INTRODUCTION

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HEME-MEDIATED REDOX REACTIONS have been suggested to contribute to organ dysfunction and/or tissue damage that occurs in some pathological states characterized by the release of hemoglobin (Hb) and myoglobin into the extracellular environment (1, 21, 29, 44). These redox reactions can interfere with the normal balance of reactive oxygen or nitrogen species (2, 36). The oxidation of Hb generates potentially cytotoxic products such as the ferryl heme intermediate (Fe⁴⁺), methemoglobin (Fe³⁺), hemichromes, and free heme or iron (26, 27, 32, 39, 47). In addition, the autoxidation of oxyhemoglobin to methemoglobin releases reactive oxygen species that in turn may lead to tissue damage. Hb is normally sequestered inside red blood cells where protective antioxidants such as superoxide dismutase (SOD), catalase, and glutathione limit its potentially harmful oxidation reactions and preserve the oxygen-carrying ability of Hb. However, cell-free Hbs are devoid of these antioxidants, and large amounts of Hb in the circulation can overwhelm plasma protective mechanisms such as haptoglobin and hemopexin. This is especially relevant because cell-free Hbs are being developed as oxygen-carrying therapeutics (2, 13).

The proposed clinical indications for hemoprotein-based oxygen carriers include surgery and resuscitation from hemorrhagic or septic shock (2, 13). Increased reactive oxygen or nitrogen species production and/or antioxidant depletion during these pathological states increases the potential for these infused hemoproteins to interact with tissue oxidants and further aggravate the oxidative burden on tissues (6, 10, 30, 51). Owing to its direct and continuous contact with circulating blood, the endothelium may be a likely target for cell-free, Hb-mediated cytotoxicity. In vitro studies suggest that Hb-catalyzed formation of free radicals and the breakdown products produced during Hb oxidation can damage endothelial cells (8, 9, 28, 42, 45). The presence of Hb was shown to induce heme oxygenase-1 (HO-1), the enzyme responsible for heme degradation, and to markedly enhance H₂O₂ cytotoxicity in endothelial cells (8, 9). Moreover, diaspirin cross-linked Hb (DBBF-Hb), a candidate oxygen therapeutic, was shown to induce hepatic heme HO-1 in rats, suggesting a protective response triggered to counter potential heme-related toxicity (50).

H₂O₂, produced by superoxide dismutation or direct enzymatic production (SOD, amine oxidase, glucose oxidase), can be generated by a variety of mammalian cells, including neutrophils, macrophages, vascular smooth muscle, and endothelial cells. The production of H₂O₂ and peroxynitrite (ONOO), a highly reactive species generated from the interaction of nitric oxide (NO) and superoxide anion (O₂·), may be increased under conditions such as ischemia-reperfusion (10, 30). H₂O₂ and ONOO have been shown to induce both apoptotic and necrotic cell death (34, 41, 49, 56). H₂O₂ can react with transition metals such as iron to generate the highly reactive hydroxyl radical, which can oxidize a variety of biological molecules. H₂O₂ can also react with Hb and other heme proteins to produce a higher oxidation state of iron, Fe⁴⁺, which can initiate membrane lipid peroxidation and oxidize other macromolecules (27, 47). The pseudoperoxidase activity of hemoproteins involving the ferryl heme intermediate is well recognized (Hb cycles between Fe³⁺ and Fe⁴⁺ as it consumes H₂O₂), and this activity has been detected in human and animal whole blood by electron paramagnetic resonance (EPR) spectroscopy (57).

Reduced glutathione (GSH), the most abundant cellular thiol (0.5-10 mM), plays an important role in regulating the intracellular redox environment (4, 15). Oxidized glutathione (GSSG) is formed via the action of glutathione peroxidase in the detoxification of H₂O₂ and other peroxides. GSSG is recycled back to GSH by NADPH-dependent glutathione reductase. The status of intracellular GSH has been shown to affect the course of apoptosis and/or necrosis induced by various stimuli in several different cell types (18, 20, 25, 43, 58).

In this study we measured intracellular GSH to assess the cellular oxidative stress induced in bovine aortic endothelial cells incubated with Hb and H₂O₂. We compared two types of cell-free Hb, ultrapure human Hb (HbA₀) and DBBF-Hb, based on their different susceptibility to oxidative modification. In addition, the role of GSH in modulating the effects of Hb/H₂O₂ interactions on the mode of cell death (apoptosis and/or necrosis) was determined in cells depleted of intracellular GSH by pretreatment with buthionine-[S,R]- sulfoximine (BSO), an inhibitor of -glutamylcysteine synthetase.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials. Endothelial growth media (EGM) and fetal bovine serum was obtained from Clonetics (San Diego, CA). HbA₀ was obtained from Hemosol (Missauga, ON, Canada). Human Hb cross-linked by bis(3,5-dibromosalicyl)fumarate (DBBF-Hb) was a kind gift from the Walter Reed Army Institute of Research, Washington, DC. Both of these Hbs were found to be free of SOD and catalase (12). Caspase inhibitors, z-VAD-fmk [benzyloxycarbonyl-Val-Ala-Asp- (OMe)-CH₂F] and z-DEVD-fmk [benzyloxycarbonyl-Asp- (OMe)-Glu(OMe)-Val-Asp(OMe)-CH₂F] were purchased from Enzyme Systems Products (Livermore, CA). Hanks' balanced salt solutions (HBSS) were obtained from Life Technologies (Grand Island, NY). H₂O₂, 5,5' dithio-bis(2-nitrobenzoic acid) (DTNB), propidium iodide (PI), 5-sulfosalicylic acid, DL-buthionine-[S,R]-sulfoximine, SOD from bovine erythrocytes, bovine liver catalase, and deferoxamine mesylate were obtained from Sigma Chemical (St. Louis, MO).

Endothelial cell culture. Bovine aortic endothelial cells (BAECs) (BW-6002, Clonetics) were grown in EGM supplemented with 10 ng/ml human recombinant epidermal growth factor, 1 µg/ml hydrocortisone, 10 µg/ml bovine brain extract, 5% fetal bovine serum, 50 µg/ml gentamicin, and 50 ng/ml amphotericin B. Cells were cultured in 100-mm petri dishes and kept in a 37°C humidified incubator with 95% air-5% CO₂. Before the experiments, cells were subcultured into six-well plates (Costar) using a 1:3 splitting ratio and were grown to confluency in 2-3 days (~1 × 10⁶ cells/well). For cell harvesting, BAECs were washed with HEPES-buffered saline and incubated with 0.025% trypsin-0.01% EDTA (Clonetics) for 5 min at 37°C. After cell detachment, trypsin-neutralizing agent was added and cells were centrifuged at 220 g for 5 min at 4°C. Cell counts were obtained using a Coulter counter (Hileah, FL). Experiments were performed with BAECs in their second to eighth passage. Phenol red-containing media was used except in experiments to assess hemoprotein oxidation.

GSH determination. GSH was measured using the DTNB colorimetric assay (19). Briefly, cells were washed with 2 ml of HBSS, and 0.6 ml of 2% wt/vol 5-sulfosalicylic was added. After the samples were centrifuged for 5 min at 10,000 g, 500-µl aliquots were mixed with 500 µl of freshly prepared DTNB solution (0.3 M sodium phosphate, 10 mM EDTA, and 0.2 mM DTNB). After 5 min, the absorbance was measured at 412 nm. Calculations were based on the extinction coefficient of 13.4 mM¹/cm derived from glutathione standards. Cell counts were measured in parallel incubations. Though this assay is not specific for GSH, it has previously been reported that GSH is the primary thiol measurable in endothelial cells with this assay (19, 24).

Membrane-associated heme determination. Cells were washed twice with 2 ml HBSS, and 0.6 ml concentrated formic acid was added to each well. The absorbance of formic acid-solubilized cells was measured at 398 nm using a double-beam Perkin Elemer Lamba 18 spectrophotometer. Calculations were based on the heme extinction coefficient of 1.56 × 10⁵ M¹/cm (45).

Analysis of Hb oxidation products. Spectral analysis of Hb oxidation products was performed using a Hewlett-Packard HP-8453 rapid scanning diode array spectrophotometer. Experiments were performed in culture media at 37°C. The percentages of oxy (Fe²⁺), met (Fe³⁺), and ferryl (Fe⁴⁺) forms of Hb were calculated as previously described (12, 64).

Annexin V binding/PI staining and flow cytometry. At the end of incubation, nonadherent and adherent cells harvested by trypsinization were pooled, washed twice in PBS, and resuspended in 1 ml of assay buffer containing (in mM) 10 HEPES/NaOH, 140 NaCl, and 2.5 CaCl₂, with pH 7.4. Annexin V-FITC (5 µl of 2 µg/ml; Pharmingen, San Diego, CA) and PI (10 µl of 5 µg/ml) were added to a 100-µl aliquot (~1 × 10⁵ cells) of this cell suspension and incubated for 15 min in the dark at room temperature. After the stained cells were diluted with 400 µl of assay buffer, 10,000 cells per sample were analyzed using a FACScan flow cytometer (Becton-Dickinson, San Jose, CA) and CellQuest analysis software. The stained cell populations were defined as: R1, viable or undamaged cells (annexin V negative, PI negative); R2, cells undergoing early apoptosis (annexin V positive, PI negative); R3, necrotic or late apoptotic cells (annexin V positive, PI positive); and R4, heme-damaged cells (annexin V negative, heme positive) (61).

Statistical analysis. Data are represented as means ± SE for replicate experiments unless otherwise stated. Statistical analysis of the data was performed by one-way ANOVA and unpaired two-tailed Student's t-test using SAS JMP (ver. 3.2) software (SAS Institute, Cary, NC).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reaction of H₂O₂ with Hb: changes in Hb absorbance spectra. Figure 1, A and B, shows typical changes in the absorbance spectra of HbA₀ and DBBF-Hb, respectively, following bolus addition of H₂O₂ at 37°C. The oxidation of Fe²⁺ Hb occurred rapidly, as shown by the decrease in the absorbance peaks at 541 and 577 nm. This was accompanied by rapid formation of the ferryl intermediate, as evidenced by the appearance of two new peaks at 545 and 588 nm and a flattened region in the visible spectrum between 600 and 700 nm (3, 12, 27). Spectral changes indicative of ferryl formation occurred within the first 2-3 min following the addition of H₂O₂ for both Hbs. As the ferryl intermediate decays, the accumulation of Fe³⁺ was clearly detectable as an absorbance increase at 630 nm. This is consistent with the known ability of Hb to undergo Fe³⁺/Fe⁴⁺ redox transition as it removes H₂O₂, also known as the pseudoperoxidase cycle (3, 12). Figure 2A shows the time course of ferryl formation following H₂O₂ addition in the presence or absence of catalase. Ferryl formation was prevented when catalase was included in the incubation mixture before the addition of H₂O₂. However, catalase added 5 min after H₂O₂ did not prevent the rapid onset of the ferryl intermediate. The rate of disappearance for ferryl DBBF-Hb over 30 min was slower than that of ferryl HbA₀, suggesting that DBBF-Hb consumes H₂O₂ at a slower rate. This observation is consistent with the calculated rates of disappearance for the ferryl forms of DBBF-Hb (12 ± 0.2 h¹) and HbA₀ (21 ± 1.4 h¹) previously reported (3, 12). HbA₀, however, undergoes excessive denaturation starting ~60 min after the addition of H₂O₂, as shown by the increase in turbidity measured at 700 nm (Figs. 1A and 2B) (39). In contrast, DBBF-Hb was more resistant to this denaturation presumably owing to stabilization of its -chains (39).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Changes in visible absorbance spectra of ultrapure human hemoglobin (Hb) (HbA0; A) or its diaspirin cross-linked derivative (DBBF-Hb; B) after addition of H2O2. Reaction mixtures were prepared containing 50 µM HbA0 or DBBF-Hb in complete media (without phenol red), and the reaction was started by adding 500 µM H2O2. Spectra were collected for 90 min at a rate of one scan every 2 min at 37°C. First spectra (t = 0) were collected before H2O2 addition when both Hbs were in the ferrous (Fe2+) state.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   A: time course for formation of ferryl intermediate after addition of 500 µM H2O2 to 50 µM HbA0 or DBBF-Hb in the presence or absence of 500 U/ml catalase (CAT). CAT was added to incubation mixture before or 5 min after H2O2 addition. First point at t = 0 was measured before addition of H2O2; arrow at 2 min indicates when H2O2 was added. B: HbA0 and DBBF-Hb denaturation after exposure to H2O2. Reaction mixtures were prepared containing 50 µM HbA0 or DBBF-Hb in complete media (without phenol red) and 500 µM H2O2, and absorbance at 700 nm, indicative of sample turbidity, was monitored for 4 h.

Reaction of H₂O₂ with Hb: effect on endothelial cell GSH. The level of GSH measured in control endothelial cells ranged between 8 and 10 nanomoles per 1 million cells, in agreement with previously reported values (19). Figure 3A shows the effects of H₂O₂ on GSH levels in the presence or absence of HbA₀ or DBBF-Hb after a 2-h incubation. Within this time frame, morphological changes or increased cell detachment were not observed with any of the treatments (data not shown). Bolus addition of 0.5 and 1 mM H₂O₂ decreased intracellular GSH by 6 ± 3% and 18 ± 1%, respectively. These modest decreases in GSH may reflect that endothelial cells possess an efficient GSH reductase system, and in the case of bolus addition, endothelial cell catalase also plays a significant role in H₂O₂ detoxification (5, 55). The combination of H₂O₂ with HbA₀ or DBBF-Hb, however, produced significantly greater decreases in GSH compared with H₂O₂ alone (Fig. 3A). Figure 3A also shows that equal concentrations of HbA₀ and DBBF-Hb (50 µM) produced the same degree of GSH loss. Increasing the concentration of DBBF-Hb from 25 to 250 µM produced even greater decreases in GSH. The drop in GSH as a function of H₂O₂ concentration appeared to follow a biphasic pattern, with a threshold of ~250 µM H₂O₂. Figure 3B shows the maximum level of ferryl intermediate formed in mixtures containing 50 µM of HbA₀ or DBBF-Hb in the presence of H₂O₂. Interestingly, the maximum level of ferryl formation also leveled off at ~250 µM H₂O₂, suggesting a possible correlation between the loss of intracellular thiol and ferryl Hb levels.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   A: measurement of reduced glutathione (GSH) after 2 h incubation with 50 µM HbA0 or DBBF-Hb (25, 50, and 250 µM) and different concentrations of H2O2. GSH values were recorded as percentage of control cells (media, no H2O2) for each experiment and reported as means ± SE for two to five independent experiments done in triplicate. B: formation of ferryl intermediate after addition of different H2O2 concentrations to cuvette mixtures containing 50 µM HbA0 or DBBF-Hb in complete media (without phenol red) at 37°C. Values represent maximum amounts of ferryl intermediate produced 2-3 min after addition of H2O2. Each point represents mean ± SE for two or three samples.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Effect of antioxidants on intracellular GSH after treatment with Hbs and H2O2. Endothelial cells were cultured for 2 h with 50 µM HbA0 or DBBF-Hb and 500 µM H2O2. For deferoxamine (DFO) treatment, cells were preincubated with 0.5 mM DFO for 3 h, washed, then treated with Hb and H2O2. Superoxide dismutase (SOD; 40 U/ml) was added to incubation mixture before 500 U/ml H2O2 was added; CAT was added before or 5 min after addition of H2O2. Values were recorded as percentages of control cells (media alone, no H2O2) for each experiment and reported as means ± SE for two to five independent experiments, each done in triplicate. *P < 0.05 vs. control.

Endothelial cell uptake of heme-associated degradation products. Figure 5A shows the uptake by endothelial cells of the heme or heme-associated degradation products, produced from the reaction of HbA₀ or DBBF-Hb with H₂O₂. A 4-h incubation with 50 µM of HbA₀ or DBBF-Hb alone produced negligible increases in heme uptake, whereas small but significant increases were noted with 200 µM (data not shown). Incubation with HbA₀/H₂O₂ caused increases in heme uptake 10- to 15-fold greater than with DBBF-Hb/H₂O₂ (note the different y-axes in Fig. 5A). Heme uptake was greater after incubation with 200 µM compared with 50 µM with both Hbs. Figure 5B shows that in the case of HbA₀ heme uptake increased with increasing concentrations of H₂O₂, due to the greater oxidant-induced denaturation of HbA₀. On the other hand, DBBF-Hb produced detectable but smaller increases in heme uptake, owing to its greater stability.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   A: measurement of membrane-associated heme in endothelial cells incubated with 1 mM H2O2 and 50 or 200 µM HbA0 or DBBF-Hb. After 4 h of incubation, cells were washed and heme or heme-containing compounds were measured at 398 nm in formic acid-solubilized cell extracts. Results are expressed as nanomoles of heme per 106 cells and reported as means ± SE for three to six independent measurements. *P < 0.05 vs. control; #P < 0.05 vs. 50 µM (note different y-axes). B: measurement of membrane-associated heme in endothelial cells after 4 h of incubation of 50 µM HbA0 or DBBF-Hb and different concentrations of H2O2.

Endothelial cell death (apoptosis vs. necrosis): annexin V binding assay. The externalization of phosphatidylserine (PS) from the inner leaflet of the plasma membrane to the outer surface of cells occurs during the early stages of apoptosis (41, 61). This event precedes the loss of membrane integrity that occurs in the later stages of cell death (41, 61). The phospholipid-binding protein annexin V can bind to exposed PS on the surface of plasma membranes, and thus can be used to detect cells undergoing early apoptosis. PI, a nucleic acid stain, enters cells with leaky plasma membranes and thus stains cells that are necrotic or in the later stages of apoptosis (61). Therefore, this assay does not distinguish between cells that have already undergone an apoptotic death and those that died only by necrosis, because in both cases the dead cells stain positive for annexin V and PI.

View larger version (46K): [in this window] [in a new window]   Fig. 6.   Analysis of phosphatidylserine externalization and membrane integrity of endothelial cells using flow cytometry and annexin V/propidium iodide (PI) staining. A: cells were incubated for 5 h with media alone (control) or 500 µM H2O2 with and without caspase inhibitor z-VAD-fmk. For inhibitor treatment, cells were preincubated for 90 min with 40 µM inhibitor and washed; then 500 µM H2O2 was added with another 40 µM of inhibitor. B: typical histograms representing annexin V staining of the PI-negative cell population (R1 and R2). Cells were incubated for 5 h with 500 µM and 1 mM H2O2. Cells were pretreated with z-VAD-fmk and z-DEVD-fmk as in A. CAT (500 U/ml) was added to incubation mixture before H2O2. Mean fluorescence of gated cell population is shown for each panel.

View larger version (32K): [in this window] [in a new window]   Fig. 7.   Analysis of phosphatidylserine externalization and membrane integrity of endothelial cells using flow cytometry and annexin V/PI staining. Cells were exposed to 500 µM H2O2 and 50 µM HbA0 or DBBF-Hb. After 5 h cells were harvested and analyzed as described in MATERIALS AND METHODS.

Endothelial cell toxicity: normal versus GSH-depleted cells. Our results clearly show that cellular GSH is consumed or decreased when exposed to hemoprotein and H₂O₂, possibly as part of a protective mechanism. Hemoprotein-induced oxidative stress may therefore be particularly damaging under certain disease states associated with decreased antioxidant status (6, 35, 48, 51, 63). To model decreased antioxidant status, we targeted the production of GSH with BSO, an irreversible inhibitor of -glutamyl-cysteine synthetase, an enzyme in the GSH synthesis pathway. Pretreatment with 1 mM BSO for 20 h decreased GSH by 80% (data not shown) (17). Figure 8 summarizes the percentage of viable, early apoptotic, necrotic/late apoptotic, and damaged cells, as determined by fluorescence-activated cell sorter analysis of annexin V/PI staining, following a 5-h incubation with H₂O₂ and H₂O₂/Hbs in normal and GSH-depleted endothelial cells. The viability of control endothelial cells was not affected by BSO pretreatment. The percentage of necrotic or late apoptotic cells in control cells (~14%) may largely reflect the unavoidable death produced during the trypsin-harvesting procedure, along with the normal basal level present in culture. Incubation with 50 µM HbA₀ or DBBF-Hb alone had no significant effect on any of the measured parameters in normal or GSH-depleted control cells (data not shown). Compared with control, H₂O₂ increased the number of early apoptotic cells (R2) by 12 and 24% in normal and GSH-depleted cells, respectively (P  0.01). There were, however, three- and twofold reductions in the number of early apoptotic cells when HbA₀ and DBBF-Hb were added to the medium with both normal and GSH-depleted cells. With regard to necrotic or late apoptotic cells (R3), H₂O₂ alone or in the presence of Hbs produced small increases, which were not significant, in cells with normal GSH levels. However, there were significant increases in necrotic or late apoptotic cells compared with control across all treatment groups in GSH-depleted cells (P  0.05). In the case of DBBF-Hb/H₂O₂, the level of necrotic or late apoptotic cells was significantly greater in GSH-depleted cells compared with the same treatment in normal cells (P  0.01). As far as those cells with heme-related damage (R4), HbA₀-H₂O₂ produced significant increases in normal and GSH-depleted cells, whereas lower but significant increases were produced in the case of DBBF-Hb/H₂O₂. Under these conditions, therefore, H₂O₂ appears to predominately increase apoptosis in GSH-depleted cells, whereas in the presence of Hbs, H₂O₂ induced less apoptosis seemingly in favor of necrosis or heme-related cell damage. In the presence of Hbs, the degree of H₂O₂-induced necrosis was not increased compared with that caused by H₂O₂ alone (see Fig. 8).

View larger version (41K): [in this window] [in a new window]   Fig. 8.   Analysis of phosphatidylserine externalization and membrane integrity or damage in endothelial cells using flow-cytometry analysis of annexin V/PI staining. Normal cells were grown in standard culture media 2-3 days before the experiment, whereas GSH-depleted cells were pretreated with 1 mM BSO for 20-24 h before the indicated treatments. Normal or GSH-depleted cells were incubated with media alone, 500 µM H2O2, 50 µM HbA0 plus 500 µM H2O2, or 50 µM DBBF-Hb plus 500 µM H2O2. After 5 h, cells were harvested and analyzed as described in MATERIALS AND METHODS. Values represent means ± SD for four to six independent experiments performed in duplicate. *P < 0.01 vs. media; **P < 0.05 vs. R3 media; #P < 0.01 vs. HbA0; +P < 0.01 vs. same treatment in normal cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Hemoprotein-mediated oxidative stress is thought to play a major role in the pathophysiology of cerebral hemorrhage, blast pressure injury, crush injury, myocardial ischemia-reperfusion injury, and rhabdomyolysis (1, 21, 29, 44). These conditions are generally characterized by the release of Hb or myoglobin owing to red blood cell hemolysis or muscle destruction, respectively. Endothelial cells are important targets for vascular damage resulting from the presence of hemoproteins in the extracellular milieu. Heme-mediated oxidative injury or dysregulation of the vasculature is also an important concern with the use of oxygen-carrying blood substitutes based on modified forms of Hb (for review, see Ref. 2). The underlying mechanisms of cytotoxicity and/or vascular dysfunction are thought to involve the interaction of heme (protein or nonprotein bound) with reactive oxygen or nitrogen species (2, 36). The balance between NO and O₂· in the vasculature can be disrupted in the presence of cell-free Hb in favor of more powerful oxidants, i.e., ONOO and H₂O₂. The ensuing oxidative reactions of Hb with ONOO, H₂O₂, or lipid peroxide (ROOH) in the vasculature may exacerbate tissue damage (2).

Chemical modifications of Hb are designed to produce cell-free proteins with oxygen-carrying characteristics similar to or in some cases better than the red blood cell. However, some site-specific modifications can produce oxygen-carrying Hbs with altered redox chemistry, as is the case with DBBF-Hb (for review see Ref. 16). The reaction of DBBF-Hb with H₂O₂ results in a more persistent ferryl intermediate in solution reflecting the reduced ability of this Hb to consume H₂O₂ (3, 12). Previously, we showed that incubation with DBBF-Hb produced negligible cytotoxicity in endothelial cells, whereas the purified ferryl form of DBBF-Hb induced DNA fragmentation and morphological changes indicative of apoptosis and necrosis (28). These observations were also applicable to polymerized forms of Hb as previously reported (28). Recently, we reported that DBBF-Hb was detected in its ferryl form in an endothelial cell model of hypoxia-reoxygenation in the absence of any exogenous H₂O₂, and that the formation of the ferryl species closely paralleled the increases in cellular lipid peroxidation (42).

In this study, we further explored the contribution of cell-free Hb to cell injury and death. Specifically, we assessed whether alterations in the redox state of cells, i.e., the level of GSH can alter the course of death, and whether Hb-mediated cytotoxicity can be attributed to heme loss and/or the oxidation state of its heme. We incubated HbA₀ (nonmodified tetramer), DBBF-Hb (chemically stabilized tetrameric form of Hb), and H₂O₂ with endothelial cells containing normal and depleted levels of GSH. Our data showed that the formation of the ferryl intermediate was closely linked to a greater loss of GSH compared with H₂O₂ alone, suggesting these cells were subjected to an oxidative burden beyond that exerted by H₂O₂ alone. The enhanced loss of GSH may be explained by the aggravated peroxidative stress induced by heme-mediated lipid peroxidation processes, or possibly the efflux of GSH from stressed cells (23, 60). Although the exact mechanism is not clear, the inhibitory effect of catalase strongly suggests the drop in GSH was dependent on an oxidative phenomenon triggered by the combination of H₂O₂ and Hbs.

Although there is no direct evidence implicating the ferryl intermediate in the in vivo cytotoxicity of DBBF-Hb, a causative role for the redox cycling of myoglobin has recently been reported in a rat model of rhabdomyolysis-induced renal failure (44). These reactions were driven by endogenous lipid hydroperoxides in the kidney tubules. Similarly, the redox cycling of myoglobin was also shown to play a role in myocardial reperfusion injury (7, 21, 31). Recently it was also shown that tert-butyl hydroperoxide-Hb interaction produced a ferryl intermediate that was cytotoxic to vascular smooth muscle cells (53).

Endothelial cells are a source of H₂O₂ and ONOO under normal conditions and to a greater extent during reperfusion after ischemia. Moreover, platelets, neutrophils, and macrophages that are attracted to injury sites are also important contributors of H₂O₂ (30). It has been shown that activated neutrophils alone can generate up to 200 µM H₂O₂ in vitro (40). Other studies have shown that the release of heme from methemoglobin induces endothelial damage, and activated neutrophils cells potentiate this effect by oxidizing Hb to methemoglobin (8). Oxidative stress or alterations in cellular redox status has been shown to induce apoptosis and/or necrosis in various cell-based systems (34, 41, 49, 56). Oxidant-induced apoptosis may play a role in organ development and maintenance of tissue homeostasis (34). H₂O₂ was shown to impact the course of apoptosis in bovine endothelial cells and human lymphoma cells via its effects on cellular ATP levels (37, 38). Low levels of H₂O₂ (100 µM) produced apoptosis without changing ATP levels, whereas higher levels of H₂O₂ (5 mM) resulted in an irreversible drop in ATP and necrotic cell death (38). The pattern of death is significant because cells that undergo necrosis release intracellular contents, which can promote inflammatory reactions and aggravate tissue dysfunction or damage (41). In our system, the presence of Hb, a scavenger of H₂O₂, seemingly altered ordered cell death in favor of other pathways of cytotoxicity. The effect of Hb may occur at two levels: 1) Hb can catalytically lower tissue H₂O₂, and thus alter the course of death; and 2) Hb can be directly toxic to cells as it undergoes redox transition into higher, more reactive oxidation states. Both Hbs consistently reduced the degree of H₂O₂-induced apoptosis in normal and GSH-depleted cells. Indeed, HbA₀ and DBBF-Hb reduced the number of early apoptotic cells by three- and twofold, respectively, in normal and GSH-depleted cells (Fig. 8). The apparent difference in the extent of apoptosis between these two proteins may reflect their different rates of H₂O₂ consumption, as previously reported in vitro and in endothelial cell culture with and without H₂O₂ (3, 12, 28, 42).

GSH depletion has been shown to occur in ischemia-reperfusion injury, sepsis, organ transplantation, and myocardial conditions (6, 35, 48, 51, 63). A variety of cell types with depleted GSH were more susceptible to the cytotoxicity of H₂O₂ and other oxidative insults (20, 25, 43, 58). In this study, the enhanced loss of GSH following incubation with Hbs and H₂O₂ suggests GSH may play an important protective role against hemoprotein oxidative stress. Our data supports this idea because hemoprotein-induced oxidative stress was more damaging in GSH-depleted endothelial cells. In fact, Hbs may have diverted H₂O₂-induced apoptosis to other forms of cytotoxicity in GSH-depleted endothelial cells. There may be a lower risk associated with the administration of cell-free modified Hbs to healthy individuals with normal redox status. However, a greater risk may be present in patients with compromised vasculatures and poor antioxidant status (e.g., diabetes, hypertension, myocardial infarction, acute ischemic stroke, and hemorrhagic shock) as was recently suggested by the clinical trial failures with DCLHb, the commercial analog of DBBF-Hb (52, 54). The primary event responsible for the microvascular effects of Hb solutions is believed to be the removal of NO by Hb. However, subsequent oxidative reactions between Hb and oxidants of the vascular system (i.e., H₂O₂ and ONOO) may potentially lead to a vascular inflammatory cascade of reactions progressing to multi-organ failure.