Abnormal glutathione transport in cystic fibrosis airway epithelia

Lin Gao1, Kwang Jin Kim1,2, James R. Yankaskas3, and Henry Jay Forman1

Departments of 1 Molecular Pharmacology and Toxicology and 2 Medicine, Will Rogers Institute Pulmonary Research Center, University of Southern California, Los Angeles, California 90033; and 3 Department of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Glutathione (GSH) is a potentially important component of antioxidant defense in the epithelial lung lining fluid. Cystic fibrosis (CF) patients have chronic inflammation in which oxidative stress can be a factor. To examine the hypothesis that the transport of GSH content was defective in CF patients, intracellular and extracellular GSH were measured by HPLC. Four cell lines were used: CFT1 cells [with defective CF transmembrane conductance regulator (CFTR), Delta F508 homozygous, two clones] and one of the CFT1 clones transfected with either normal CFTR (CFTR repleted) or beta -galactosidase. GSH content in the apical fluid was 55% lower in CFTR-deficient cultures than in CFTR-repleted cells (P < 0.001). In contrast, intracellular GSH content was similar in CFT1 cells and CFTR-repleted cells. gamma -Glutamyl transpeptidase activity, which degrades extracellular GSH, did not account for differences in apical GSH. Rather, GSH efflux of CFTR-deficient cells was lower than that of CFTR-repleted cells. These studies suggested that decreased GSH content in the apical fluid in CF resulted from abnormal GSH transport associated with a defective CFTR.

gamma -glutamyl transpeptidase; epithelial lung lining fluid; cystic fibrosis transmembrane conductance regulator; thiol


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CYSTIC FIBROSIS (CF) is a systemic disorder primarily manifested in the lung, pancreas, and intestinal tract. CF is caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. About 70% of mutations of the CFTR gene in the Caucasian population display deletion of phenylalanine at position 508 (Delta F508). CFTR is an apical membrane glycoprotein that functions as a cAMP-dependent chloride channel. In addition to regulating chloride transport of epithelia, CFTR also appears to modulate sodium transport (1).

Lung dysfunction is a major cause of death in CF patients. The major clinical manifestations affecting the respiratory tract are mucus accumulation, Pseudomonas aeruginosa infection, and chronic inflammation. Replacement gene therapy may eventually provide a cure for CF (18). Improvement in delivery of the CFTR gene is the crucial step for such gene therapy. In the meantime, pharmacological remedies for correcting the defect may be attempted. For example, 4-phenylbutyrate has been found to restore CFTR function in vitro (23). More immediately, CF therapy is targeted at treating the resulting physiological and pathological manifestations, such as chronic inflammation.

Inflammation presents an oxidative challenge to the lung. Invading neutrophils produce superoxide, hydrogen peroxide, and hypochlorous acid. Glutathione (GSH) is a major component of cellular defense against oxidative injury. GSH is normally present at 2-10 mM concentrations inside cells. Most cells depend on de novo GSH synthesis to maintain their intracellular GSH content. gamma -Glutamylcysteine synthetase (GCS) and cysteine are the rate-limiting enzyme and substrate, respectively, for the de novo synthesis of GSH. On the other hand, gamma -glutamyl transpeptidase (GGT), which is located on the outer surface of the plasma membrane, can degrade extracellular GSH. GCS and GGT constitute part of a system for transport of GSH between organs and its recycling between the extracellular and intracellular compartments. As a consequence of GSH export by epithelial cells, GSH is found in high concentration in some extracellular fluids, such as the epithelial lung lining fluid (ELF; see Refs. 3, 25). Normal human ELF contains a high GSH concentration (i.e., ~400 µM) that is 140-fold higher than that in the plasma (3). Extracellular GSH can serve as a scavenger of carbon-centered free radicals produced by lipid peroxidation and hypochlorous acid produced by neutrophils during inflammation. Therefore, a decrease in GSH content in ELF may increase the susceptibility of the lung epithelium to chronic inflammation such as occurs in CF. Evidence of oxidative stress has also been found in CF patients along with pulmonary dysfunction (2, 12). GSH content is decreased in the lung lavage fluid from adult CF patients (22). However, GSH content is either unchanged in the lavage fluid from CF children without inflammation or tends to decrease but is not statistically significant from CF children with inflammation (12). Because of the technical difficulties in making an accurate measurement of GSH, especially in damaged lungs, there may be an advantage in studying cells derived from CF patients and controls in vitro.

In hepatocytes and kidney, GSH transport has been found to be membrane potential dependent (9, 14, 15). In hepatocytes, GSH transport has also been shown to be carrier mediated (20). Another study has also suggested GSH transport via vesicle secretion in myeloma cells and Xenopus laevis oocytes (4). Whether similar GSH transporters exist in lung is still not clear.

Recently, CFTR has been suggested to be permeable to several organic anions and GSH (16, 17). Furthermore, mutations of CFTR cause abnormal chloride and sodium transport, which may cause changes in the fluid composition in ELF as well as in plasma membrane potential. Both changes in membrane potential and mutations of CFTR might affect GSH transport and homeostasis in CF lung epithelium. Therefore, the purposes of the present study were to examine 1) whether intracellular/extracellular GSH balance in cells with defective CFTR was abnormal; 2) whether this biochemical imbalance could be corrected by stable transfection with the normal CFTR gene; and 3) the possible mechanisms of any abnormality in GSH balance.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals. Acivicin, 2,4-dinitrofluorobenzene, and a somatic cell ATP assay kit were purchased from Sigma (St. Louis, MO). gamma -Glutamyl-glutamic acid was purchased from Bachem Bioscience (Torrance, CA). All chemicals used were at least analytical grade.

Cell culture and treatment. CFT1 clone 2 (CFT1-c2) and clone 9 (CFT1-c9) cells are immortalized human tracheal epithelial cells from a homozygous Delta F508 CF patient (27). CFT1-LC3 cells are CFT1-c2 cells transfected with beta -galactosidase as transfection control, and CFT1-LCFSN cells are CFT1-c2 cells transfected with normal CFTR gene (19). HBE1 cells are immortalized human bronchial epithelial cells from a normal individual (27). Cells were grown in serum-free Ham's F-12 medium supplemented with seven additives (5 µg/ml insulin, 3.7 µg/ml endothelial cell growth supplement, 25 ng/ml epidermal growth factor, 3 × 10-8 M triiodothyronine, 1 × 10-6 M hydrocortisone, 5 µg/ml transferrin, and 10 ng/ml cholera toxin) in T-75cm2 collagen-coated flasks. Cells were passaged two times without cholera toxin to eliminate its effects.

Subsequently, cells (0.5 × 106 cells/4.2 cm2) were seeded on collagen-coated permeable supports. One milliliter of F-12 medium was added to the inner well (apical side), and 2 ml medium were added to the outer well (basolateral side). After 24 h, cells were exposed to a medium mixture (50% F-12-50% NIH/3T3 cell-conditioned DMEM that contained 2% FBS). Transepithelial resistance (Rt) was measured to monitor the development of tight junctions. When cultures developed a maximum Rt (in ~7-10 days; see RESULTS), the medium was removed and replenished with 0.5 and 1.5 ml of fresh medium mixture on the apical and basolateral sides, respectively. In some studies, 0.2 mg/ml acivicin was added to medium to inhibit GGT. Media and cells were collected at different time points for GGT activity, ATP content, and GSH content measurement.

GSH content measurement. At different time points, 0.5 ml of apical and basolateral media were collected and centrifuged to spin down any detached cells that might be present. The cells were washed one time with ice-cold PBS and scraped into the same buffer. Protein from either media or cells was precipitated in 10% perchloric acid containing 2 mM EDTA and 10 nmol/ml gamma -glutamyl-glutamic acid as an internal standard. On centrifugation, GSH, glutathione disulfide (GSSG), and cysteine glutathione disulfide (Cys-SG) content in the supernatant was derivatized with 2,4-dinitrofluorobenzene and determined by HPLC, as previously described by Fariss and Reed (8). Protein concentration was measured with the bicinchoninic acid protein assay kit (Pierce, Rockford, IL).

GGT activity assay. Cells were scraped into ice-cold PBS buffer. After centrifugation, the cell pellets were suspended in PBS containing 0.1% Triton X-100 and sonicated briefly on ice. GGT activity was measured with the fluorescent substrate gamma -glutamyl-7-amino-4-methyl-coumarin, as described previously (11).

ATP assay. To determine the cytotoxic effect of acivicin, intracellular ATP content was measured with a somatic cell ATP assay kit. After the cells were treated with 0.2 mg/ml of acivicin for 18 h, cells grown on the permeable support were washed one time with PBS. ATP was released with 0.5 ml of somatic cell ATP-releasing solution. ATP concentration was determined by firefly luciferin-luciferase reaction with a Perkin-Elmer LS-5 fluorescence spectrophotometer in the phosphorescence mode. The ATP content was calculated based on the ATP standard curve and was normalized by protein concentration.

Statistics. Data are expressed as means ± SE and were evaluated by one-way ANOVA; n is the number of observations. Statistical significance was then determined by Tukey's test. P < 0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Apical medium GSH content was decreased with CFTR-deficient cells. Cells grew to confluence at ~7-8 days after seeding. Both CFT1-c2 and CFT1-c9 cells developed a higher Rt (>1,000 Omega  · cm2) compared with other cells (~20 Omega  · cm2). Both apical and basolateral media and cells were collected 18 h after media were changed. Intracellular GSH content in both CFT1-c2 and CFT1-c9 cells was not different from that in CFT1-c2 cells stably transfected with either beta -galactosidase (CFT1-LC3) or normal CFTR (CFT1-LCFSN; P > 0.05; Fig. 1). Intracellular GSH content in HBE1 cells was similar to that of CFT1-LCFSN cells (88.3 ± 7.1% CFT1-LCFSN; P > 0.05). Thus intracellular GSH content was not significantly affected by the defect in CFTR function.


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Fig. 1.   Total intracellular glutathione (GSH) content. CFT1-c2, CFT1 clone 2 cells; CFT1-c9, CFT1 clone 9 cells; CFT1-LC3, CFT1-c2 cells transfected with beta -galactosidase as transfection control; CFT1-LCFSN, CFT1-c2 cells transfected with normal cystic fibrosis transmembrane conductance regulator (CFTR) gene. Cells were collected 18 h after medium was changed. Both GSH and glutathione disulfide (GSSG) contents were determined by HPLC. No cysteine glutathione disulfide (Cys-SG) was present. Total intracellular GSH content in CFT1-LCFSN cells was 9.19 ± 0.69 nmol/mg protein. Data are expressed as means ± SE (n >=  3 observations) and were evaluated by one-way ANOVA (P > 0.05).

In contrast, GSH found on the apical surface of both CFT1-c2 and CFT1-c9 cells was markedly lower than that of the "CFTR-repaired" CFT1-LCFSN cells (P < 0.001; Fig. 2). CFT1-c2 cells transfected with beta -galactosidase (CFT1-LC3) had no significant difference versus CFT1-c2 cells (Fig. 2). GSH content in the apical fluid of HBE1 cells was similar to that of CFT1-LCFSN cells (113.4 ± 8.5% CFT1-LCFSN; P > 0.05). GSH concentration on the basolateral side was variable but lower than that of apical GSH concentration for each cell line (data not shown). Because of the high cystine content in cell culture medium, most GSH in the medium reacted with cystine and formed Cys-SG. Therefore, extracellular GSH content was calculated from GSH equivalents (= GSH + 2 GSSG + Cys-SG).


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Fig. 2.   Total apical GSH content. Apical media were collected 18 h after media were changed. GSH, GSSG, and Cys-SG contents were determined by HPLC. Glutathione equivalents (glutathioneeq) = GSH + 2 GSSG + Cys-SG. Total apical glutathioneeq of CFT1-LCFSN (CFTR- transfected) cells was 58.13 ± 5.45 nmol/mg cellular protein. Data are expressed as means ± SE (n >=  3 observations) and were evaluated by one-way ANOVA. Statistical significance was then determined by Tukey's test. * Statistically significant difference (P < 0.05) from CFT1-LCFSN cells.

Increased GGT activity did not explain the abnormal apical GSH content in CFTR-deficient cells. To study whether the low apical GSH content in CFT1 and CFT1-LC3 cells was caused by a rapid degradation of GSH outside the cells, GGT activity was measured. GGT activity in CFT1-c9 cells was higher than in all other cells tested (P < 0.001), as shown in Table 1. Nonetheless, GGT activity in CFT1-c2 cells was not different from either CFT1-c2 cells transfected with beta -galactosidase (CFT1-LC3) or CFT1-c2 cells transfected with normal CFTR gene (CFT1-LCFSN; P > 0.05). GGT activity in HBE1 cells was similar to that of CFT1-LCFSN cells (91.8 ± 15.0% CFT1-LCFSN; P > 0.05).

                              
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Table 1.   Relative gamma -glutamyl transpeptidase activity

When cells were incubated for 18 h with 0.2 mg/ml acivicin to inhibit GGT activity, GGT activity decreased to <1.1 pmol · min-1 · mg protein-1 in all cells tested. As expected, inhibition of GSH degradation by GGT with acivicin caused a slight increase in apical GSH content in all cell lines tested. However, apical GSH content of CFT1-c2, CFT1-c9, and CFT1-LC3 cells was still substantially lower than for CFT1-LCFSN cells, as shown in Fig. 3. Apical GSH content of HBE1 cells was higher than that for CFT1-LCFSN cells (146.5 ± 6.2% CFT1-LCFSN; P < 0.05). With acivicin treatment, the pattern of intracellular GSH content in all cell lines did not change compared with that without acivicin treatment. These results suggested that the differences in GGT activity among the cell lines studied here did not significantly contribute to the decrease in apical GSH content in CFTR-deficient cells. Intracellular ATP content was not decreased after acivicin treatment for 18 h in all cell lines tested, suggesting that acivicin was not toxic to the cells under the present condition (data not shown).


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Fig. 3.   Total apical GSH content with acivicin inhibition of gamma -glutamyl transpeptidase. Apical media were collected 18 h after treatment with acivicin (0.2 mg/ml). GSH, GSSG, and Cys-SG contents were determined by HPLC. Glutathioneeq = GSH + 2 GSSG + Cys-SG. Total apical glutathioneeq of CFT1-LCFSN (CFTR-transfected) cells was 59.08 ± 5.44 nmol/mg cellular protein. Data are expressed as means ± SE (n >=  3 observations) and were evaluated by one-way ANOVA. Statistical significance was then determined by Tukey's test. * Statistically significant difference (P < 0.05) from CFT1-LCFSN cells.

Decreased GSH secretion from CFTR-deficient cells. Another possible mechanism that was examined as a possible explanation for the differences in the apical GSH content was the release of cellular GSH. GSH efflux into the apical fluid was examined in the presence of the GGT inhibitor acivicin to inhibit any extracellular GSH degradation (Fig. 4). The efflux rates (in pmol · min-1 · mg cellular protein-1) were 20 ± 3 from CFT1-c9 cells, 29 ± 2 from CFT1-LC3 cells (CFT1-c2 cells transfected with beta -galactosidase), 50 ± 2 from CFT1-LCFSN cells (CFT1-c2 cells transfected with normal CFTR), and 60 ± 2 from HBE1 cells. The efflux rate of CFT1-c2 cells was similar to that of CFT1-LC3 cells. Three separate experiments of GSH efflux were done, and similar results were observed. Thus the rate of efflux of GSH to the apical medium was significantly impaired by the CFTR defect and was markedly elevated by transfection with normal CFTR.


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Fig. 4.   Apical GSH efflux. Apical media were collected at different time points after treatment with acivicin (0.2 mg/ml). GSH, GSSG, and Cys-SG contents were determined by HPLC and are presented as nmol/mg cellular protein. Glutathioneeq = GSH + 2 GSSG + Cys-SG. CFT1 cells were CFT1 clone 9 cells. Data are expressed as means ± SE (n = 3 observations). Rates of GSH efflux were provided in text.

Basolateral GSH accumulation was slower than for apical GSH accumulation. In contrast to the accumulation in the apical medium, which was linear for >10 h, there was a lag in the accumulation of GSH in the basolateral fluid. Initially, the GSH concentration was very low, and there was no clear difference in accumulation between the cell lines. After 2 h, the basolateral GSH concentration began to approach the apical concentration. The lag in basolateral accumulation may result from paracellular transport.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Antioxidants in ELF are important in protection of the integrity of the lung epithelium against oxidative damage. In CF, patients are exposed to chronic infection and inflammation. Therefore, maintenance of sufficient antioxidant levels in ELF in CF patients is crucial. The most abundant antioxidants in ELF are GSH, ascorbic acid, and uric acid (6). GSH content has been reported to be decreased in plasma and lavaged ELF of CF patients (22). Yet, others have found no such change in the GSH content in lung lavage from pediatric patients with CF, especially those without inflammation (12). One major problem with determining the extracellular GSH in CF lungs by lavage is what it represents. The bronchotracheal surface is very small compared with the alveolar surface such that what is obtained by filling a lung segment probably largely represents GSH content on the alveolar rather than tracheobronchial surface. Another problem is that a small amount of damage to tissue can result in release of significant GSH during cell lysis. Recently, Linsdell and Hanrahan (17) demonstrated that CFTR itself might transport GSH using isolated membrane from kidney cells. This further raised the question of whether the CFTR defect affected GSH transport and GSH content in ELF from CF patients.

In the present study, GSH content was determined to be lower in the apical medium of CFTR-deficient cells than CFTR-sufficient cells (Fig. 2). This was the first study to use confluent airway epithelium and show such differences in the extracellular GSH content. By growing cells on a permeable support, cells were allowed to form tight junctions, as determined by Rt. This mimicked the physiological condition in the lung. In addition, these cells were derived from the human airway where inhaled oxidants such as nitrogen dioxide and ozone have their initial effects (5) and where the consequence of inflammation in CF patients probably originates. Lung damage in CF patients mainly results from ion transport abnormalities in airway epithelia that lead to mucus accumulation and bacterial colonization (13). Although a recent paper suggested that CFTR is widely expressed, including bronchi, distal airway, and alveoli of human lungs (7), rat alveolar type II cells have relatively low CFTR (28). Thus the question of the involvement of the alveolus as a primary site of damage from the CFTR defect remains unresolved. Apical GSH is physiologically important because GSH in the epithelial lining layer serves as an initial defense against oxidative stress. Some oxidants are so insoluble (i.e., ozone and nitrogen dioxide) or so reactive (i.e., hypochlorous acid, peroxynitrite, ozone, and nitrogen dioxide) that they would most rapidly react with the extracellular fluid contents or outer leaflet of the cell membrane. This does not imply that these toxicants cannot cause damage by entering cells but suggests the value of extracellular GSH in lessening that possibility. GSH reacts directly and rapidly with hypochlorite to produce GSSG and chloride (10) and with peroxynitrite to form GSSG and nitrate (21). Although reaction with the oxidant gases may be more controversial, GSH will rapidly reduce carbon-centered radicals formed by their reactions (24).

We explored three possible explanations for the lower apical surface GSH content of the CFT1 cells compared with the CFTR-transfected CFT1 cells. These are increased extracellular degradation, decreased intracellular GSH and decreased export of GSH. The last is considered the most likely for the following reasons.

One possible explanation for reduced extracellular GSH content that was considered was degradation of GSH outside the cells. GGT activity was found to be higher in CFT1-c9 cells than in CFT1-LCFSN cells (Table 1). However, when GGT activity was inhibited, GSH content in apical medium was still lower in CFT1 and CFT1-LC3 (beta -galactosidase-transfected) cells than in CFT1-LCFSN cells (Fig. 3). This suggested that such increased GGT activity in CFT1-c9 cells did not result in the abnormally low GSH content in the apical medium. Furthermore, the GGT activity of the CFT1-c9 cells was actually small relative to the apical GSH content. Indeed, if GGT was acting at its maximum rate and no GSH continued to be released from the cells, it would have required ~13 h to have degraded enough of the GSH found in the CFT1-LCFSN fluid to the level found in the CFT1-c9 apical fluid. An explanation for the high GGT activity in CFT1-c9 cells may have been clonal variation.

A second possibility for the lower GSH on the apical surface of CFT1 cells that was examined was decreased intracellular GSH by either decreased GCS activity or decreased cysteine availability. Decreased GSH biosynthesis seems unlikely, as the intracellular GSH content in CFT1 cells was not decreased.

On the basis of the evidence provided here, the most likely mechanism for the difference in apical GSH content between CFTR-deficient and -sufficient cells was abnormally low GSH export by CFT1 cells. As shown in Fig. 4, GSH efflux from CFTR-deficient cells was lower than that from CFTR-restored cells. Therefore, the differences in apical GSH content among the cell lines were probably caused by differences in GSH efflux. A recent study has shown that CFTR was permeable to GSH and to a lesser extent to GSSG (17). From the present study, it appeared that the CFTR mutation directly affected GSH transport in CF cells. It is also possible that GSH transport was indirectly regulated by CFTR. In rat hepatocytes and isolated kidney membrane, GSH transport has been shown to be dependent on membrane potential (9, 14, 15). It is possible that the GSH transporter in lung is similar to that in hepatocytes in being membrane potential dependent. Therefore, such decreased CFTR function, which affects the membrane potential, could indirectly regulate GSH transport.

Studies have demonstrated a contribution of oxidative stress to the pathophysiology of CF (26). Because GSH is an antioxidant, the decrease in apical GSH content would make CF cells more susceptible to oxidative stress as found in the chronic inflammation associated with CF. Potentially, modulation of GSH content in ELF might provide a partial remedy to alleviate inflammation in CF patients.


    ACKNOWLEDGEMENTS

We thank Jinah Choi and Drs. Rui-Ming Liu, Huanfang Zhou, Beth Schomer, and Timothy Robison for technical and other expert advice. We also thank Ronald Kim (University of North Carolina) for assistance with establishing the cell lines in our laboratory.


    FOOTNOTES

This work was supported by the Berger-Webb Foundation and National Institutes of Health Grants ES-05511, HL-38658, and HL-46943.

Part of this work was presented at the North American Cystic Fibrosis Conference in Montreal, Canada, 1998.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: H. J. Forman, Dept. of Molecular Pharmacology and Toxicology, School of Pharmacy, PSC 516, Univ. of Southern California, 1985 Zonal Ave., Los Angeles, CA 90033 (E-mail: hforman{at}hsc.usc.edu).

Received 27 August 1998; accepted in final form 12 March 1999.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Lung Cell Mol Physiol 277(1):L113-L118
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