Role of Oxidant Stress in Lawsone-Induced Hemolytic Anemia

David C. McMillan1, Snehal D. Sarvate, John E. Oatis, Jr. and David J. Jollow

Department of Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29425

Received July 1, 2004; accepted September 22, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lawsone (2-hydroxy-1,4-naphthoquinone) is the active ingredient of henna (Lawsonia alba), the crushed leaves of which are used as a cosmetic dye. Application of henna can induce a severe hemolytic anemia, and lawsone is thought to be the causative agent. Administration of lawsone to rats has been shown to induce a hemolytic response that is associated with oxidative damage to erythrocytes. However, direct exposure of isolated erythrocytes to lawsone did not provoke oxidative damage, suggesting that lawsone must undergo extra-erythrocytic bioactivation in vivo. In the present study, the survival of rat 51Cr-labeled erythrocytes in vivo after in vitro exposure to lawsone and its hydroquinone form, 1,2,4-trihydroxynaphthalene (THN) has been examined. Neither lawsone nor THN were directly hemolytic or methemoglobinemic, even at high concentrations (>3 mM). Lawsone had no effect on erythrocytic GSH levels, whereas THN (3 mM) induced a modest depletion (~30%). Cyclic voltammetry revealed that the lack of hemotoxicity of lawsone was associated with a poor capacity to undergo redox cycling. In contrast, ortho-substituted 1,4-naphthoquinones without a 2-hydroxy group, such as 2-methyl- and 2-methoxy-1,4-naphthoquinone, were redox active, were able to deplete GSH, and were direct-acting hemolytic agents. An oxidant stress-associated hemolytic response to lawsone could be provoked, however, if it was incubated with GSH-depleted erythrocytes. The data suggest that lawsone is a weak direct-acting hemolytic agent that does not require extra-erythrocytic metabolism to cause hemotoxicity. Thus, the hemolytic response to henna may be restricted to individuals with compromised antioxidant defenses.

Key Words: hemolytic anemia; lawsone; redox cycling; glutathione; erythrocyte; oxidative stress.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Henna, a natural product obtained from the crushed leaves of Lawsonia alba, has for centuries been used as a cosmetic agent to dye the skin, hair, and nails of people in many Middle Eastern countries, and large amounts of it are imported for this purpose into the United States. Application of henna has resulted in life-threatening episodes of hemolytic anemia, particularly in individuals with a genetic deficiency in erythrocytic glucose-6-phosphate dehydrogenase (G6PD) activity (Raupp et al., 2001Go; Zinkham and Oski, 1996Go). The active dye ingredient, lawsone (2-hydroxy-1,4-naphthoquinone), which constitutes about 1% of the dry weight of the leaves, has been implicated in the causation of henna-induced hemolytic anemia because of its structural similarity to other ortho-substituted 1,4-naphthoquinones (Fig. 1), such as menadione (2-methyl-1,4-naphthoquinone), that are known to induce oxidative injury within red cells. 1,4-Naphthoquinones are thought to induce oxidative damage as a consequence of their ability to undergo redox cycling (Munday et al., 1994Go), with the generation of reactive oxygen species (ROS). In support of this concept, oxidative responses to in vitro exposure of red cells to menadione (i.e., hydrogen peroxide formation, GSH depletion, methemoglobin production, and the presence of Heinz bodies) have been shown to correlate with hemolytic activity in vivo (Munday et al., 1991Go).



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FIG. 1. Structures of lawsone and its putative hydroquinone metabolite, THN. The structures of the ortho-substituted 1,4-naphthoquinones, menadione and 2-methoxy-1,4-naphthoquinone are also shown.

 
Previous studies on lawsone have shown that it can provoke an anemic response when administered in vivo to rats (Munday et al., 1991Go), and this response has been characterized as oxidative in nature due to the presence of Heinz bodies in the circulating erythrocytes. However, lawsone has been reported to cause little or no oxidative effects when incubated with rat erythrocytes in vitro (Kruger-Zeitzer et al., 1990Go), though evidence for a direct effect of lawsone on erythrocyte survival in vivo has not been reported. On the other hand, lawsone has been reported to deplete GSH and increase methemoglobin formation in red cells isolated from G6PD-deficient humans (Zinkham and Oski, 1996Go), although high concentrations of lawsone (>3 mM) were necessary to achieve these effects.

One potential explanation for the discordance between the in vitro versus in vivo data is that the hemolytic response to lawsone may not be the result of a direct effect on erythrocytes, but may be mediated by an extra-erythrocytic metabolite of lawsone formed in vivo. In support of this possibility, Munday et al. (1999aGo,bGo) have shown that lawsone hemotoxicity in rats can be enhanced by inducers of DT diaphorase (NADPH:quinone oxidoreductase 1). DT diaphorase is known to catalyze a two-electron reduction of lawsone to its hydroquinone, 1,2,4-trihydroxy-naphthalene (THN). THN has been shown to auto-oxidize rapidly in vitro with concomitant generation of ROS (Munday, 1997Go). An alternate hypothesis for the lack of in vitro effects is that red cells with normal G6PD activity are not susceptible to the direct hemolytic action of lawsone because of weak redox activity due to the stabilizing effect of its acidic 2-hydroxy group.

To examine the basis for the discrepancy between the in vitro and in vivo activity of lawsone, we have compared its direct hemolytic activity and electrochemical behavior with ortho-substituted 1,4-naphthoquinone derivatives that are known to induce direct oxidative damage to isolated erythrocytes; viz., menadione and 2-methoxy-1,4-naphthoquinone. In addition, we have synthesized and examined the hemolytic potential of THN. Lastly, we have investigated the hemolytic potential of lawsone and THN in GSH-depleted rat erythrocytes, which we have used as an experimental model for the enhanced sensitivity of human G6PD-deficient erythrocytes to drug-induced oxidative stress (Bolchoz et al., 2002Go).

We report that direct exposure of rat 51Cr-labeled erythrocytes to lawsone or THN does not reduce their survival when the labeled cells are returned to the circulation of isologous rats, and that lawsone is only weakly redox active. In contrast, both menadione and 2-methoxy-1,4-naphthoquinone were direct-acting hemolytic agents in erythrocytes, and were electrochemically active and capable of forming fully reversible redox couples. However, lawsone was able to produce an oxidant pressure within rat erythrocytes, as evidenced by its enhancement of hexose monophosphate (HMP) shunt activity. Stimulation of HMP shunt activity was not accompanied by depletion of GSH, suggesting that ROS generation was not sufficient to overcome the antioxidant defenses in these red cells. Impairment of this protective system by prior depletion of erythrocytic GSH revealed profound hemolytic responses by both lawsone and THN. We conclude that extra-erythrocytic metabolism of lawsone is not obligatory for hemolytic activity and that hemotoxicity is most marked when antioxidant defenses are impaired, as happens in G6PD deficiency.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and materials. Lawsone, menadione, and 2-methoxy-1,4-naphthoquinone were purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification. Rabbit anti-rat hemoglobin IgG, diethyl maleate, and D-glucose-1–14C (1 mCi/ml) were also obtained from Sigma. in sterile saline (1 mCi/ml, pH 8) was purchased from New England Nuclear (Billerica, MA). Carbo-Sorb E was purchased from Perkin Elmer Life Sciences (Boston, MA). All other reagents were of the best commercially available grade.

THN was synthesized from lawsone in the following manner: Lawsone (316 mg) was suspended/dissolved in 20 ml hot H2O, and Na2S2O4 was added until the color of the solution changed from orange/red to a pale yellow, and until further addition of Na2S2O4 caused no further color change. The reaction mixture was cooled under argon, then extracted under argon with three volumes of diethyl ether (10 ml). The combined extracts were washed with a saturated NaCl solution (10 ml) containing a small amount of Na2S2O4, then dried over MgSO4, and the solvent removed at reduced pressure to yield 237 mg of a gray solid. NMR analysis showed no starting material or other impurities. 1H NMR (DMSO-d6): {delta} 6.59 (s, 1, H-3), 7.15 (m, 1, H6), 7.30 (m, 1, H-6), 7.87 (m, 1, H-8), 7.89 (m, 1, H-5), 7.99 (s, 1, OH-1), 9.02 (s, 1, OH-2), 9.41 (s, 1, OH-4). 13C NMR: {delta}102.1(C-2), 119.9 (C-10), 121.2 (C-8), 121.7 (C-6), 122.5 (C-5), 125.6 (C-7), 127.2 (C-9), 130.2 (C-1), 141.3 (C-2), 146.7 (C-4).

Animals and incubation conditions. Male Sprague-Dawley rats (100–120 g) were purchased from Harlan Laboratories (Indianapolis, IN) and maintained on food and water ad libitum. Animals were acclimated for 1 week to a 12-h light-dark cycle prior to their use. Erythrocytes were collected from anesthetized rats into heparinized tubes and washed in isotonic phosphate-buffered saline (pH 7.4) supplemented with 10 mM D-glucose (PBSG) to remove the plasma and buffy coat. The red cells were resuspended to a 40% hematocrit and used the same day they were collected. Experiments were carried out by addition of various concentrations of lawsone or THN dissolved in PBSG to the erythrocyte suspensions (2 ml), which were allowed to incubate at 37°C for up to 2 h. Menadione and 2-methoxy-1,4-naphthoquinone were dissolved in acetone (15 µl).

For experiments using GSH-depleted red cells, washed erythrocyte suspensions were titrated with diethyl maleate (750 µM initial concentration), and the loss of GSH was followed by HPLC with electrochemical detection as described previously (Bolchoz et al., 2002Go). Diethyl maleate pretreatment typically caused about a 95% depletion of erythrocytic GSH.

Electrochemistry experiments. Cyclic voltammetry experiments were performed using a Bioanalytical Systems (West Lafayette, IN) CV-27 voltammograph, C-1 A/B cell stand, and a Model RXY recorder. Standard solutions of the test compounds (75 µM) were prepared in argon-purged PBSG. Samples were scanned at a rate of 150 mV/sec under argon atmosphere at room temperature using a carbon paste working electrode, and a Ag/AgCl reference electrode.

NMR. Proton and carbon NMR spectra were obtained on a Varian Inova spectrometer operating at 400 and 100 MHz, respectively. Some of the proton and carbon resonances were assigned using gradient versions of the heteronuclear single quantum coherence (HSQC) and heteronuclear multibond correlation (HMBC) experiments. In the HSQC 128 fids were acquired, linear prediction increased the points in F1 to 512, Gaussian weighted, then Fourier transformed. In the HMBC 400 fids were acquired, linear prediction increased the points in F1 to 1200, sinebell weighted, then Fourier transformed. The remainder of the proton and carbon assignments are based on the NOESY-1D pulse sequence employing an on resonance sech180 shaped pulse for spin population perturbation with a 500 msec mixing time.

Determination of the hemolytic response. The survival of 51Cr-labeled red cells in vivo after in vitro exposure to the test compounds was determined as described previously (Harrison and Jollow, 1986Go). After the incubation, the red cells were washed and resuspended (40% hematocrit), and an aliquot (0.5 ml) was administered iv to isologous rats. T0 blood samples were taken from the orbital sinus 30 min after administration of the labeled cells. Additional blood samples were taken at 48-h intervals for up to 14 days. At the end of the experiment the samples were counted in a well-type gamma counter, and the data were expressed as a percentage of the T0 blood sample. Statistical significance was determined with the use of Student's t test.

Determination of oxidative stress responses in vitro. The amount of GSH, glutathione disulfide (GSSG), and glutathione-protein mixed disulfides (PSSG) in aliquots of red cell incubates was determined by HPLC-EC as noted above. The amount of sulfhydryl present in the samples was determined by comparison of peak areas to prepared standards. Methemoglobin formation in red cell incubates was determined as described previously (Harrison and Jollow, 1987Go). HMP shunt activity in red cell incubates treated with lawsone was estimated by measuring the evolution of 14CO2 from 14C-1-glucose as described previously (Grossman et al., 1995Go). The presence of cytoskeletal membrane-bound hemoglobin in erythrocyte ghosts was also determined as described previously (McMillan et al., 1995Go).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Survival of Lawsone-Treated Erythrocytes In Vivo
Lawsone has been shown to induce an oxidant stress-related hemolytic anemia when administered to rats (Munday et al., 1991Go); however, unlike other 1,4-naphthoquinones, lawsone does not appear to elicit oxidant stress-type responses when incubated directly with rat red cells (Kruger-Zeitzer et al., 1990Go), suggesting a distinctly different mode of action. To determine if direct exposure of erythrocytes to lawsone could affect their viability in vivo, isolated rat red cells were tagged with 51Cr and incubated for 2 h at 37°C with various concentrations of lawsone. The erythrocytes were then washed and returned to isologous rats. Survival of the tagged cells was assessed by measurement of radioactivity in serial blood samples collected at intervals over the subsequent 14 days. Figure 2A shows the effect of in vitro exposure to 3 mM lawsone on in vivo survival of the rat erythrocytes. The data indicate that the rate of removal of the lawsone-treated red cells was not significantly different from that of control red cells incubated in PBSG alone. In additional experiments, incubation of red cells with higher concentrations of lawsone (up to 15 mM) did not affect their viability (data not shown).



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FIG. 2. (A) Survival of rat 51Cr-labeled erythrocytes in vivo after in vitro incubation with indicated concentrations of lawsone and THN for 2 h at 37°C. Control cells were incubated with the vehicle alone. After incubation, the erythrocytes were washed and administered iv to rats. (B) Survival of rat 51Cr-labeled erythrocytes in vivo after in vitro incubation with the indicated concentrations of menadione and 2-methoxy-1,4-naphthoquinone. Data points are means ± SD (n = 3).

 
To examine the hypothesis that the hydroquinone metabolite of lawsone, THN, mediates the hemolytic response to lawsone in vivo, THN was synthesized and its direct hemolytic activity was determined as described above for lawsone. As shown in Figure 2A, incubation of the radiolabeled red cells with THN (3 mM) for 2 h at 37°C had no effect on erythrocyte survival when the treated red cells were returned to the circulation of isologous rats.

For the purpose of comparison, Figure 2B shows the effect of in vitro exposure to menadione or 2-methoxy-1,4-naphthoquinone on erythrocyte survival in vivo. In contrast to lawsone and THN, both of these compounds induced concentration-dependent increases in the rate of removal of the radiolabeled red cells as compared to the controls. Of note, the hemolytic activity of the 2-methoxy derivative appeared to be greater than that of menadione.

Methemoglobin Formation and GSH Depletion
A plausible explanation for the inactivity of lawsone in vitro is that its acidic ortho-hydroxy group renders the molecule incapable of continuous redox cycling due to the stabilizing influence of keto-enol tautomerization. This effect could dampen redox cycling, thus preventing lawsone from generating ROS at rates sufficient to cause cellular damage. To examine this possibility, we compared the oxidative effects (i.e., hemoglobin and GSH oxidation) and electrochemical behavior of lawsone with that of menadione and 2-methoxy-1,4-naphthoquinone, in which the ortho-hydroxy group is modified to eliminate tautomerization.

The ability of these compounds to induce methemoglobin formation in rat red cells as a function of time is shown in Figure 3A. Hemolytic concentrations of both menadione and 2-methoxy-1,4-naphthoquinone (500 µM) induced significant methemoglobin formation. In both cases, methemoglobin levels rose rapidly and remained elevated throughout the 4-h incubation period. Again, 2-methoxy-1,4-napthoquinone was the more potent methemoglobin-forming agent as compared to menadione.



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FIG. 3. (A) Methemoglobin, (B) GSH, and (C) PSSG levels versus time in rat erythrocytes treated with ortho-substituted 1,4-naphthoquinones. Erythrocyte suspensions were incubated with the vehicle ({circ}) or the indicated concentrations of lawsone ({blacksquare}), THN ({diamondsuit}), menadione ({blacktriangleup}), and 2-methoxy-1,4-naphthoquinone (•). Aliquots were taken at the indicated time points and assayed for methemoglobin, GSH, and PSSG as described in Materials and Methods. Values shown are means ± SD (n = 3).

 
The levels of methemoglobin in red cell incubates exposed to lawsone and THN (3 mM) were not significantly different from controls up to 2 h (Fig. 3A). After longer incubation time (4 h), a modest but statistically significant elevation in methemoglobin levels in the cells was observed for both compounds. The reason for this longer-term effect is unclear but may reflect a decrease in production of NADH in the cell as glucose in the medium becomes exhausted. As with the hemolytic response, even higher concentrations (up to 15 mM) of lawsone and THN were without additional effect on methemoglobin formation (data not shown).

The ability of these 1,4-napthoquinones to deplete erythrocytic GSH is shown in Figure 3B. Incubation of rat erythrocytes with hemolytic concentrations of menadione and 2-methoxy-1,4-naphthoquinone (500 µM) induced a rapid and complete depletion of erythrocytic GSH. The loss of GSH was matched by a corresponding increase in PSSG (Fig. 3C); GSSG levels remained low and constant during the incubation period (data not shown). In comparison, incubation of red cells with lawsone (3 mM) did not deplete GSH from the cells at any time point. Similar results were obtained when the concentration of lawsone was increased to 15 mM (data not shown). In contrast to lawsone, treatment of red cells with THN (3 mM) induced a rapid but modest depletion (~30%) of GSH, that was also accompanied by the formation of PSSG. The depletion was transient in that GSH levels returned to near normal within an hour of incubation. These data suggest that THN has some oxidative activity, which, though modest as compared with menadione and 2-methoxy-1,4-naphthoquinone, appeared to be greater than that of lawsone.

Electrochemical Activity of Lawsone
In view of the lack of in vitro hemotoxicity of lawsone as compared to its 2-methyl and 2-methoxy derivatives, it was of interest to examine the electrochemical behavior of these compounds in order to determine whether they had the inherent ability to undergo reversible oxidation-reduction reactions. Thus, stock solutions of these compounds (75 µM) were prepared in argon-purged PBSG (pH 7.4) and analyzed by cyclic voltammetry. Figure 4A shows the baseline electrochemical activity of the buffer when a reducing potential scan was initiated at +0.55 V and then reversed in the positive (oxidizing) direction at –0.70 V. The potential scan of lawsone (Fig. 4B) under the same conditions showed a small reduction peak (a) at –0.32 V (ipc 0.8 µA), and when the scan was reversed, a peak (b) at –0.10 V (ipa 0.4 µA) corresponding to oxidation of the product formed at the electrode surface. The potential scans of menadione (Fig. 5C) and 2-methoxy-1,4-naphthoquinone (Fig. 5D) showed that reduction of these quinones occurred at similar negative potentials (–0.32 and –0.38 V, respectively), however, their cathodic (ipc 5.5 and 3.4 µA, respectively) and anodic (ipa 3.0 and 0.9 µA, respectively) response currents were of much greater magnitude, which is indicative of higher rates of reduction of these compounds at the electrode surface. Interestingly, the 2-methoxy hydroquinone appeared to be somewhat less electrochemically active than the hydroquinone of menadione.



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FIG. 4. Cyclic voltammograms of (A) PBSG (pH 7.4), (B) lawsone, (C) menadione, and (D) 2-methoxy-1,4-naphthoquinone. The compounds (75 µM) were dissolved in argon-purged PBSG at room temperature. Working electrode, carbon-paste; reference electrode, Ag/AgCl; auxiliary electrode, platinum; scan rate, 150 mV/sec. Cathodic peak (reduction), a; anodic peak (oxidation), b. Scans were initiated in the negative potential direction.

 


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FIG. 5. HMP shunt activity in GSH-normal rat erythrocytes treated with lawsone (3 mM). Erythrocyte suspensions in PBSG containing 14C-1-glucose were sealed and allowed to incubate at 37°C for 45 min. At the end of the incubation period, the amount of 14CO2 collected in CarboSorb E was counted. Values are means ± SD (n = 3).

 
Taken together, the data are consistent with both menadione and 2-methoxy-1,4-naphthoquinone undergoing facile 2-electron reduction at physiological pH to the hydroquinone species, followed by rapid oxidation back to the quinone. Lawsone also appears to have the capacity to redox cycle in this fashion, but at a much-reduced rate as compared with its hemolytic analogs.

Effect of Lawsone on HMP Shunt Activity
Although lawsone had no direct hemolytic activity in rat erythrocytes, analysis of lawsone by cyclic voltammetry indicated at least some inherent ability to redox cycle. This observation, along with the moderate depletion of GSH induced by THN, suggested that lawsone can provoke an oxidative "pressure", if not an oxidative stress, within erythrocytes. That is, lawsone may indeed be causing GSH oxidation in these erythrocytes, but not at a rate sufficient to overcome the activity of glutathione reductase, and hence GSH depletion is not observed. Since NADPH provides reducing equivalents for glutathione reductase, then its utilization would release the negative feedback control on G6PD activity, which should be reflected by a stimulation of the HMP shunt pathway.

To determine if lawsone could provoke an oxidative pressure in rat red cells, the ability of lawsone to stimulate the production of NADPH through the HMP shunt was examined. As shown in Figure 5, addition of lawsone (3 mM) to erythrocyte suspensions caused a significant stimulation of HMP shunt activity, as measured by the evolution of 14CO2 derived from 14C-1-glucose. The stimulatory effect of lawsone corresponded to about a five-fold increase in activity as compared to the control. These data indicate that lawsone is redox active within the normal rat red cell and that this redox cycling results in the production of ROS.

Hemotoxicity of Lawsone in GSH-Depleted Erythrocytes
The failure of lawsone to inflict hemolytic damage to rat erythrocytes implies that the rate of lawsone-induced ROS production is less than the capacity of the GSH-dependent protective capacity to deal with it. It follows that if the GSH-protective capacity was reduced, as in human G6PD deficiency, toxicity should result. To test this hypothesis, the protective capacity of rat erythrocytes was impaired by depleting GSH with diethyl maleate prior to exposure to lawsone. Previous studies have shown that titration of GSH levels to 1–5% of normal mimics the G6PD-deficient situation without effect on the rate of removal of saline-treated control cells (Bolchoz et al., 2002Go; Bowman et al., 2004Go).

As shown in Figure 6, depletion of ca. 96% of GSH in the red cells used in the present studies did not cause a decrease in erythrocyte survival (i.e., GSH-depleted control cells vs. normal control cells). However, when lawsone or THN was included in the incubation medium, the GSH-depleted red cells showed a marked hemolytic response, as indicated by the decreased survival of the treated 51Cr-tagged red cells when they were reinfused into isologous rats. In striking contrast to the data presented in Figure 2A, where both compounds were inactive in GSH-normal cells, even at concentrations as high as 15 mM, the hemolytic concentration-dependence in GSH-depleted cells was in the micromolar range. In the presence of a markedly reduced GSH-dependent protective capacity, lawsone and THN showed a potency in GSH-depleted red cells generally similar to that of menadione and 2-methoxy-1,4-naphthoquinone in GSH-normal erythrocytes.



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FIG. 6. Survival of GSH-depleted 51Cr-labeled erythrocytes in vivo after in vitro exposure to lawsone and THN. Radiolabeled red cells were titrated with DEM to deplete intracellular GSH (>90%). The cells were then incubated with the vehicle or the indicated concentrations of lawsone or THN. For 2 h at 37°C. Data points are means ± SD (n = 4).

 
In view of the appearance of hemotoxicity of lawsone in GSH-depleted red cells, it was of interest to determine whether a corresponding increase in methemoglobin formation could also be observed. Thus, lawsone (3 mM) was added to normal and GSH-depleted erythrocyte suspensions, and methemoglobin formation was monitored as a function of time. As shown in Figure 7, the levels of methemoglobin in the GSH-depleted red cells were not significantly different from the levels in GSH-normal red cells throughout the 2-h incubation period.



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FIG. 7. Methemoglobin formation versus time in GSH-normal versus GSH-depleted rat erythrocytes treated with lawsone (3 mM). Values are means ± SD (n = 3).

 
Effect of Lawsone on Membrane Skeletal Proteins
We have shown previously that the hemolytic response to redox-active metabolites of hemolytic agents is associated with oxidative damage to the membrane cytoskeleton, principally in the form of disulfide-linked hemoglobin adducts and monomeric and polymeric forms of hemoglobin (McMillan et al., 1995Go, 2001Go; Bolchoz et al., 2002Go). To determine if lawsone hemotoxicity was associated with this type of oxidative damage, erythrocyte ghost proteins from lawsone-treated GSH-normal and GSH-depleted red cells were resolved on nonreducing SDS-PAGE gels and probed with an anti-hemoglobin antibody. As shown in Figure 8, membrane ghosts from GSH-normal erythrocytes contained only a small amount of membrane-bound hemoglobin monomer (lane 1; 16 kDa). Membrane ghosts from GSH-depleted control erythrocytes (lane 2) contained a slightly increased amount of hemoglobin monomer but were otherwise unaffected. In GSH-normal red cells treated with 3 mM lawsone (lane 3), the amount of hemoglobin monomer was not different from the GSH-normal control. In GSH-depleted red cells treated with 250 µM lawsone (lane 4), the amount of membrane-bound hemoglobin monomer was enhanced as compared to the GSH-depleted control. In addition, these membrane ghosts contained hemoglobin-cytoskeletal protein adducts. The pattern of hemoglobin adduct formation in GSH-depleted red cells treated with lawsone was remarkably similar to that of GSH-normal red cells treated with 500 µM menadione (lane 5), though the amount of membrane-bound hemoglobin in the menadione-treated red cells was considerably greater. These data are consistent with an oxidative stress-type mechanism for lawsone and suggest that its hemolytic activity in GSH-depleted erythrocytes is not the result of some other (i.e., nonspecific) effect.



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FIG. 8. Effect of lawsone on membrane skeletal proteins in GSH-normal and GSH-depleted rat erythrocytes. GSH-normal erythrocytes were incubated with the vehicle (lane 1), 3 mM lawsone (lane 3), and 500 µM menadione (lane 5); GSH-depleted erythrocytes (96%) were incubated with the vehicle (lane 2) and 250 µM lawsone (lane 4) for 2 h at 37°C. After exposure the erythrocytes were washed, and membrane ghosts were prepared and washed extensively to remove unbound hemoglobin. The ghosts were solubilized in SDS and subjected to PAGE. The proteins were then transferred to a PVDF membrane and stained with rabbit anti-rat hemoglobin.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Henna has been used for more than 4000 years as a cosmetic by Mediterranean, Middle Eastern, and Asian cultures. In many circumstances, the dye is applied over extensive areas of the body to create a variety of designs, and henna is frequently applied to newborn infants for ceremonial purposes (Kandil et al., 1996Go). The widespread use of henna clearly indicates that it is considered to be safe for external application. On the other hand, there are numerous case reports of adult and pediatric hemolytic crises following henna application, some of which have had fatal outcomes (Valaes, 1994Go). Of importance, susceptibility to the hemotoxicity of henna appears to be confined to G6PD-deficient individuals (Kandil et al., 1996Go; Raupp et al., 2001Go). The underlying basis for the discrepancy between normal and G6PD-deficient individuals in regard to henna toxicity is unknown, but it may be analogous to favism, which occurs in individuals with low-activity variants of G6PD-deficiency (e.g., the Mediterranean variant) who ingest fava beans (Beutler, 1978Go).

Lawsone is considered to be the active ingredient in henna responsible for its hemotoxicity. In support of this viewpoint, lawsone has been shown to provoke a hemolytic response when administered to rats, as evidenced by decreased hematocrits, reduced levels of hemoglobin, and increased spleen/body weight ratios (Munday et al., 1991Go). Furthermore, the appearance of circulating erythrocytes containing Heinz bodies suggests that the hemolytic response to lawsone in vivo is the result of oxidative injury to the red cell. On the basis of these observations, lawsone can be classified as an oxidant stress-type hemolytic agent.

However, unlike the other 1,4-napthoquinones, in vitro incubation of lawsone with isolated erythrocytes failed to provoke any oxidative effects (i.e., GSH depletion, hemoglobin oxidation, or hydrogen peroxide generation) that have been observed when ortho-substituted alkyl-1,4-naphthoquinones, such as menadione, are exposed to red cells (Munday et al., 1994Go). This observation has led to the hypothesis that lawsone causes erythrocyte damage in vivo by a mechanism that is distinctly different from that of other 1,4-napthoquinones.

Munday et al. (1999a)Go observed that pretreatment of rats with doses of butylated hydroxyanisole that induced DT diaphorase activity in extra-erythrocytic tissues (i.e., liver and GI tract) also caused an exacerbation of lawsone hemotoxicity. To reconcile the difference between the in vivo versus in vitro responses, these investigators postulated that lawsone is hemotoxic in vivo but not in vitro because it is reduced to its hydroquinone by DT diaphorase outside of the erythrocyte. Rapid auto-oxidation of the hydroquinone then occurs and results in the generation of ROS (Munday, 1997Go), which subsequently attack the red cell. In contrast, menadione is thought to be reduced to its hydroquinone within the erythrocyte through direct interaction with oxyhemoglobin (Munday et al., 1994Go), a reaction which has been reported not to occur with lawsone (Kruger-Zeitzer et al., 1990Go).

Although lawsone had been shown not to induce oxidative changes in erythrocyte suspensions, the effect of in vitro exposure to lawsone on erythrocyte survival in vivo had not been examined. Thus, we reinvestigated the hemolytic potential of lawsone using the in vitro 51Cr-labeled erythrocyte exposure/in vivo survival assay. The data presented in this report confirm that direct exposure to lawsone does not provoke a hemolytic response in normal rat erythrocytes (Fig. 2A), even at extremely high incubation concentrations. The lack of direct hemolytic activity of lawsone was also associated with an inability to cause methemoglobin accumulation (Fig. 3A) or to deplete erythrocytic GSH by oxidation to PSSG (Figs. 3B and 3C). Similar responses were seen with THN, indicating that the hemotoxicity of lawsone in the intact animal is not due solely to extra-erythrocytic reduction of lawsone to THN.

The apparent lack of oxidative activity of lawsone has been explained by the stabilizing influence of its acidic 2-hydroxy group, which is thought to impede its ability to redox cycle. Comparison of the structure-redox activity of these ortho-substituted 1,4-naphthoquinone derivatives using cyclic voltammetry (Fig. 4) showed that, while lawsone was relatively sluggish in its ability to redox cycle as compared with menadione and 2-methoxy-1,4-naphthoquinone, it did have some capacity in this regard. The importance of the 2-hydroxy group was confirmed by methylation of the 2-hydroxy group, which yielded a quinone with a much greater ability to redox cycle.

Although THN was not hemolytic in normal rat erythrocytes, it did have a modest ability to deplete GSH (Fig. 3B), which is consistent with the notion that it can complete one half of a redox cycle; i.e., it auto-oxidizes within the erythrocyte to lawsone with the release of ROS. These data raised the possibility that the lack of hemotoxicity of lawsone and THN might be due not to their inability to redox cycle per se, but because the rate at which they do so might not be sufficient to overcome the cellular GSH-dependent protective mechanism. Measurement of HMP shunt activity (Fig. 5) demonstrated that lawsone enhances the demand for NADPH in normal rat erythrocytes. Since NADPH is utilized primarily within the red cell to support glutathione reductase activity, and hence glutathione peroxidase-mediated detoxification of ROS, it follows that lawsone must be generating ROS secondary to redox cycling. Consistent with this view, depletion of erythrocytic GSH prior to incubation of the cells with lawsone or THN as a means to impair the protective mechanism revealed a massive potentiation of lawsone and THN hemolytic activity (Fig. 6). In the GSH-normal erythrocytes, a concentration as high as 15 mM elicited no hemotoxicity, which is in striking contrast to the almost complete removal of GSH-depleted erythrocytes from the circulation of rats after exposure to 250 µM lawsone or THN. As is appropriate for a redox pair, the hemolytic activities of lawsone and THN were identical.

Although depletion of GSH had a profound effect on the hemolytic activity of lawsone, GSH depletion had no effect on the formation of methemoglobin (Fig. 7). Interestingly, lawsone is the first pro-oxidant compound we have examined with hemolytic activity but apparently no methemoglobinemic activity. Since GSH depletion does not affect NADH production, the lack of induction of methemoglobinemia indicates that GSH depletion had no effect on the stability of lawsone in the erythrocyte or its ability to redox cycle.

Given the massive potentiation of lawsone hemotoxicity in GSH-depleted erythrocytes, there was concern that the hemolytic response might be caused by a nonspecific effect of lawsone. We think that this is unlikely inasmuch as no frank lysis of the GSH-depleted erythrocytes was observed following a 2-h incubation with lawsone. Furthermore, previous studies on redox-active, pro-oxidant drug metabolites have shown a strong correlation between the hemolytic response and the formation of disulfide-linked hemoglobin-skeletal proteins adducts (Jollow and McMillan, 2001Go). We have shown previously that these adducts arise secondarily to the generation of glutathione and hemoglobin thiyl free radicals (Bradshaw et al., 1995Go, 1997Go). In the present studies, hemoglobin-skeletal protein adducts were detected in lawsone-treated GSH-depleted red cells (Fig. 8), indicating that the hemolytic response of these cells was associated with hemoglobin-thiol oxidation. Moreover, the pattern of adduct formation induced by lawsone was remarkably similar to that of menadione, suggesting that the mechanisms underlying their hemolytic responses are not radically different.

Collectively, the data indicate that the absence of hemolytic activity in normal rat red cells exposed to lawsone in vitro is not due to extra-erythrocytic metabolism of lawsone, but instead is related to the relatively sluggish redox cycling of this compound within erythrocytes. Thus, lawsone cannot generate ROS at a rate sufficient to exceed the capacity of the cells to remove them, and hence no toxicity results. The situation is quite different in erythrocytes with reduced protective capacity; under these conditions, lawsone appears to be capable of inflicting ROS-associated hemolytic damage to erythrocytes in a manner similar to that of its better-known analog, menadione. This concept appears to offer an explanation for the clinical conundrum; the long-term societal acceptance of the safety of henna coupled with the clinically recognized profound, and sometimes life-threatening, hemolytic episodes in G6PD-deficient individuals. Since the hallmark of G6PD-deficiency is the inability to maintain adequate GSH levels when challenged by an oxidative stress, it is likely that human G6PD-deficient and rat GSH-depleted red cells will react to lawsone in a similar fashion.

While these observations offer a plausible explanation for the susceptibility of G6PD-deficient individuals to lawsone hemotoxicity, several important questions remain to be answered, including the following: (1) Why is the extent of exacerbation lawsone hemotoxicity so large? The exacerbating effect of GSH depletion observed for primaquine metabolites was about 3–5 fold (Bowman et al., 2004Go). In contrast, a greater than 100-fold exacerbation, based on extrapolation from the present data, was observed for lawsone. (2) Why does lawsone induce a hemolytic response in vivo but not after in vitro exposure, regardless of the concentration used in the suspension medium, and (3) why did induction of DT diaphorase in extra-erythrocytic tissues exacerbate the hemolytic response to lawsone? The data suggest that lawsone is a direct-acting hemolytic agent, which induces an oxidant stress, and are thus consistent with the clinical observation of enhanced susceptibility to henna by G6PD-deficient individuals.


    ACKNOWLEDGMENTS
 
This work was supported by NIH grant AI46424. The authors would like to thank Jennifer M. Schulte for her technical assistance in the preparation of this manuscript. The authors also acknowledge the Medical University of South Carolina NMR Resource Facility.


    NOTES
 

1 To whom correspondence should be addressed at Dept. of Pharmacology, Medical University of South Carolina, 173 Ashley Ave., Charleston, SC 29425. Fax: (843) 792-2475. E-mail: mcmilldc{at}musc.edu.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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