Ascorbate interacts with reduced glutathione to scavenge phenoxyl radicals in HL60 cells

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Free Radical Biology & Medicine 44 (2008) 1637 – 1644 www.elsevier.com/locate/freeradbiomed

Original Contribution

Ascorbate interacts with reduced glutathione to scavenge phenoxyl radicals in HL60 cells Sarah L. Cuddihy, Amber Parker, D. Tim Harwood, Margreet C.M. Vissers, Christine C. Winterbourn ⁎ Department of Pathology, University of Otago Christchurch, Christchurch, New Zealand Received 12 November 2007; revised 11 January 2008; accepted 18 January 2008 Available online 7 February 2008

Abstract We have compared the abilities of ascorbate and reduced glutathione (GSH) to act as intracellular free radical scavengers and protect cells against radical-mediated lipid peroxidation. Phenoxyl radicals were generated in HL60 cells, through the action of their myeloperoxidase, by adding H2O2 and phenol. Normally cultured cells, which contain no ascorbate; cells that had been preloaded with ascorbate; and those that had been depleted of GSH with buthionine sulfoximine were investigated. Generation of phenoxyl radicals resulted in the oxidation of ascorbate and GSH. Ascorbate loss was much greater in the absence of GSH, and adding glucose gave GSH-dependent protection against ascorbate loss. Ascorbate, or glucose metabolism, had little effect on the GSH loss. Glutathionyl radical formation was detected by spin trapping with DMPO in cells lacking ascorbate, and the signal was suppressed by ascorbate loading. Addition of phenol plus H2O2 to the cells caused lipid peroxidation, as measured with C11-BODIPY. Peroxidation was greatest in cells that lacked both ascorbate and GSH. Either scavenger alone gave substantial inhibition but optimal protection was seen with both present. These results indicate that GSH and ascorbate can each act as an intracellular radical scavenger and protect against lipid peroxidation. With both present, ascorbate is preferred and acts as the ultimate radical sink for phenoxyl or glutathionyl radicals. However, GSH is still consumed by metabolically recycling dehydroascorbate. Thus, recycling scavenging by ascorbate does not spare GSH, but it does enable the two antioxidants to provide more protection against lipid peroxidation than either alone. © 2008 Elsevier Inc. All rights reserved. Keywords: Free radicals; Glutathione; Ascorbate; Myeloperoxidase; Radical scavenging antioxidant; HL60 cells; Spin trapping

Reduced glutathione (GSH) and ascorbate are well-established physiological antioxidants. A major contributor to their antioxidant protection is their ability to scavenge free radicals. There is no doubt that both are excellent scavengers in vitro. However, it is less clear whether one predominates intracellularly or whether they have independent or synergistic roles. Radical scavenging by GSH leads to the generation of glutathionyl radicals, which react successively with the thiolate of glutathione and then oxygen to produce superoxide (reactions (1)–(3)) [1–3]:

U U PhO þ GSH V PhOH þ GS

U U GS þ GS V GSSG 

ð1Þ ð2Þ

⁎ Corresponding author. Fax: +64 3 364 1083. E-mail address: [email protected] (C.C. Winterbourn). 0891-5849/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2008.01.021

U þ O

GSSG

2

U

Y GSSG þ O2

ð3Þ

U

2O2 þ 2Hþ Y H2 O2

ð4Þ

U

U

PhO þ AH Y PhOH þ AH

U

ð5Þ

U

GS þ AH þ Hþ Y GSH þ AH

ð6Þ

U

2AH Y AH þ DHA

ð7Þ

2GSH þ DHA Y GSSG þ AH

ð8Þ

U

U

(PhOH, phenol; PhO , phenoxyl radical; GS , glutathionyl U radical; GSSG −, glutathione disulfide radical anion; AH−, U ascorbate; AH , ascorbyl radical; and DHA, dehydroascorbate).

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In this way superoxide acts as a radical sink, and scavenging by GSH coupled with superoxide dismutase activity provides a cellular mechanism for radical removal [1]. However, H2O2 is generated (reaction (4)), with the possibility of amplifying prooxidant effects. On the other hand, scavenging by ascorbate forms ascorbyl radicals (reactions (5) and (6)), which dismutate (reaction (7)) so there is no subsequent formation of superoxide or H2O2. Although the ascorbate radical may itself react with superoxide to form hydrogen peroxide, due to the relative rate constants this can occur only under conditions of simultaneous low superoxide dismutase and high superoxide [4]. Ascorbate, therefore, could provide more complete antioxidant protection. Ascorbate is a stronger reductant than GSH. It has therefore been argued on kinetic grounds that when both are present, scavenging of glutathionyl and other radicals by ascorbate (reactions (6) and (7)) is favored and ascorbate is the more likely radical sink [5,6]. Further synergism between the two antioxidants is possible through the recycling of dehydroascorbate to ascorbate by GSH (reaction (8)). Although the direct reduction by GSH is slow, mammalian cells contain glutaredoxins [7] and plant cells dehydroascorbate reductases [8], which catalyze the reaction. It is clear from animal studies by Meister and colleagues [9] that the functions of ascorbate and GSH overlap. Ascorbate ameliorates the adverse effects of inhibiting GSH synthesis, and administration of glutathione ester delays the scorbutic effects of ascorbate deficiency. Studies of mammalian cells in which the scavenging roles of the two antioxidants have been compared directly are relatively few. This is partly because when these cells are cultured, they generally contain no ascorbate. Also, when ascorbate is simply added to culture medium, it undergoes autoxidation to generate hydrogen peroxide (H2O2), creating a prooxidant environment that complicates any antioxidant role [10]. Where the effects of manipulating GSH and ascorbate levels on the cytotoxicity of oxidants have been examined, each antioxidant individually was not as protective as the combination [11–13]. However, these studies did not establish mechanisms of cell death and protection or whether free radical scavenging was involved. We have investigated radical scavenging and interactions between ascorbate and GSH in cultured HL60 cells. These human myelocytic cells provide a good model for studying intracellular free radical reactions because they contain myeloperoxidase (MPO) [14,15], an enzyme capable of oxidizing numerous peroxidase substrates, including phenols, to their respective radicals (reaction (9)). Ascorbate is also a good substrate for the enzyme [16,17]: MPO

U

2PhOH þ H2 O2 Y 2PhO þ 2H2 O

ð9Þ

HL60 cells have been used extensively by Kagan and colleagues to show that adding H2O2 plus substrates such as phenol or etoposide leads to MPO-dependent generation of the substrate radical, with consequent oxidation of GSH, protein thiol loss, and lipid peroxidation [18–22]. These studies provide direct evidence for radical scavenging by intracellular GSH. However, the situation may be different for ascorbate-replete cells. We have investigated the fate of radicals generated in cultured HL60 cells with and without a physiological complement

of ascorbate and GSH. This was achieved using a combination of ascorbate preloading and inhibition of GSH synthesis with buthionine sulfoximine (BSO). We measured MPO-dependent GSH and ascorbate consumption on adding H2O2 with or without phenol and monitored glutathionyl radical formation with 5,5′ dimethyl-1-pyrroline N-oxide (DMPO) and lipid peroxidation as an example of radical damage to the cells. We found ascorbate to be the preferred radical scavenger. However, GSH was important for sparing ascorbate, and the greatest protection against lipid peroxidation was seen when both antioxidants were present. Materials and methods Cell culture and ascorbate loading HL60 cells were grown in suspension culture in RPMI 1640 medium supplemented with 10% fetal bovine serum, 25 units/ml penicillin, and 25 μg/ml streptomycin at 37°C in a 5% CO2 atmosphere. Low-passage-number cells were used. Cells were harvested during exponential growth when they were at densities of 0.5–1 × 106 cells/ml, washed, and suspended at 2 × 106/ml in phosphate-buffered saline (PBS; 10 mM phosphate buffer, pH 7.4, with 140 mM NaCl, 0.5 mM MgCl2, and 1 mM CaCl2). Cell counts were performed using a hemocytometer. To load the cells with ascorbate [23], dehydroascorbate was generated by treating ascorbic acid (2 mM in 10 mM phosphate buffer, pH 5.6, containing 140 mM NaCl) with ascorbate oxidase (1 unit/ml) for 5 min. This solution (60 μl/ml) was immediately added to the cell suspension along with 50 μM dithiothreitol (final concentration, to ensure complete intracellular reduction of imported dehydroascorbate). The cells were incubated for 20 min at 37°C, washed twice with PBS, recounted, and suspended at 1 × 106/ml in PBS with no glucose. Unloaded cells were subjected to the same incubation procedure but with no ascorbate/ascorbate oxidase. GSH depletion Cells were depleted of GSH by culturing in medium containing 0.5 mM BSO for 2 days [24]. They were then treated or loaded with ascorbate as for control cells. Treatment of HL60 cells with hydrogen peroxide Aliquots of cell suspension were treated with H2O2 alone or were preincubated for 10 min with 1 mM phenol before the requisite concentration of H2O2 was added. After 20 min at 37°C, cells were pelleted at 1000 g for 5 min and analyzed for ascorbate, glutathione, and oxidative markers. The H2O2 was standardized using ɛ240 = 43.6 M−1 cm−1. GSH analysis Cells (1 × 106) were suspended in 400 μl of PBS adjusted to pH 8.0 with KOH and treated with 1 mM monobromobimane for 20 min in the dark at room temperature [25]. Proteins were precipitated with trichloroacetic acid (5% w/v final concentration) and supernatants were subjected to high-performance

S.L. Cuddihy et al. / Free Radical Biology & Medicine 44 (2008) 1637–1644

liquid chromatography (HPLC) using a SPHERI-5 ODS 5μ column (Brownlee, Applied Biosystems, San Jose, CA, USA) with fluorescence detection (excitation 394 nm and emission 480 nm). Retention times and peak areas were compared with GSH standards derivatized by the same process. GSH and glutathione oxidation products were in some cases measured by stable isotope dilution liquid chromatography/mass spectrometry with selective reaction monitoring (LC/MS/MS) as described [26], using an LCQ DECA XPplus ion trap mass spectrometer (ThermoFinnigan, San Jose, CA, USA). Cells were treated with 4 mM N-ethylmaleimide to derivatize the GSH before lysis with 80% ethanol. Isotopically labeled internal standards for GSH and GSSG were added and lysates dried down and resuspended in PBS before injection. A control experiment (data not shown) showed no difference in cellular GSH content measured by this and the monobromobimane assay. Ascorbate analysis Pelleted cells (1 × 106) were lysed in 50 μl 0.54 M perchloric acid containing 0.25 mM diethylenetriamine pentaacetic acid, diluted with 50 μl water, and centrifuged to pellet protein. Supernatants were separated by HPLC using a Phenomenex Aqua 5μ C18 column with electrochemical detection (ESA Coulochem II detector) [27]. Retention times and peak areas were compared with ascorbic acid standards. Detection of spin trapped glutathionyl radicals Glutathionyl radicals were trapped as adducts with the spin trap DMPO and analyzed by HPLC as previously described [18] but with LC/MS/MS detection. Unloaded or ascorbate-loaded HL60 cells were resuspended at 2.5× 106 cells/ml and incubated for 3 min with 100 mM DMPO, in the presence or absence of phenol. Hydrogen peroxide was added and after 20 min at 37°C, the cells were pelleted; lysed in 200 μl of a solution containing 0.7 mM ascorbate, 5 mM DTT, and 500 U/ml catalase; and sonicated briefly. Supernatants were spun through C18 SPE columns equilibrated with the same solution. Eluates were analyzed using a Luna C18 column (150 × 2.0 mm) and a mobile phase of aqueous formic acid (0.1%) followed by a linear gradient from 0 to 70% methanol. The GS–DMPO nitrone adduct was observed at the requisite mass as a [M + H]+ion (m/z 419) eluting at 14.4 min. For semiquantitative analysis, a fragment ion (m/z 290) was monitored when undergoing collision-induced dissociation. This fragment represents a loss of pyroglutamate (129 Da) from the glutathione portion of the molecule, which is a common loss for glutathionylated species.

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Hydrogen peroxide consumption H2O2 consumption by HL60 cells was measured by analyzing samples of the incubation mixture by the ferrous sulfate/ xylenol orange assay [29]. MPO activity HL60 cells (106 in 200 μl) were lysed by adding NP-40 (final concentration 1%) and sonicating on ice for 1 min. Debris was removed by centrifugation, and MPO activity was measured using tetramethylbenzidine as substrate [30]. Materials All biochemicals were from Sigma Chemical Co. (St Louis, MO, USA), except for C11-BODIPY(581/591), from Invitrogen (Carlsbad, CA, USA), and reagent hypochlorite from Sarah Lee (Auckland, NZ). Results Interdependence of GSH and ascorbate loss in H2O2-treated HL60 cells Cultured HL60 cells (containing GSH but no ascorbate) and cells that had been depleted of N95% of their GSH by culturing in the presence of BSO were investigated. Both preparations were also studied after preloading with ascorbate. This was achieved by incubation with dehydroascorbate, which is taken up via glucose transporters and reduced intracellularly [31]. Based on preliminary measurements of time- and concentration-dependent increases in intracellular ascorbate (not shown), incubation conditions were selected to give a concentration similar to that in neutrophils. Ascorbate was not detectable in unloaded cells and increased to ∼20% of the GSH concentration after loading (Table 1). Ascorbate loading did not affect the GSH concentration. Cells were incubated with phenol and H2O2 for 20 min. Phenol oxidation has been shown to cause GSH consumption in normally cultured HL60 cells, via a mechanism involving MPO-dependent generation of the phenoxyl radical (reaction (9)) and scavenging by GSH [19]. In agreement with this, we observed that addition of phenol caused H2O2-dependent GSH loss (Fig. 1A). Ascorbate loading did not affect GSH loss, but the ascorbate became Table 1 Baseline concentrations of GSH and ascorbate in HL60 cells

Lipid peroxidation

Cell type

GSH (nmol/106 cells)

Ascorbate (nmol/106 cells)

GSH: ascorbate

Lipid peroxidation was assessed using C11-BODIPY(581/ 591). HL60 cells (1 × 106/ml in HBSS without glucose) were preloaded with 5 μM C11-BODIPY for 30 min as described [28] before exposure to H2O2. Peroxidation was quantified by flow cytometry (Beckman Coulter FC500, Fullerton, CA, USA), monitoring red fluorescence of unoxidized C11-BODIPY and green fluorescence of its oxidation product.

HL60 HL60–ascorbate loaded HL60–BSO treated, ascorbate loaded Neutrophil

3.02 ± 0.77 3.24 ± 0.58 n.d.

n.d. 0.58 ± 0.20 0.53 ± 0.12

5.5

1.32 + 0.20

0.42 + 0.06

3.2

Results for the HL60 cells are means + SD for 4–14 independent analyses. Values for neutrophils prepared from six different donors are given for comparison. n.d., not detectable.

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Fig. 1. Loss of (A, C) GSH and (B, D) ascorbate after treatment of HL60 cells with phenol and varying concentrations of hydrogen peroxide (A, B) or with hydrogen peroxide alone (C, D). Normally cultured cells (○, G), ascorbate-loaded cells (●, G + A), and cells that had been depleted of GSH and then loaded with ascorbate (□, A) were pretreated or not with 1 mM phenol and then reacted with H2O2 for 20 min and analyzed for GSH and ascorbate. The H2O2 was all consumed in that time. Phenol alone had no effect on control cells (not shown). Results are means ± SD for one to five separate experiments in which each analysis was carried out in duplicate or triplicate.

oxidized to a greater extent than GSH (Fig. 1B). Ascorbate loss was considerably greater in BSO-treated cells, indicating a sparing effect of GSH. Losses of ascorbate and GSH were not due to efflux from cells, as only trace amounts were detected in the supernatants (data not shown). Trypan blue staining after 20 min did not reveal any loss of viability. Analysis of glutathione oxidation products by LC/MS showed that only 7.5% of the GSH was converted to GSSG and 0.4% to glutathione sulfonamide, one of the products of the reaction of GSH with HOCl [26,32]. No other products were evident. GSH and ascorbate were also consumed when the cells were treated with H2O2 in the absence of phenol (Figs. 1C and D). GSH consumption was about fourfold less than with phenol present, whereas the decrease in ascorbate was only halved (∼50% loss with 20 μM H2O2 in Fig. 1B compared with 40 μM in Fig. 1D). As described elsewhere (A. Parker et al., submitted for publication), this consumption of ascorbate can be explained by its acting as a direct substrate for the MPO present in the HL60 cells. GSH loss was slightly less for cells containing no ascorbate (Fig. 1C) and there was much greater loss of ascorbate from GSHdepleted cells (Fig. 1D). The previous experiments were performed without added glucose. The presence of glucose gave substantial protection against ascorbate losses but did not affect GSH consumption (Table 2). Its effect was due to energy-dependent recycling of endogenous ascorbate rather than interference with ascorbate transport via glucose transporters, as the nonmetabolizable glucose analogue 2deoxyglucose (5 mM) gave no protection (70% of ascorbate consumed compared with 62% with no glucose shown in Table 2).

Evidence for glutathionyl radical formation and inhibition by ascorbate To determine if glutathionyl radicals were produced, the spin trap DMPO was added and the products were analyzed by LC/ MS. Others have shown that although DMPO traps the glutathionyl radical initially as a nitroxide, this is rapidly metabolized to EPR-silent species, including a stable nitrone [18]. We identified the GS-DMPO nitrone on the basis of its UV spectrum (λmax 258 nm) and by the protonated mass of 419 Da by full scan LC/MS. Treatment of HL60 cells with phenol and H2O2 produced a large GS-DMPO nitrone peak (Fig. 2). A significant but

Table 2 Effects of glucose on GSH and ascorbate consumption in HL60 cells Cell treatment and antioxidant content

Ascorbate (% consumption)

GSH (% consumption)

−glucose

+glucose

− glucose

+glucose

85 ± 2 N/A 91 ± 4

47 ± 16 N/A 79 ± 3

26 ± 7 16 ± 2 N/A

13 ± 3 16 ± 11 N/A

61 ± 13 N/A 93 ± 7

4±3 N/A 65 ± 6

29 ± 5 41 ± 13 N/A

28 ± 6 39 ± 6 N/A

a

H2O2 only GSH + ascorbate GSH only Ascorbate only H2O2 + phenolb GSH + ascorbate GSH only Ascorbate only

HL60 cells were normally cultured (GSH only), preloaded with ascorbate (GSH+ ascorbate), or pretreated with BSO (ascorbate only) and then treated with a80 nmol H2O2/106 cells or b1 mM phenol plus 20 nmol H2O2/106 cells. Results are means+ SD from three to eight independent experiments. N/A, not applicable.

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with cells containing ascorbate. Adding ascorbate did not affect the signal of preformed GS-DMPO (not shown). Lipid peroxidation and protection by GSH and ascorbate

Fig. 2. Detection of glutathionyl radical formation in HL60 cells by spin trapping with DMPO. Cells were preincubated with DMPO and then treated with either H2O2 alone (32 nmol/106 cells) or phenol (1 mM) and H2O2 (8 nmol/106 cells) for 20 min. Extracts were analyzed by LC/MS and areas corresponding to the glutathione-DMPO nitrone (GS-DMPO) determined. Values are expressed relative to the peak measured for cells incubated with H2O2 and phenol from the same experiment. Control cells incubated without phenol or H2O2 gave no detectable signal. Results are means and range for one or two independent experiments involving duplicate analyses.

very much reduced signal was observed when the cells were treated with H2O2 only (note that four times more H2O2 was added in the absence of phenol), and essentially no GS-DMPO was formed in control cells. The nitrone was scarcely detectable

Lipid peroxidation was investigated using C11-BODIPY as a reporter molecule. C11-BODIPY is a lipophilic polyunsaturated compound that localizes to membranes and changes fluorescence from red to green on oxidation. It has been shown to be a sensitive indicator of cellular lipid peroxidation [33]. Analysis by flow cytometry showed that addition of H2O2 alone to C11-BODIPYloaded HL60 cells caused a small increase in green fluorescence that was considerably enhanced in the presence of phenol (Fig. 3A). The abilities of ascorbate and GSH to protect against C11-BODIPY peroxidation in the presence (Fig. 3B) and absence (Fig. 3C) of phenol were examined using a combination of BSO treatment and ascorbate loading. Cells depleted of GSH with BSO had slightly higher baseline fluorescence and showed a substantially greater response to H2O2 than those containing GSH. Ascorbate gave near-complete protection of the BSO-treated cells at the lower H2O2 concentrations but less at higher concentrations at which it was all consumed. Greatest protection was seen when both antioxidants were present. No C11-BODIPY oxidation was

Fig. 3. Detection of lipid peroxidation in HL60 cells using C11-BODIPY. (A) Flow cytometry analyses of green fluorescence in control cells (gray shaded) or cells treated with 40 nmol H2O2/106 cells without phenol (solid trace) and with phenol (dashed trace). (B and C) Dependence of green fluorescence increase on H2O2 concentration in the presence (B) or absence (C) of phenol. Data points represent mean fluorescence of the sample relative to the untreated control from the same experiment. (▼) Normally cultured cells (containing only GSH); (●) ascorbate-loaded cells (GSH and ascorbate); (○) BSO-treated cells (no GSH or ascorbate); (▽) BSO-treated, ascorbate-loaded cells (ascorbate only). Results are means and SEM of four to six independent experiments, each carried out in duplicate or triplicate. The red fluorescent peak, corresponding to unoxidized C11-BODIPY, was comparable for all treatments. This indicates comparable loadings and that only a small fraction of the probe was oxidized.

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seen when high-passage-number HL60 cells that had lost their MPO were treated with H2O2 and phenol (not shown), indicating that lipid peroxidation was MPO-dependent. Discussion The objective of our study was to determine how intracellular ascorbate and GSH interact as free radical scavengers, using an HL60 cell model in which phenoxyl radicals are formed through the action of MPO. We observed consumption of GSH and ascorbate when the cells were exposed to both H2O2 and phenol. Consumption of GSH was substantially enhanced compared with cells treated with H2O2 alone, whereas the increase in ascorbate oxidation was more modest. As reported elsewhere (A. Parker et al., submitted for publication), ascorbate consumption is MPOdependent and is most likely due to direct ascorbate peroxidase activity of the enzyme. Ascorbate is known to be a good MPO substrate, with rate constants for the reaction of ascorbate with Compounds I and II similar to those for phenols such as tyrosine [16,17]. The low level of GSH oxidation in the absence of phenol, with glutathione sulfonamide formation as a minor product, could be explained by MPO converting a small amount of the added H2O2 to HOCl. When cells lacking ascorbate were treated with phenol and H2O2, spin trapping showed a substantial amount of glutathionyl radical formation. This is consistent with other studies [18,19] of phenoxyl radical scavenging by GSH in these cells. Small but detectable amounts of glutathionyl radicals were trapped in the absence of phenol. As GSH is an extremely poor MPO substrate [34], this suggests a very low level of radical generation from endogenous substrates under these conditions. The trapped GSDMPO nitrone signal was almost undetectable in cells containing ascorbate.1 This result is consistent with the ascorbate scavenging either the glutathionyl or the phenoxyl radical. However, it must be interpreted cautiously as ascorbate is able to reduce nitroxides to hydroxylamines [35]. An alternative explanation could be that reduction of the GS-DMPO nitroxide prevented its oxidation to the nitrone, the stable product detected by HPLC. However, the major route for nitroxide reduction in cells is proposed to be via microsomal reductases [36], which would be independent of ascorbate concentration. In addition, oxidation to the nitrone should be favored in HL60 cells as it is promoted by ferryl intermediates of heme proteins such as MPO [37]. On these grounds, it seems unlikely that reduction of the nitroxide by ascorbate could account for the near-complete inhibition of nitrone formation that we observed with ascorbate. Therefore, we 1 Attempts to detect ascorbyl radicals directly by EPR in our system were unsuccessful (J. M. May, personal communication). This was also the case when similar experiments were carried out by Kagan et al. [21] with Trolox instead of phenol, although they did observe a signal in the presence of aminotriazole. They attributed this to enhanced Trolox radical formation due to aminotriazole inhibiting catalase but not MPO. However, it is not obvious why inhibition of catalase would be necessary to see ascorbyl radicals in our situation, as it was not required to see ascorbate oxidation or MPO-dependent glutathionyl radical formation. There is also the possibility of involvement of semidehydroascorbate reductase activity, although this activity has not been documented for HL60.

conclude that at least some of this inhibition represents radical scavenging and that in HL60 cells containing physiological concentrations of GSH and ascorbate, ascorbate is favored as the ultimate radical sink. In related studies, Kagan and coworkers showed that peroxidase-generated phenoxyl radicals react preferentially with ascorbate in either cell lysates [38] or ascorbate-loaded HL60 cells [21]. However, their studies were performed with hindered phenols (etoposide and a vitamin E analogue) that react poorly or not at all with GSH. Our results with a more reactive phenoxyl radical in intact cells provide more direct comparison between the two scavengers. We observed a complex interplay between ascorbate and GSH oxidation. There was more oxidation of both species in cells treated with H2O2 and phenol than with H2O2 alone, but the changes were qualitatively similar. Ascorbate loading of the cells resulted in substantial ascorbate oxidation and suppression of the GS-DMPO signal. Scavenging of glutathionyl radicals would regenerate GSH, yet ascorbate did not decrease the extent of GSH oxidation. However, GSH did protect the ascorbate, as losses were much greater in BSO-treated cells. Furthermore, adding glucose to the cells prevented most of the ascorbate loss and had little effect on GSH. Interestingly, and consistent with the low recovery of GSH in the presence of glucose, GSSG was not a major product. There was scarcely any export of GSH or formation of glutathione sulfonamide, a product of HOCl oxidation [26,32], and most of the GSH lost was unaccounted for. Possible explanations include the formation of protein mixed disulfides and perhaps degradation. Our results imply that, whereas GSH loss was largely irreversible, the cells were able to metabolically recycle dehydroascorbic acid. The greater ascorbate losses and the limited protection by glucose in GSH-depleted cells point to recycling being GSH-dependent. These findings shed light on the mechanism of dehydroascorbate reduction in HL60 cells. In other cell types, such as erythrocytes [39] or endothelial cells [40], reduction is mainly GSH-dependent, with thioredoxin reductase playing a minor role. Direct reduction by GSH is slow, however, and the reaction is most likely catalyzed by glutaredoxin [7]. For HL60 cells, Guaiquil et al. [23] observed that dehydroascorbate was reduced when loaded into GSH-depleted cells and concluded that GSH is not required. Our loading studies agree with theirs. However, when the cells were stressed with H2O2 (±phenol), ascorbate loss was much greater in the absence of GSH. It seems, therefore, that there is a maintenance level of dehydroascorbate reduction independent of GSH, but increased recycling of ascorbate under oxidative stress requires GSH-dependent reduction. This interpretation is consistent with the impaired ascorbate reduction associated with glutathione reductase deficiency in mature neutrophils [41]. To examine radical-mediated cell damage to the HL60 cells, we measured oxidation of the fluorescent probe, C11-BODIPY, as an index of lipid peroxidation. Cells treated with the substrates for phenoxyl radical generation underwent MPO-dependent C11BODIPY oxidation. Peroxidation was less, although still significant, in cells treated with H2O2 alone, suggesting that endogenous MPO substrates, such as tyrosine derivatives or nitrite, may be oxidized to their radicals to initiate the process. Peroxidation was highest in cells lacking GSH and ascorbate, with

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Scheme 1. Proposed reactions initiated by H2O2 reacting with MPO in HL60 cells. PhOU, phenoxyl radical; AscU, ascorbyl radical; GSU, glutathionyl radical. Reactions of radicals generated from endogenous MPO substrates would be similar to those of PhOU and have not been shown. The small proportion of the H2O2 reacting with MPO and chloride to form HOCl, suggested from our results, is not shown.

either antioxidant providing substantial protection (in the case of ascorbate, until it was all consumed). This implies that GSH and ascorbate are both effective radical scavengers for protecting against cellular lipid peroxidation. It is noteworthy that the greatest protection was seen with a combination of ascorbate and GSH. This could be because the combination gives the greatest scavenging efficiency, or perhaps more likely because ascorbate concentrations are maintained more effectively through recycling by GSH. Thus our results show that GSH and ascorbate can to some extent substitute for one another as radical scavengers, but optimal protection involves synergy between the two. In apparent contradiction to our observation that GSH protected against lipid peroxidation in HL60 cells, Borisenko et al. [22] concluded that glutathionyl radicals propagate oxidative stress. They also examined cells treated with H2O2 and phenol and showed that scavenging glutathionyl radicals with Ac-Tempo decreased the extent of lipid peroxidation. However, whereas their results imply that glutathionyl radicals can cause some lipid peroxidation, our finding that peroxidation was many fold higher in cells lacking GSH implies that they have low promotional activity compared with other radicals. Therefore, although not absolute, GSH can still be protective in regard to lipid peroxidation. It is, however, interesting to note recent work indicating that thiyl radicals are capable of promoting cis–trans isomerization of lipids, the biological consequences of which are only just being explored [42]. A mechanism that explains our results and the interplay between GSH and ascorbate in MPO-containing HL60 cells is given is Scheme 1. Addition of H2O2 to cells containing GSH but no ascorbate results in limited oxidation of endogenous one-electron substrates of MPO, giving rise to low levels of glutathionyl radicals and lipid peroxidation. With phenol added, phenoxyl radicals are generated and lipid peroxidation and glutathionyl radical formation are much enhanced. More radicals react with GSH than induce lipid peroxidation as peroxidation is much greater when no GSH is present. There is little GSSG or metabolic recycling of oxidized GSH. When the cells contain ascorbate, it acts both as a substrate for MPO and as a radical scavenger and is readily consumed. Phenoxyl radicals are scavenged either directly

by ascorbate or indirectly via GSH, making ascorbate the main radical sink. The ascorbyl radicals generated in this process dismutate to give dehydroascorbate, which is metabolically recycled by a GSH-dependent mechanism. Thus, GSH protects against ascorbate loss but ascorbate, even though inhibiting radical-mediated GSH loss, does not preserve GSH. Radical scavenging by ascorbate inhibits lipid peroxidation, with the greatest protection seen when GSH is present to recycle dehydroascorbate. These findings should be applicable to other cell situations in which radicals are generated. We predict that ascorbate should be favored over GSH as the main radical sink. However, the situation should change and scavenging by GSH become more prominent if ascorbate is depleted or if compartmentalization restricts its access to the radicals. It is also noteworthy that cultured cells generally lack ascorbate and will therefore be more reliant on GSH as a scavenger. A major difference between the two mechanisms is that ascorbate radicals terminate the chain by dismutation, whereas glutathionyl radicals can lead to secondary generation of superoxide and hydrogen peroxide [1]. We observed protection against lipid peroxidation by GSH, but adverse effects of these secondary products are a possibility. Acknowledgments This work was supported by the Health Research Council of New Zealand, National Research Centre for Growth and Development. References [1] Winterbourn, C. C. Superoxide as an intracellular radical sink. Free Radic. Biol. Med. 14:85–90; 1993. [2] Wardman, P.; von Sonntag, C. Kinetic factors that control the fate of thiyl radicals in cells. Methods Enzymol. 251:31–45; 1995. [3] Pichorner, H.; Metodiewa, D.; Winterbourn, C. C. Generation of superoxide and tyrosine peroxide as a result of tyrosyl radical scavenging by glutathione. Arch. Biochem. Biophys. 323:429–437; 1995. [4] Scarpa, M.; Stevanato, R.; Viglino, P.; Rigo, A. Superoxide ion as active intermediate in the autoxidation of ascorbate by molecular oxygen. J. Biol. Chem. 258:6695–6697; 1983.

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