From the Department of Molecular Cell Biology, Sylvius Laboratory, Leiden University, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands
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ABSTRACT |
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The impermeable oxidant ferricyanide is reduced
by the plasma membrane redox system of HL-60 cells. The rate of
reduction is strongly enhanced by ascorbate or dehydroascorbate. The
aim of this study was to determine the mechanism by which ascorbate and
dehydroascorbate accelerate ferricyanide reduction in HL-60 cells.
Addition of ascorbate or dehydroascorbate to cells in the presence of
ferricyanide led to the intracellular accumulation of ascorbate.
Control experiments showed that extracellular ascorbate was rapidly
converted to dehydroascorbate in the presence of ferricyanide. These
data suggest that intracellular ascorbate originates from extracellular
dehydroascorbate. Accumulation of ascorbate was prevented by inhibitors
of dehydroascorbate transport into the cell. These compounds also
strongly inhibited ascorbate-stimulated ferricyanide reduction in HL-60
cells. Thus, it is concluded that the stimulation of ferricyanide
reduction is dependent on intracellular accumulation of ascorbate.
Changing the -tocopherol content of the cells had no effect on the
ascorbate-stimulated ferricyanide reduction, showing that a
nonenzymatic redox system utilizing
-tocopherol was not involved.
p-Chloromercuribenzenesulfonic acid strongly affected
ferricyanide reduction in the absence of ascorbate, whereas the
stimulated reaction was much less responsive to this compound. Thus, it
appears that at least two different membrane redox systems are
operative in HL-60 cells, both capable of reducing ferricyanide, but
through different mechanisms. The first system is the ferricyanide
reductase, which uses NADH as its source for electrons, whereas the
novel system proposed in this paper relies on ascorbate.
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INTRODUCTION |
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Many eukaryotic cells contain a redox system in their plasma membrane capable of reducing extracellular substrates using electrons from intracellular NADH (1). The system efficiently reduces the impermeable substrate ferricyanide. This is not the natural substrate for the redox system, but as yet, there has been no conclusive evidence for the substrates of this system or for its primary function. It has been suggested that the system is involved in the maintenance of the redox state of sulfhydryl residues in membrane proteins (1), the neutralization of oxidative stressors outside the cell (2), or the uptake of iron through a non-transferrin pathway (3, 4).
This redox system deserves special attention because of its possible involvement in regulation of growth and differentiation. Activation of the redox system results in stimulation of growth in serum-limited HeLa cells (5) and HL-60 cells (6). Induction of differentiation in HL-60 cells has been associated with transient changes in reductase activity (7). In other cells, activation of the redox system has been shown to modulate protein kinase C activity (8). These findings raise an interest in the role of the plasma membrane reductase in these cells and the mechanisms through which it operates.
The reduction of ferricyanide by the plasma membrane reductase can be greatly stimulated by the addition of ascorbate and the oxidized form of ascorbate, dehydroascorbate (DHA).1 This has been found both in K562 cells (9), a leukemic cell line, and in human erythrocytes (10). Although several mechanisms have been proposed, the exact mechanism of this enhancement by ascorbate and DHA remains to be elucidated. For K562 cells, it was suggested that the stimulation of ferricyanide reduction by ascorbate was due to a plasma membrane-localized ascorbate free radical (AFR) reductase (9). This enzyme is supposed to catalyze the reduction of external ascorbate free radical using intracellular NADH. It was proposed that ferricyanide reacts with ascorbate to ferrocyanide and the ascorbate free radical. The latter would then be regenerated by the AFR reductase to ascorbate, which can subsequently again react with ferricyanide.
Another mechanism is based on studies on erythrocytes and involves the accumulation of ascorbate in cells, where it may serve as an intracellular electron donor for a plasma membrane reductase (10). In many cells, accumulation of ascorbate is achieved through a facilitative glucose transporter, GLUT-1 (11, 12). This transporter efficiently transports DHA into the cell, but not ascorbate itself. Inside the cell, DHA is reduced to ascorbate. This metabolic trapping mechanism enables the cell to accumulate ascorbate at concentrations far exceeding that of its environment. However, recently, it was found that, in erythrocytes, the enhancement of ferricyanide reduction by DHA was not affected by an inhibitor of the GLUT-1 transporter (13). This led to the conclusion that ascorbate was closely involved in the redox reaction, but that it acted both intra- and extracellularly. It was concluded that DHA could be regenerated to ascorbate independent of its cellular location. Thus, controversy exists on the mechanism of enhancement of ferricyanide reduction by ascorbate and DHA.
We have studied the effect of DHA and ascorbate on ferricyanide reduction by HL-60 cells. It has been reported that HL-60 cells contain a ferricyanide reductase and an AFR reductase (14, 15). In this study, the possible role of both enzymes in the enhanced ferricyanide reduction was investigated. It is concluded that ascorbate-stimulated ferricyanide reduction does not involve an AFR reductase. Rather, it involves a mechanism where ascorbate is internalized and where intracellular ascorbate is involved in the reduction of extracellular ferricyanide by a novel redox system, different from the NADH-driven ferricyanide reductase.
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EXPERIMENTAL PROCEDURES |
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L-Ascorbic acid and L-dehydroascorbic
acid were purchased from Aldrich, and potassium ferricyanide and
p-chloromercuribenzenesulfonic acid (pCMBS) were
from Fluka. Cytochalasin B and -tocopherol were from Sigma;
L-buthionine-(RS)-sulfoximine was from Acros Chimica; and phloretin was from ICN.
HL-60 myeloid leukemic cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Life Technologies, Inc.), 100 units/ml penicillin G, 100 µg/ml streptomycin, 2.5 µg/ml amphotericin B, and 3 mM L-glutamine. Culture flasks were kept in a humidified atmosphere with 5% CO2 at 37 °C. Cells were harvested when the culture had reached a density of ~9 × 105 cells/ml and were washed twice in 20 mM Tris and 150 mM NaCl at pH 7.4 (Tris/NaCl). Subsequently, the cells were suspended in this buffer for further use.
Ferricyanide reduction was determined as follows. Cells (4 × 106/ml) in Tris/NaCl were incubated at 37 °C in a closed shaking water bath. The reaction was started by the addition of 1 mM ferricyanide and various concentrations of ascorbate or DHA and was followed for at least 60 min. Aliquots were centrifuged, and supernatants were assayed for ferrocyanide using the bathophenanthroline-disulfonic acid assay (16). The rate of ferrocyanide generation was used as a measure for the activity of the reductase.
Accumulation of ascorbate in cells was determined by incubating 4 × 106 cells/ml in Tris/NaCl in the presence of ascorbate or DHA, with or without 1 mM ferricyanide. The suspension was incubated at 37 °C in a closed shaking water bath, and 1-ml aliquots were taken at set time points. After centrifugation, cells were washed twice in ice-cold Tris/NaCl containing a 100 µM concentration of the GLUT-1 inhibitor phloretin (11, 13). The cell pellet was extracted with 600 µl of methanol, which was diluted to 1 ml with water and 1 mM EDTA (end concentration). After centrifugation, the supernatants were analyzed for ascorbate on an HPLC system with a Partisil SAX column (10 µm, 250 × 4.6 mm), eluting with a gradient starting at 7 mM potassium phosphate (pH 4.0) and changing to 0.25 M potassium phosphate and 0.5 M KCl (pH 5.0). Ascorbate was detected at 265 nm using a Jasco 870-UV detector. Extraction and HPLC analysis were validated using [14C]ascorbate (Amersham Pharmacia Biotech). The extraction yielded over 95% of the cell-associated radioactivity, and HPLC analysis revealed that 90% of the intracellular radioactivity corresponded with ascorbate.
The stability of ascorbate and DHA was tested by incubating a 25 µM concentration of either compound with 4 × 106 cells/ml in Tris/NaCl, with or without 1 mM ferricyanide, at 37 °C in a closed shaking water bath. At set times, 100-µl aliquots were mixed with 50 µl of 0.1 M dithiothreitol, 600 µl of methanol, and 250 µl of water. The extracts were spun down, and the ascorbate concentration in the supernatant was determined by HPLC.
Glutathione (GSH/GSSG) depletion was achieved by incubating cells with L-buthionine-(RS)-sulfoximine (17). Cells were incubated at a density of 3.5 × 105/ml in growth medium supplemented with 0 or 500 µM L-buthionine-(RS)-sulfoximine. After 2 days at 37 °C, cells were centrifuged and washed twice in Tris/NaCl. Glutathione levels in cells, i.e. the total amount of GSH/GSSG, were measured using an enzyme cycling assay (18).
-Tocopherol depletion was achieved by growing cells for at least six
generations in culture medium in which fetal bovine serum was replaced
by 5 µg/ml transferrin, 5 µg/ml insulin, and 0.5% bovine serum
albumin (19).
-Tocopherol supplementation of cells was achieved by
growing cells for 2 days at 37 °C in culture medium supplemented
with 0, 30, or 100 µM
-tocopherol at an initial cell
density of 3.5 × 105 cells/ml.
-Tocopherol was extracted and analyzed essentially as described by
Thurnham et al. (20). Briefly, 5 × 106
cells were spun down and lysed with 10 mM SDS and ethanol
containing
-tocopherol acetate as an internal standard. The lysate
was extracted using heptane, which was subsequently evaporated under
N2. The samples were dissolved in the mobile phase of the
HPLC system (methanol/acetonitrile/chloroform, 47:47:6 (v/v)), injected
onto a Spherisorb ODS-2 column (250 × 4.6 mm), and eluted
isocratically. Both
-tocopherol and
-tocopherol acetate were
detected at 292 nm.
ESR spectra were recorded on a JEOL-RE2X spectrometer operating at 9.36 GHz with a 100-kHz modulation frequency and equipped with a TM110 cavity. Samples were transferred to a quartz flat cell using a sampling device, which allowed sampling within seconds after mixing. ESR spectrometer settings were as follows: microwave power, 40 milliwatts; modulation amplitude, 1 G; time constant, 0.1 s; and scan rate, 6 G/min.
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RESULTS |
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Ascorbate and DHA Metabolism-- Several reports mention the rapid irreversible degradation of DHA in solution (11, 21), yielding 2,3-diketo-L-gulonic acid (22). Therefore, the stability of ascorbate and DHA in cell suspensions was first determined. DHA can be measured by the addition of dithiothreitol, which converts DHA to ascorbate, after which the ascorbate concentration can be determined by its absorbance at 256 nm. Samples were mixed with dithiothreitol and methanol to determine the total, i.e. intra- and extracellular, amount of ascorbate and DHA. As expected, it was found that DHA was rapidly degraded in a suspension at pH 7.4 (Fig. 1), with a half-life of 8-9 min. Ascorbate remained stable for at least 90 min. Ferricyanide had no effect on the rate of DHA degradation (Fig. 1). Addition of ferricyanide to ascorbate, on the other hand, resulted in a rapid decrease in ascorbate, with similar kinetics as found for DHA.
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Enhancement of Ferricyanide Reduction-- Subsequently, the effect of ascorbate and DHA on ferricyanide reduction by HL-60 cells was investigated. Without ascorbate and DHA, the reduction of ferricyanide to ferrocyanide was linear for at least 90 min (Fig. 3). In the presence of ascorbate and DHA, the rate of reduction was higher, and the same linear kinetics were found for both compounds. It should be noted that, with ascorbate, a sudden increase in ferrocyanide levels could be observed within seconds after mixing of ferricyanide and ascorbate (Fig. 3). This increase, which also occurred in the absence of cells, had a stoichiometry of two ferrocyanide molecules formed per molecule of ascorbate added and resulted from the direct reaction between ascorbate and ferricyanide, generating DHA and ferrocyanide.
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Formation of Ascorbate Free Radical-- The ascorbate free radical has been proposed as an intermediate in the accelerated reduction of ferricyanide by K562 cells (9). It is relatively stable and results from the one-electron oxidation of ascorbate. To test its involvement in the accelerated reduction of ferricyanide, the formation of the radical was measured under various experimental conditions (Fig. 6). The ascorbate free radical can readily be observed in a 25 µM ascorbate solution in buffer, with an ESR spectrum consisting of a doublet with hyperfine splitting, aH4 = 1.8 G (Fig. 6A). Transition metal ions, which are always present in buffer solutions, mediate this formation of the ascorbate free radical (25). In the presence of 1 mM ferricyanide, no radical signal could be detected (Fig. 6B). The ascorbate free radical was also detected in the presence of HL-60 cells, although the signal intensity was lower than in the absence of cells (Fig. 6C). This may be caused by a decreased ascorbate degradation, due to chelation of transition metal ions, or by an increased regeneration by an AFR reductase. However, this was not further investigated, as it was considered to be outside the scope of this paper. Again, in the presence of ferricyanide, no radical signal could be detected (Fig. 6D).
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Involvement of GLUT-1 Transporter-- Extracellular DHA is taken up by HL-60 cells through a facilitative process, catalyzed by the GLUT-1 glucose transporter (11, 12). To test whether the activity of the GLUT-1 transporter was also involved in ascorbate- or DHA-mediated ferricyanide reduction, the effect of several GLUT-1 substrates and inhibitors was studied. Table I shows that glucose and its analogs 2-deoxyglucose and 6-deoxyglucose strongly inhibited the ascorbate-stimulated ferricyanide reduction as well as the accumulation of ascorbate. Cytochalasin B, which inhibits GLUT-1 (11), was also an effective inhibitor. Ferricyanide reduction in the absence of ascorbate was hardly affected by these compounds, showing that they did not have an effect on the ferricyanide reductase.
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Mechanism of Enhanced Ferricyanide Reduction-- Previous studies on erythrocytes suggest that ascorbate may serve as an intracellular electron donor for the plasma membrane reductase (26). The accumulation of ascorbate in HL-60 leads to a similar concept, in which the reduction of ferricyanide and the oxidation of ascorbate are coupled. Comparison of the data in Figs. 2A and 3 shows that the amount of ferrocyanide formed exceeded the amount of ascorbate in the cells: after 60 min, 12 nmol of ferricyanide was converted by 106 cells, whereas they contained 0.8 nmol of ascorbate. As one molecule of ascorbate can reduce two molecules of ferricyanide, this amount of ascorbate accounted only for the reduction of 1.6 nmol of ferricyanide. Thus, the presence of a recycling mechanism for ascorbate is required to explain the amount of ferricyanide reduced.
There are several ways by which intracellular ascorbate can reduce ferricyanide. It can either remain inside the cell and donate electrons to a redox system for ferricyanide reduction or, alternatively, leave the cell to react with ferricyanide directly. In the latter case, DHA will be formed outside the cells and has to re-enter the cell to be recycled to ascorbate. Thus, with a reuptake step being essential, the stimulation of ferricyanide reduction should remain sensitive to GLUT-1 inhibitors throughout the entire incubation period. As shown in Table I, this was not the case. Cytochalasin B inhibited only when added before the addition of ascorbate. When 5 µM cytochalasin B was added 20 min after ascorbate, no inhibition was observed. This indicates that ascorbate remains in the cell and that intracellular ascorbate mediates the accelerated reduction of ferricyanide. Replotting the data in Figs. 2B and 4 results in Fig. 7, which shows the relation between the intracellular concentration of ascorbate and the rate of ferricyanide reduction. This rate correlated to the intracellular concentration in a saturable manner, resembling Michaelis-Menten kinetics. A fit of the data resulted in an apparent Km of 0.29 nmol/106 cells, which corresponds to 0.55 mM (assuming a cell diameter of 10 µm).
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DISCUSSION |
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Ascorbate and DHA strongly increased the rate of ferricyanide reduction in HL-60 cells, as has previously been found for K562 cells and erythrocytes (9, 10). For K562 cells, it was suggested that the stimulation of ferricyanide reduction by ascorbate was due to a plasma membrane-localized AFR reductase (9). However, it is highly unlikely that this mechanism explains our results. The reaction between ferricyanide and ascorbate is very fast, and the stoichiometry of the reaction amounted to 2 mol of ferrocyanide formed per mol of ascorbate (Fig. 3). This indicates that the oxidation of ascorbate does not stop at the level of the ascorbate free radical, but that it continues to DHA. This was confirmed by ESR spectroscopy (Fig. 6). Addition of excess ferricyanide to a solution of ascorbate, as used in our experiments, resulted in the formation of DHA, not the ascorbate free radical. In the experiments with K562 cells, a similar excess of ferricyanide was used, and it is therefore also unlikely that, in K562 cells, the enhanced reduction of ferricyanide by ascorbate is mediated by an AFR reductase (9).
Another mechanism that has been proposed was based on studies on erythrocytes and involves the accumulation of ascorbate in cells, where it may serve as an intracellular electron donor for the plasma membrane reductase (10). The data presented in this paper also strongly suggest that intracellular accumulation of ascorbate in the cell is an essential part of the mechanism by which ascorbate or DHA has its effect on ferricyanide reduction. We found that only conditions that allowed the accumulation of ascorbate resulted in an accelerated reduction of ferricyanide (Fig. 2). In many cells, DHA is transported by a facilitative process, which is mediated by the GLUT-1 glucose transporter (11). Ascorbate, on the other hand, is not transported over the plasma membrane of HL-60 cells. Our experiments corroborate this view, as ascorbate was accumulated in the cells only when DHA was present in the extracellular medium (Fig. 2). Several substrates and inhibitors of the GLUT-1 transporter blocked the accumulation of ascorbate, showing the involvement of this transport system (Table I).
Intracellularly, DHA is reduced to ascorbate, as followed from control experiments using 14C-labeled ascorbate. This conversion was also found in erythrocytes incubated with DHA (10). However, on incubation with ferricyanide, erythrocytes showed an efflux of DHA. As DHA is transported by the facilitative GLUT-1 transporter, it can be transported in both directions following a concentration gradient. Apparently, in erythrocytes, ferricyanide induced the formation of intracellular DHA from ascorbate, which could subsequently leave the cell. However, no significant efflux of DHA from HL-60 cells could be observed, indicating the presence of an efficient regenerating system (Fig. 2). The major part of the DHA generated inside the cells was thus recycled to ascorbate, before it had a chance to be transported or hydrolyzed. Recently, Guaiquil et al. (12) reported that HL-60 cells have an efficient ascorbate-regenerating system that does not require intracellular glutathione. The lack of effect of glutathione depletion on ascorbate accumulation found in our study corroborates this view. It was concluded that uptake of DHA into the cell and its accumulation in the cell as ascorbate are essential steps in the enhancement of ferricyanide reduction.
The stimulation of ferricyanide reduction by ascorbate was inhibited by GLUT-1 inhibitors or competing substrates (Table I). This is in clear contrast to data recently obtained for erythrocytes, where it was found that inhibition of DHA transport by cytochalasin B did not impair the stimulation of ferricyanide reduction by ascorbate (13). This led to the conclusion that ascorbate was closely involved in the redox reaction, but that it acted both intra- and extracellularly. It was supposed that DHA could be regenerated to ascorbate independent of its cellular location. In HL-60 cells, this is clearly not the case. In these cells, the GLUT-1 transporter is involved in the stimulation of ferricyanide reduction by ascorbate and DHA.
The redox equivalents represented by intracellular ascorbate were exceeded many times (at least by a factor 8) by the total amount consumed by ferricyanide. This shows that ferricyanide reduction only lasts because ascorbate is continuously regenerated. Once ascorbate was accumulated in the cells, the addition of GLUT-1 inhibitors no longer had any effect on the rate of ferricyanide reduction (Table I). Thus, the effect of ascorbate or DHA on ferricyanide reduction cannot be explained by an excretion and reabsorption of ascorbate from the medium. Instead, the data show that ascorbate has its effect inside the cell, serving either as a direct substrate for a redox system or as an activator of such a system.
What redox system is responsible for the effect of ascorbate? It is possible that ascorbate-stimulated ferricyanide reduction uses the same system as the basal reduction of ferricyanide. However, when the effect of the inhibitor pCMBS on both the basal and ascorbate-stimulated reductions is compared, this seems to be unlikely. The basal reduction is much more sensitive to inhibition by pCMBS than the accelerated reduction (Table I). This indicates that the basal and accelerated reductions of ferricyanide are actually two separate processes, resulting in an additive reduction of extracellular ferricyanide. Summarizing our data, a model for the reduction of ferricyanide by HL-60 cells is proposed in Fig. 8. At least two membrane redox systems are present in HL-60 cells, both capable of reducing ferricyanide. The first system is the ferricyanide reductase, which is thought to use NADH as its source for electrons. The second system is the one proposed in this paper, and it relies on ascorbate for its reducing equivalents. It is interesting to note that, under normal cell culture conditions, there is only a limited supply of ascorbate to feed this system since serum contains very low levels of ascorbate (30). Under physiological conditions, however, ascorbate is present at much higher concentrations. Therefore, this redox system may be a formidable addition to the capability of the cell to counter oxidative processes at its surface, far exceeding the capacity of the NADH-dependent reductase.
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The proteinaceous character of the basal ferricyanide reductase seems
well established (24, 31-33), but the nature of the ascorbate-dependent system is not clear. It could involve
either direct chemical reactions or a membrane-based enzyme (2, 28). There have been reports in the literature suggesting a nonenzymatic route of electron transport. In liposomes, it was found that
-tocopherol, a natural antioxidant present in membrane lipids,
mediated ferricyanide reduction by ascorbate without intervention of an
enzyme system (2). Also, for erythrocytes, it was observed that
-tocopherol could augment ascorbate-induced ferricyanide reduction,
but the involvement of a membrane-localized enzyme system could not be excluded (2). Conversely, the present results show that, in HL-60
cells, the supplementation or depletion of
-tocopherol had no
significant effect on the efficacy of ascorbate in the stimulation of
ferricyanide reduction (Table II). Even in the total absence of
-tocopherol, HL-60 cells remain fully responsive to the addition of
ascorbate. This unequivocally shows that
-tocopherol does not play a
significant role in the stimulation of ferricyanide reduction by
ascorbate and DHA in HL-60 cells.
The intracellular level of ascorbate appeared to have a saturable dose-response relation with the observed rate of ferricyanide reduction, indicating that the reaction has Michaelis-Menten kinetics toward ascorbate. pCMBS inhibitor studies suggest the involvement of a protein since this compound is well known to interfere with protein functions through its reactivity toward sulfhydryl groups. Thus, these experiments support the view that ascorbate-driven ferricyanide reduction proceeds through a redox system containing a proteinaceous component. However, further experiments will be needed to establish the exact nature of the system by which intracellular ascorbate can donate electrons to extracellular ferricyanide.
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FOOTNOTES |
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* 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. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Molecular
Cell Biology, Leiden University, P. O. Box 9503, 2300 RA Leiden, The
Netherlands. Tel.: 31-71-5276057; Fax: 31-71-5276125; E-mail: dune{at}dds.nl.
1 The abbreviations used are: DHA, dehydroascorbate; AFR, ascorbate free radical; pCMBS, p-chloromercuribenzenesulfonic acid; HPLC, high performance liquid chromatography.
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REFERENCES |
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