suggests that membrane rigidification may be a result of lipid peroxidation. Membrane rigidification is known to affect membrane permeability w20x, to alter the activity of membrane bound enzymes such as the Naq,Kq-ATPase w20,30x and to uncouple oxidative phosphorylation in mitochondria w6x. The increase in anisotropy preceded cell lysis as indicated by LDH release. DMTU inhibited the rise in anisotropy and protected astrocytes from GD injury. Various pathways have been shown to lead to the generation of free radicals during stress. Such pathways include the activation of nitric oxide synthetase w9x, the metabolism of arachidonic acid w16x and the formation of xanthine oxidase w17x. Inhibitors of these mechanisms of free radical generation failed to protect astrocytes from GD. It is likely that during GD free radical production is similar to that found in healthy cells. Normally free radicals are primarily formed as by-products of mitochondrial function w10x. The decrease in the level of GSH, and

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therefore radical scavenging capability, which occurs during GD may permit these free radicals to cause damage. In a neural cell line, depletion of GSH led to increased levels of free radicals and caused cell death within 40 h w13x. In this case, loss of additional scavenging ability or additional generation of radicals is likely since GSH depletion alone using BSO did not cause the same extent of injury seen with glucose deprivation. The macromolecular synthesis inhibitors partially prevented the fall in GSH that occurred during GD. Inhibition of transcription and translation may allow high energy phosphates and amino acid precursors to be used for the maintenance of GSH. This is consistent with data from cortical neurons where macromolecular synthesis inhibitors were found to protect from free radical damage by shunting cysteine from protein synthesis to the formation of GSH w25x. These drugs may also protect by preventing synthesis of proteins that contribute to cell death. In this paper, we show that GD leads to progressive, severe depletion of GSH which is associated with astrocyte injury. Increasing or decreasing GSH levels prior to the insult resulted in a corresponding decrease or increase in injury. The increase in fluorescence anisotropy and the detection of increased free radicals with cis-PnA observed during GD are consistent with oxidative membrane rigidification. Increasing cellular levels of GSH, or adding the free radical scavengers DMTU or trolox, reduce cell death. Thus during GD and in conditions where energy depletion may occur, GSH levels are reduced, which in turn permits oxidative damage to injure and kill astrocytes. Glucose deprivation may provide a useful model for investigating the role of redox modulation and glutathione levels on transcriptional gene regulation and second messenger systems. Free radical injury of astrocytes may impair their ability to regulate extracellular glutamate or Kq, reduce their ability to defend against oxidative stress and therefore increase the likelihood of neuronal loss.

Acknowledgements We would like to thank Xiao Yun Sun for her expert technical assistance. This work was supported in part by NIH grant GM 49831 to R.G.G.

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