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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2010 Nov;161(5):1186–1199. doi: 10.1111/j.1476-5381.2010.00962.x

Dapsone hydroxylamine induces premature removal of human erythrocytes by membrane reorganization and antibody binding

Luciana Bordin 1, Cristina Fiore 2, Francesco Zen 3, Michael D Coleman 4, Eugenio Ragazzi 5, Giulio Clari 1
PMCID: PMC2998697  PMID: 20662842

Abstract

BACKGROUND AND PURPOSE

N-hydroxylation of dapsone leads to the formation of the toxic hydroxylamines responsible for the clinical methaemoglobinaemia associated with dapsone therapy. Dapsone has been associated with decreased lifespan of erythrocytes, with consequences such as anaemia and morbidity in patients treated with dapsone for malaria. Here, we investigated how dapsone and/or its hydroxylamine derivative (DDS-NHOH) induced erythrocyte membrane alterations that could lead to premature cell removal.

EXPERIMENTAL APPROACH

Erythrocytes from healthy donors were subjected to incubation with dapsone and DDS-NHOH for varying times and the band 3 protein tyrosine-phosphorylation process, band 3 aggregation, membrane alteration and IgG binding were all examined and compared with erythrocytes from two patients receiving dapsone therapy.

KEY RESULTS

The hydroxylamine derivative, but not dapsone (the parent sulphone) altered membrane protein interactions, leading both to aggregation of band 3 protein and to circulating autologous antibody binding, shown in erythrocytes from patients receiving dapsone therapy. The band 3 tyrosine-phosphorylation process can be used as a diagnostic system to monitor membrane alterations both in vitro, assessing concentration and time-dependent effects of DDS-NHOH treatment, and in vivo, evaluating erythrocytes from dapsone-treated patients, in resting or oxidatively stimulated conditions.

CONCLUSIONS AND IMPLICATIONS

DDS-NHOH-induced alterations of human erythrocytes can be directly monitored in vitro by tyrosine-phosphorylation level and formation of band 3 protein aggregates. The latter, together with antibody-mediated labelling of erythrocytes, also observed after clinical use of dapsone, may lead to shortening of erythrocyte lifespan.

Keywords: DDS-NHOH, human erythrocyte membrane alterations, band 3 Tyr-phosphorylation, band 3 aggregates, autologous IgG binding

Introduction

Dapsone has been used for over half a century in the treatment of leprosy, for anti-inflammatory conditions and, in the chlorproguanil-dapsone and artesunate–dapsone–proguanil combinations, for treating malaria. It is also a second-line treatment for AIDS-related Pneumocystis pneumonia (Sangiolo et al., 2005), and is increasingly applied to a variety of immunologically related conditions (Bahadir et al., 2004; Ujiie et al., 2006), despite its well-documented toxicity, which is closely related to its routes of biotransformation. Mono- and diacetylated metabolites of dapsone (MADDS and DADDS) are not associated with toxicity (Coleman et al., 1991), although N-hydroxylation of the parent drug and MADDS lead to the formation of the toxic hydroxylamines DDS-NHOH and MADDS-NHOH (Israili et al., 1973; Coleman et al., 1989). These species, formed either by one isoform of the cytochrome P450 (CYP) family, CYP2C9 (Winter et al., 2000), or other oxidative enzyme systems, are linked with several immune-mediated hypersensitivity reactions (Vyas et al., 2006). The hydroxylamines are also responsible for the clinical methaemoglobinaemia associated with dapsone therapy (Israili et al., 1973; Schiff et al., 2006). DDS-NHOH can also induce erythrocyte removal in rat (Grossman et al., 1995; McMillan et al., 1995) and morphological alteration in human erythrocytes (McMillan et al., 1995). The drug has long been associated with shortening of the erythrocytic lifespan in man (Cream, 1970; Byrd and Gelber, 1991) and this remains a clinical problem (Wertheim et al., 2006). Indeed, anaemia is already a major cause of mortality and morbidity in areas where dapsone is used to treat malaria (Gonazales et al., 2000).

DDS-NHOH undergoes a coupled oxidation-reduction reaction with haemoglobin and molecular oxygen yielding methaemoglobin and reactive oxygen species (ROS) (ferryl haem and hydroxyl radicals), respectively (Bradshaw et al., 1997). To date, no direct evidence of the mechanism whereby DDS-NHOH shortens the erythrocytic lifespan has been reported. Only a possible effect of DDS-NHOH on the integrity of the erythrocytic lipid bilayer has been excluded, since neither lipid peroxidation nor phosphatidylserine externalization has ever been detected (McMillan et al., 1998; 2005;).

In human erythrocytes, tyrosine phosphorylation (Tyr-P) of membrane proteins mainly involves the band 3 protein. This is the most abundant membrane protein of red blood cells (RBC) and is divided into three regions: an external domain, enriched in glycosyl chains that probably allow band 3 protein to be recognized as a specific antigen (Bratosin et al., 1998); a transmembrane domain, representing the anionic exchanger of cells; and a cytosolic portion (Wang, 1994) containing all the sites of potential phosphorylation. Although serine/threonine (Ser/Thr)-phosphorylation of the band 3 cytosolic domain has been demonstrated to regulate the anion flux rate (Baggio et al., 1993a,b;), Tyr-P is involved in multiple functions, including regulation of glycolysis (Low et al., 1993), alteration of erythrocyte morphology (Bordin et al., 1995) and volume (Musch et al., 1999), and senescence (Bordin et al., 2009; Pantaleo et al., 2009).

We have previously demonstrated that the level of band 3 Tyr-P can be useful as a diagnostic system in analysing erythrocyte membrane status. When triggered by oxidative (diamide) or hyperosmotic stress, the band 3 Tyr-P level can predict both pathological and particular physiological conditions in red blood cells. In glucose-6-phosphate dehydrogenase (G6PD) deficiency, the higher band 3 Tyr-P level, compared with normal control cells, correlates well with chronic impairment of cell anti-oxidative defences (Bordin et al., 2005b); conversely, the lower band 3 Tyr-P level observed in pregnancy is synonymous of characteristically increased anti-oxidative defences (Bordin et al., 2006).

Our present data indicated that DDS-NHOH induced progressive alterations in erythrocytes, starting from cytosol, where it induces methaemoglobin formation (Israili et al., 1973; Schiff et al., 2006), to glutathione oxidation initial impairment of Tyr-protein kinase and then impairment of phosphatase activity. Later, the effect of DDS-NHOH progressed, with reorganization of membrane proteins, as shown by enzyme recruitment and the formation of high molecular weight aggregates (HMWA) of band 3 protein. Lastly, general membrane reorganization was achieved, with protein relocation from the Triton-soluble compartment to the cytoskeleton and with autologous antibody recognition. Notably, the same membrane reorganizations, as observed in cells treated in vitro, were found in the membranes of cells from two dapsone-treated patients, more markedly in the patient with glucose 6 phosphate dehydrogenase (G6PD) deficiency.

Methods

Treatment of erythrocytes

Following informed consent, erythrocytes were obtained from fresh blood collected from healthy volunteers, and from two middle-aged male patients, who were considered for dapsone therapy in the hospital's dermatological department. The choice of therapy was not influenced by the study design; the two patients were diagnosed as suffering from dermatitis herpetiformis according to skin biopsies and cell surface deposition of IgA, and were given oral dapsone. At admission, both had normal blood and urine samples. Their treatment started with 100 mg·day−1 dapsone, the usual dose (Leonard and Fry, 1991).

Patient 1 remained successfully in treatment for the length of the study; blood was withdrawn before and during dapsone administration (after 14 days' treatment). (Relative blood values are listed in Table S1)

Patient 2 was hospitalized for a haemolytic episode following 3 days of 100 mg·day−1 dapsone (P2100). His laboratory tests revealed that he had a deficiency in G6PD, class II, according to the WHO directive (Betke et al., 1967). G6PD residual activity in red cells was <10%, measured spectrophotometrically at 340 nm on a Sigma diagnostic kit (Sigma-Aldrich, Italy). Dapsone was discontinued for a month, after which laboratory test results had returned to normal range. Dapsone treatment was later re-administered, starting with 2 days with 30 mg·day−1, and then 50 mg·day−1, with partial relief but not total remission of symptoms.

Blood samples from both patients were taken before and during treatments. Samples from patient 1 were called P1 and P1100 to indicate samples before administration and during 100 mg·day−1 dapsone treatment; RBC from patient 2 were called P2, P230 and P250 to indicate samples withdrawn before and after 2 days at 30 mg·day−1, or after 3 days at 50 mg·day−1 dapsone, respectively.

Human erythrocytes were recovered and pelleted at 3000×g for 3 min (Brunati et al., 2000). After removal of the supernatant, packed RBC were washed three times at 3000×g for 3 min in nine volumes of Dulbecco's phosphate-buffered saline, containing 5 mM glucose (D-PBS), to avoid contamination by leukocytes and platelets.

For in vitro evaluation of the effects of dapsone and DDS-NHOH on normal erythrocytes, packed cells (50 µL) were resuspended (at 20% haematocrit) in D-PBS and incubated at 35°C for varying times in the presence or absence of increasing concentrations (from 0.15 to 0.6 mM) of dapsone or DDS-NHOH (or acetone as solvent). Parallel experiments were carried out in the same conditions but at 50% haematocrit in platelet poor-plasma (P-PP), diluted to 66% in D-PBS. In this case, blood was centrifuged at 180×g for 10 min, and the supernatant was further centrifuged at 1500×g for 15 min to obtain P-PP (Ciccoli et al., 2004); RBC were purified as described above.

After incubation, each sample was centrifuged at 3000×g, and packed cells were washed in D-PBS and subjected to haemolysis in 1.5 mL of hypotonic buffer containing 5 mM sodium phosphate, pH 8; 0.02% NaN3, 30 µM phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium orthovanadate, and a protease inhibitor cocktail.

For evaluation of RBC from patients receiving dapsone therapy, packed cells were incubated for 30 min at 35°C in D-PBS (20% haematocrit) in the presence or absence of 0.3 mM diamide, and then subjected to haemolysis as described above.

Membranes were separated from the cytosol by centrifugation (17 000 × g for 20 min) and washed once in hypotonic buffer. Aliquots of membranes (10 µg) were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE; 10% gels) in reducing conditions unless otherwise indicated, transferred to nitrocellulose membranes, and immunostained with the appropriate antibody.

Preparation of band 3 proteolytic fragment (cdb3)

The 40/45-kDa fragment of the cytoplasmic domain of band 3 (cdb3) was obtained by α-chymotrypsin-promoted breakdown of inverted membrane vesicles derived from ghosts and isolated by DE 52 chromatography, according to Bennett and Stenbuck, (1980).

[32P]-cdb3 was prepared by incubating 30 µg cdb3 for 10 min at 30°C, in 300 µL of the same incubation mixture as described below for the kinase assay except that the cell lysates were substituted by Syk immunoprecipitated (Syk-IP) protein from 3 mL of normal cell cytosol (Brunati et al., 2000). [32P]-cdb3 was recovered as supernatant after centrifuging 6 min at 6000× g, lyophilized, resuspended in imidazole buffer (25 mM, pH 7, mercaptoethanol 10 mM, dithiothreitol (DTT) 4 mM), and dialysed with the same buffer.

Protein kinase assay

Packed treated erythrocytes were subjected to several freeze–thaw cycles to yield a homogeneous cell lysate. 3 µL of each were assayed for tyrosine kinase activity at 30°C for 10 min in a 30-µL incubation mixture containing 50 mM Tris HCl, pH 7.5, 10 mM MnCl2, 30 µM (γ−32P)ATP (specific activity 1000 cpm·pmol−1), 10 µM vanadate and 3 µg of cdb3 as tyrosine kinase activity substrate. After incubation, the reaction was stopped by the addition of 1% SDS and 1% β-mercaptoethanol (final concentrations), and heated for 5 min at 100°C. Solubilized proteins were subjected to SDS/(10%)PAGE, and the gels were stained with Coomassie blue and treated with 2 M NaOH for 60 min at 55°C to highlight Tyr-P.

Dried gels were autoradiographed for 2 days. Bands corresponding to [32P]-cdb3 were then excised and counted in a liquid scintillation counter.

Protein phosphatase activity

Three µL of the total cell lysate obtained as above were assayed for tyrosine protein phosphatase activity in a 30-µL incubation mixture containing imidazole buffer and 3 µg [32P]-cdb3, at 30°C for 10 min.

The reaction was stopped as described above and solubilized proteins were subjected to SDS-PAGE (10% gels). The gels were stained, dried and autoradiographed, and [32P]-cdb3 was counted in a liquid scintillation counter.

Preparation of membrane skeletal and soluble fractions

Membranes, obtained from 200 µL of treated and untreated erythrocytes and recovered as described above, were extracted with 2 volumes of buffer A containing 50 mM Tris, pH 7.5, 1% (final) Triton X-100, 1 mM vanadate and protease inhibitor cocktail for 1 h at 4°C. After removal of one aliquot for Western blot analysis, the remainder was microfuged at 80 000×g for 40 min. Both supernatant, corresponding to the Triton-soluble fraction, and pellet, corresponding to the Triton-insoluble fraction (cytoskeleton), were then collected, and the pellet was resuspended to the same soluble fraction volume with buffer A. 10 µg of total membrane and the corresponding soluble and cytoskeleton fractions were then subjected to Western blot analysis and revealed with anti-band 3, anti-Syk or anti-SHP-2 antibodies.

Quantitative determination of total glutathione (GSSG+GSH) and oxidized glutathione (GSSG) content in erythrocytes

Total glutathione was determined according to the method of Tietze (1969). Briefly, 10 µL of cytosol, obtained from differently treated erythrocytes, was added to 2 mL of reaction mixture containing 1.9 mL of phosphate 0.1 M/ EDTA 0.6 mM buffer, pH 7.4, 30 µL of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) 10 mM, 100 µL of NADPH 5 mM and glutathione reductase 10 µg, and analysed spectrophotometrically at 412 nm. The GSSG content was evaluated in 10-µL cytosol incubated in the glutathione assay mixture, to which 3 µL of 2-vinylpyridine (2-VP) was added (Teare et al., 1993). The level of GSH was determined by calculating the difference between the two determinations.

Data analysis

Data are presented as means ± SEM of at least three individual experiments from six different blood donors. One-way or two-way anova was used to analyze the variables under investigation. Following a significant anova result, post hoc statistical analyses were carried out by the Tukey's honestly significant difference (HSD) test (Ruxton and Beauchamp, 2008). Differences were considered significant at P < 0.05.

Materials

For in vitro work, dapsone was supplied by Aldrich Chemistry (Milano, Italy), whereas for dapsone therapy, it was supplied by St. Antonio Bissone SA Pharmacy (Bissone/TI, Switzerland). D-PBS (Dulbecco's phosphate-buffered saline, containing 5 mM glucose), anti-P-Tyr and anti-Syk monoclonal antibodies were purchased from Sigma (Milan, Italy) and Upstate (Lake Placid, NY), respectively. Rabbit anti-SHP-2 (C-18) polyclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Protease inhibitor cocktail was obtained from Calbiochem (Darmstadt, Germany). [γ-32P]-ATP was purchased from Amersham Pharmacia Biotech (Little Chalfont, UK), and dapsone hydroxylamine (DDS-NHOH) from Toronto Research Chemicals Inc. (North York, Ontario). Anti-mouse and anti-rabbit secondary antibodies conjugated with horseradish peroxidase (HRP) were from Bio-Rad Laboratorories (Hercules, California), anti-human IgG-HRP was purchased from Biodesign (TEMA Ricerca, Bologna, Italy). All other reagents were from Sigma.

Results

The action of DDS-NHOH was tested by incubating human erythrocytes with increasing concentrations of the hydroxylamine for 30 min. In these conditions, the membrane proteins, mainly band 3, exhibited Tyr-P, which increased concentration dependently up to 0.3 mM DDS-NHOH and drastically decreased to the control level at higher concentrations. However, the parent compound, dapsone, was not able to trigger erythrocyte band 3 Tyr-P at any concentration (Figure 1).

Figure 1.

Figure 1

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Erythrocytes were incubated with increasing concentrations of dapsone (0.15–0.6 mM), all ineffective in triggering band 3 Tyr-P (and thus only shown in one lane) or DDS-NHOH. Membranes (10 µg), obtained as described in Methods, were analysed by Western blotting and revealed with anti-actin for loading control and, successively, with anti-P-Tyr antibodies. Membrane band 3 P-Tyr pattern obtained from erythrocytes treated with 1 mM diamide (Diam) was used to compare band 3 Tyr-P level. Lane C.B. Coomassie Blue staining gel. Lane Anti-band 3 shows the last lane after being stripped and reprobed with anti-band 3 antibody. Results shown are representative of six separate experiments.

Comparing the ability of DDS-NHOH to induce band 3 Tyr-P with that of another oxidant, diamide, the maximum Tyr-P-level for DDS-NHOH (at 0.3 mM) was close to that obtained with 1 mM diamide.

Band 3 P-Tyr level and enzyme recruitment

To better characterize DDS-NHOH-induced alterations in human erythrocytes, we tested the same hydroxylamine concentrations described above at increasing times of incubation. As shown in Figure 2, DDS-NHOH induced band 3 Tyr-P which peaked after 30 min at 0.3 mM, and was completely reversed after 45 min of incubation. This response was distinguishable from that previously observed with diamide, represented by concentration-dependent increase of band 3 Tyr-P (Bordin et al., 2005a). A relatively low concentration of DDS-NHOH (0.15 mM) yielded effects after 15 min, but to a lower extent, and decreased more rapidly in 30 min of incubation. 0.6 mM only induced a slight band 3 Tyr-P level that almost completely disappeared in 30 min of incubation (panel A).

Figure 2.

Figure 2

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Concentration- and time-dependent effect of DDS-NHOH on the membrane Tyr-phosphorylative process. Erythrocytes were treated with increasing DDS-NHOH concentrations and incubated at 35°C. Aliquots were taken at various incubation times, and membranes (10 µg), obtained as described in Methods, were analysed by Western blotting and revealed with anti-P-Tyr (panel A), anti-Syk (panel B) or anti-SHP-2 (panel C) antibodies. Densitometric analysis. Bands corresponding to band 3-Tyr-P, Syk and SHP-2 were counted in a densitometer. Amounts of Syk and SHP-2 in unstimulated erythrocytes were chosen as 100% value. For band 3-Tyr-P evaluation, an arbitrary unit was chosen, corresponding to band 3-Tyr-P level obtained with 1 mM diamide. Results represent means ± SEM of four separate experiments in duplicate. In each experiment, data analysis was carried out by two-way anova, giving significant differences among concentrations in the time course of experiment (P < 0.001). Post-hoc Tukey's test was performed; filled symbols indicate significant difference versus respective basal value (P < 0.01).

However, when analysed with anti-Syk (panel B) or anti-SHP-2 (panel C) antibodies, membranes revealed concentration and time-dependent recruitment of both enzymes (Figure S1). The content of both enzymes, after incubation with 0.6 mM, were almost four times greater than those in the control.

DDS-NHOH effect on enzyme activities

Tyr-kinase and phosphatase activities expressed by the cells after these treatments are shown in Table 1. For this purpose, erythrocytes were incubated with increasing DDS-NHOH concentrations and incubations were stopped at various incubation times. Cells were subjected to several freeze–thaw cycles as described in the Methods, and activities were assayed with cdb3 or 32P-cdb3 as substrates for Tyr-kinases or Tyr-P-phosphatases, respectively. We found that both Tyr-kinase and phosphatase activities were promptly inhibited by DDS-NHOH, both concentration- and time-dependently and total inactivation was reached in both after 60 min incubation with 0.15 and 0.3 mM. At 0.6 mM, DDS-NHOH treatment produced almost complete inhibition after only 15 min of incubation.

Table 1.

Tyr-protein kinase and phosphatase activities

Tyr-kinase activity (% cpm on cdb3) Tyr-phosphatase activity (% on 32P-cdb3)
Control 100 100
0.15 mM DDS-NHOH 15 min 95 ± 2 (ns) 90 ± 5 (ns)
 30 min 80 ± 4* 75 ± 7*
 60 min n.d. n.d.
 2 h30 min n.d. n.d.
0.3 mM DDS-NHOH 15 min 70 ± 2* 74 ± 3*
 30 min 53 ± 4* 50 ± 5*
 60 min n.d. n.d.
 2 h30 min n.d. n.d.
0.6 mM DDS-NHOH 15 min 13 ± 4* 15 ± 4*
 30 min n.d. n.d.
 60 min n.d. n.d.
 2 h30 min n.d. n.d.
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n.d. not detected.

Erythrocytes were treated with increasing concentrations of DDS-NHOH and recovered by centrifuging at 3000 g for 3 min. Packed cells were subjected to several freeze–thaw cycles and aliquots (3 µL) were incubated with purified cdb3 in presence of (γ-32P)ATP for kinase activity or [32P]-cdb3 for PTP-ase activity, as described in Methods. Values are expressed as % means ± SEM of corresponding control (n = 3).

*

P < 0.05, significantly different versus control (anova followed by Tukey's test); ns, not significant.

Effect of DDS-NHOH on band 3 aggregation

In a previous study, we proposed band 3 alterations as biomarkers able to reveal the status of membranes. Band 3 aggregated in high molecular weight aggregate (HMWA) complexes following diamide treatment (oxidative stress), but disaggregated in hypertonic conditions (hyperosmotic stress) (Bordin et al., 2006). We therefore analysed membranes from DDS-NHOH treated erythrocytes with anti-band 3 antibodies.

As shown in Figure 3, DDS-NHOH induced net band 3 reorganization, shown by the appearance of a band at 180–200 kDa, forming at DDS-NHOH concentrations higher than 0.15 mM (panel A). In addition, this rearrangement became more marked as incubation time was prolonged (panel B), thus partially confirming the hypothesis of a progression of the DDS-NHOH effect.

Figure 3.

Figure 3

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Erythrocytes were treated with increasing concentrations of DDS-NHOH or acetone (control) for 30 min (panel A), or for various incubation times (panel B). Membranes (1 µg), recovered as described in Methods, were analysed by SDS/8%PAGE in non-reducing conditions, blotted to nitrocellulose paper and revealed with anti-band 3 antibodies (anti-actin as loading control). A is representative of six separate experiments. B. Densitometric scanning of band 3 HMWA provided the values shown, expressed as fold-increase over the amount of band 3 HMWA in unstimulated cells. Results represent means ± SEM of six separate experiments in duplicate. Data analysis was carried out by two-way anova, indicating significant differences among concentrations in the time course of experiment (P < 0.001). Post-hoc Tukey's test was performed; filled symbols indicate significant difference versus respective basal value (P < 0.01).

DDS-NHOH-induced reorganization of cytoskeleton

To better evaluate band 3 aggregation, band 3 HMWA location in membrane organization was studied. Cells were treated with 0.3 mM DDS-NHOH for 30 min – conditions which induced the highest band 3 Tyr-P level – and for prolonged incubation times – when band 3 Tyr-P level was absent. Corresponding membranes were extracted with Triton X-100 and the membrane-soluble compartment was separated from the Triton-insoluble cytoskeleton (Bordin et al., 2005a). The pattern of Figure 4 shows that, following 30 min DDS-NHOH treatment, the HMWA content of band 3 markedly increased (about five times that of the control), but only in the Triton-soluble fraction, whereas the corresponding cytoskeleton (Triton-insoluble fraction) showed the same amount of band 3 HMWA as the control. By prolonging incubation time, not only was the content of band 3 HMWA further increased (up to about seven times the control), but complete membrane reorganization also occurred, the cytoskeleton containing almost all the band 3 HMWA complexes formed, including some of those previously present in the soluble fraction.

Figure 4.

Figure 4

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Erythrocytes were treated with 0.3 mM DDS-NHOH or solvent, for 30 min or prolonged time incubation (2 h30 min and 24 h, which showed similar patterns) as indicated. Membranes (10 µg) were extracted with Triton X-100. Membranes, Triton-soluble or –insoluble (cytoskeleton) fractions, collected by ultracentrifugation (see Methods) were analysed by SDS/8%PAGE in non-reducing conditions and revealed with anti-band 3 antibodies. Results shown are representative of five separate experiments.

When erythrocytes were treated with 0.6 mM DDS-NHOH the band 3 HMWA were detected almost completely in the cytoskeleton even after 30 min incubation (data not shown).

Effect of DDS-NHOH on enzyme redistribution

Following the rearrangement of band 3 HMWA complexes within RBC membranes, we also analysed enzyme distribution between Triton-soluble and insoluble fractions of the membranes (Figure 5). Interestingly, both Syk (panel A) and SHP-2 (panel B) were engaged by DDS-NHOH to the cytoskeleton, while the amounts of both enzymes present in the membrane extract (Triton-soluble fraction) remained similar to that present in the control Triton-soluble fraction. Only treatment with 0.3 mM DDS-NHOH for 30 min incubation showed an increase (about 30% more than in the control) of the amount of Syk present in the Triton-soluble fraction. As these were the conditions for the maximum level of band 3 Tyr-P, the favoured substrate-kinase combination resulting in the band 3 Tyr-phosphorylating process may be ascribed to this Syk recruitment to the soluble fraction.

Figure 5.

Figure 5

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Enzyme distribution following DDS-NHOH treatment. Erythrocytes were treated with increasing DDS-NHOH concentrations or solvent (control), for 30 min or prolonged incubation (2 h30 min and 24 h patterns being similar are shown in same panel). Membranes (10 µg) were extracted with Triton X-100. Membranes, Triton-soluble (extracted) or insoluble (cytoskeleton) fractions, collected by ultracentrifugation (see Methods) were analysed by SDS-PAGE and revealed with anti-Syk (panel A) or anti-SHP-2 (panel B) antibodies. Bands corresponding to Syk and SHP-2 were counted in a densitometer. Amounts of Syk and SHP-2 in unstimulated cells were chosen as 100% value. Results are means ± SEM of three separate experiments in duplicate. Data analysis was carried out by one-way anova, showing significant differences in means among different treatments for membrane and cytoskeleton fractions (P < 0.001); for extract fraction, no significant difference was found. Post-hoc Tukey's test was performed versus corresponding basal value. *P < 0.01, significant difference versus corresponding basal value considered as control.

DDS-NHOH-induced autologous antibody binding

Removal of human erythrocytes involves cells undergoing significant alterations, induced either by normal ageing or by toxic/disease injury, and is mediated by antibody recognition (Lutz et al., 1987; Bratosin et al., 1998; Arese et al., 2005; Kay, 2005).

When we analysed membranes from erythrocytes exposed to concentration- and time-dependent DDS-NHOH treatments in the presence of autologous plasma (P-PP), immunostaining with anti-human IgG revealed net enhancement of the number of autologous antibodies with increasing both DDS-NHOH concentration (Figure 6, panel A) and incubation time (panel B).

Figure 6.

Figure 6

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Erythrocytes were incubated in D-PBS-P-PP with increasing DDS-NHOH concentrations and incubated at 35°C for 24 h (panel A), or for varying incubation times (panel B). Membranes (15 µg), obtained as described in Methods, were analysed by Western blotting under non-reducing conditions and revealed with anti-human IgG HRP (or anti-actin as loading control). A: Lane Ctrl- represents erythrocytes incubated in D-PBS; Ctrl+ was obtained by incubating erythrocytes in D-PBS-P-PP in presence of 2 mM diamide. Results shown are representative of three separate experiments. B. Band corresponding to autologous antibody was counted in a densitometer. Amount of IgG bound to unstimulated cells was chosen as arbitrary unit. Results are means ± SEM of six experiments in duplicate. Data analysis was carried out by two-way anova, indicating significant differences among concentrations in the time course of experiment (P < 0.001). Post-hoc Tukey's test was performed; asterisks indicate significant difference (*P < 0.01) versus basal value considered as control.

Interestingly, in this case too, the progression of the DDS-NHOH effect was clearly distinguishable, with an initial slight antibody recognition which occurred only at 0.3 and 0.6 mM, and was increased greatly after 24 h treatment.

Membrane reorganization, IgG binding and phosphorylative process in RBC from patients receiving dapsone therapy

In order to confirm that the DDS-NHOH-induced membrane reorganization described above was the mechanism effectively leading to RBC denaturation/removal in vivo, we analysed membranes from two patients receiving dapsone therapy for dermatitis herpetiformis. Quantitative analysis obtained by densitometric scanning of the anti-band 3 and anti-IgG immune-blots, evidenced that dapsone therapy in patient 1 (P1) induced a slight increase in band 3 HMWA (12.3 ± 0.9%) (Figure 7, lane b compared with lane a, representing P1 before dapsone), which was correlated with an increase in bound IgG (11 ± 0.8%) (Figure 7, anti-human IgG panel). Instead, patient 2, characterized by G6PD deficiency, was clearly intolerant to the same 100 mg·day−1 oral dapsone (Table S1) so that the dose was reduced to 30 mg·day−1 and then to 50 mg·day−1. However, even when corrected and reduced, dapsone was not completely well tolerated by P2, who showed slight signs of intolerance (Table S1). RBC membranes from patient 2 showed a higher level of band 3 HMWA (Figure 7, anti-band 3 panel) even before dapsone (P2) (lane c). This band 3 aggregation increased (22 ± 0.8%) during the 30 mg·day−1 dapsone (P230, lane d), but reached a dramatic level at 50 mg·day−1 (P250, lane e). The effect was correlated with a 30% increase in bound IgG in P230 and with more than 200% in P250 (Figure 7, anti-human IgG panel, lanes d, e, compared with lane c, corresponding to P2 before dapsone).

Figure 7.

Figure 7

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RBC were collected from Patient 1 before (P1) and during 100 mg·day−1 of dapsone therapy (P1100), and patient 2 before (P2) and after 30 mg·day−1 (P230) and during 50 mg·day−1 (P250) of dapsone therapy. Membranes (1 µg), recovered as described in Methods, were subjected to Western blotting in non-reducing conditions and revealed with anti-band 3 antibodies. For analysis of human IgG, the same membranes (10 µg) were subjected to separate Western blotting in non-reducing conditions and revealed with anti-human IgG. Anti-actin panel, representing loading control, is referred to anti-human IgG panel. Results shown are representative of two separate experiments in duplicate.

In addition, when analysed for their Tyr-P level extent, membranes from RBC of both patients showed that the basal level of band 3 Tyr-P was negligible (Figure 8, lanes a, c, e–g). Successive analysis of glutathione content (Table 2) disclosed that dapsone treatment induced a decrease in total GSH content in both patients. However, whereas P1100 maintained about 85% of total glutathione in the reduced form (GSH), P2 showed progressive depletion of glutathione, with an alarming rise in GSSG which, at P250, reached almost 60% of total glutathione. When erythrocytes from both patients were subjected to 0.3 mM diamide to induce oxidative stress, P1100 showed a reduction in total glutathione content and a rise of GSSG. P2 and P230 also displayed a net reduction in the amount of total glutathione which at P250 was only 50%, compared with the glutathione content of P2. The diamide-induced net increase in the GSSG form reached almost 100% glutathione at P250.

Figure 8.

Figure 8

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RBC from patients 1 and 2 receiving different doses of dapsone (100, 50 or 30 mg) were incubated in absence or presence of 0.3 mM diamide (diam). Membranes (10 µg), obtained as described in Methods, were subjected to Western blotting and revealed with anti-P-Tyr (uppermost panel), anti-Syk or anti-SHP-2 (successive panels) antibodies. Anti-actin panel represents loading control (lowest panel).

Results shown are representative of two separate experiments in duplicate.

Table 2.

Glutathione content

Blood samples Glutot (mM) basal GSSG (mM) Glutot (mM) 0.3 mM diamide GSSG (mM)
DDS-NHOH (mM): 0 2.15 ± 0.03 0.15 ± 0.01
0.15 1.87 ± 0.02a 0.25 ± 0.02*
0.3 1.58 ± 0.02a 0.35 ± 0.02*
0.6 1.25 ± 0.02a 0.65 ± 0.02*
P1 2.12 ± 0.03 0.15 ± 0.01 1.92 ± 0.03 0.26 ± 0.01
P1100 1.89 ± 0.02† 0.25 ± 0.02† 1.68 ± 0.02† 0.35 ± 0.02†
P2 1.83 ± 0.03 0.22 ± 0.04 1.56 ± 0.03 0.35 ± 0.04
P230 1.36 ± 0.03† 0.45 ± 0.03† 1.18 ± 0.03† 0.73 ± 0.03†
P250 0.92 ± 0.03† 0.52 ± 0.03† 0.76 ± 0.03† 0.67 ± 0.03†
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Control erythrocytes were incubated with increasing DDS-NHOH concentrations, haemolysed, cytosol was recovered and total glutathione (Glutot = GSH + GSSG) and oxidized glutathione (GSSG) were determined, as described in Methods.

*

P < 0.01, significant effect of DDS-NHOH (anova followed by Tukey's test).

When patients receiving dapsone therapy were analysed, erythrocytes, withdrawn at different stages of therapy, were haemolysed, both untreated or following treatment with 0.3 mM diamide. Cytosol was recovered and total glutathione (GSH + GSSG) and oxidized glutathione (GSSG) determined. Values are means ± SEM of six separate experiments.

†

P < 0.01, significant effect of dapsone treatment (anova followed by Tukey's test) versus corresponding values before dapsone treatment.

Interestingly, when RBC were incubated with 0.3 mM diamide (conditions that triggered band 3 Tyr-P in G6PD deficient patients, but not in normal subjects; (Bordin et al., 2006), P1100 exhibited a slight trace of band 3 Tyr-P. In contrast, P2 showed net band 3 Tyr-P (Figure 8, lane h) – as expected, due to his G6PD deficiency, which dramatically escalated on increasing dapsone treatment (lanes i, j). Syk and SHP-2 content in membranes from P2 also rose after dapsone, in both the absence (lanes e-g) and presence (lanes h-j) of diamide incubation.

Discussion and conclusions

Oxidative stress occurs when ROS, which are intermediaries of normal oxygen metabolism, are produced faster than the endogenous antioxidant defence systems can neutralize them. The excessive production of ROS can damage proteins, lipids, nucleic acids and matrix components in a number of cells, and is often related to both metabolic and inflammatory diseases. We have recently shown that, in G6PD deficient erythrocytes, characterized by chronic lowering of anti-oxidant defences, membrane status seems similar to that of control cells, but diamide-induced oxidative assault results in an abnormal increase in the Tyr-P level of membrane band 3 (Bordin et al., 2005b). Similarly, in endometriosis, typified by excessive ROS production due to inflammatory status, analysis of the diamide-induced abnormally higher erythrocyte band 3 Tyr-P level can discriminate endometriotic from non-endometriotic patients. In addition, the sensitivity of this parameter also allows it to detect post-surgical systemic improvement, also represented by decreased diamide-induced Tyr-P of band 3 (Bordin et al., 2010).

Diamide is known to trigger band 3 Tyr-P by partially inhibiting Tyr-protein phosphatases (Bordin et al., 2005a) and by inducing disulfide bond formation, leading to band 3 aggregation (Fiore et al., 2008). The band 3 Tyr-phosphorylative mechanism has also been considered in the evaluation of DDS-NHOH-induced erythrocyte alterations. That DDS-NHOH did not act as an oxidant like diamide was indicated by the fact that SHP-2 recruitment (Figure 2C) was not Tyr-P-dependent. This observation represents a novel event for SHP-2 recruitment because we had previously observed that diamide-induced SHP-2 translocation to membranes occurred only after Lyn-catalysed phosphorylation of band 3 residue 359, which is the docking site for this phosphatase (Bordin et al., 2002). DDS-NHOH modifies membranes in such a way as to uncover new docking sites for SHP-2. Together with the massive Syk translocation, which is indifferent to Tyr-P level (Bordin et al., 2005a), SHP-2 relocation can also be used as a further parameter of the altered status of membranes.

That this compound affected oxidative status of the cell was previously demonstrated by its ability to induce oxyhaemoglobin, GSH content decrease and conversion to oxidized glutathione (GSSG) and GS-S-protein (Table 2) through a ROS-generating system, yielding nitrosoarene and methaemoglobin (McMillan et al., 1995; 2005;). A number of active oxygen species and sulphur-free radicals are produced as a consequence of this redox cycle and are certainly more or less involved in the erythrocyte damage leading to their removal from circulation. This damage is indicated by the time- and concentration-dependent increase of IgG binding to the membranes, as an indicator of reduced erythrocyte lifespan (Lutz et al., 1987; Arese et al., 2005; Kay, 2005).

We observed that DDS-NHOH triggered Tyr-P of band 3 protein in intact erythrocytes. This was expected by an oxidant, such as diamide, directly oxidising cysteine located in the catalytic domain of the enzyme (Hecht and Zick, 1992) and thus inhibiting protein tyrosine phosphatase activities. In 30 min incubation, DDS-NHOH induces band 3 aggregation in HMWA mainly located in the Triton-soluble part of the membrane.

With increasing incubation times, the effects of DDS-NHOH were clearly differentiated from those of diamide. These effects of DDS-NHOH were amplified, leading to further increases in band 3 HMWA, but, more interestingly, also to their total relocation within the membrane, accompanied by reorganization of both recruited Tyr-protein kinases and phosphatases, which were markedly inhibited (Table 1). This new membrane organization was easily recognized and marked by autologous IgG, and is representative of damaged cells.

In addition, since band 3 Tyr-P gradually tails off within the first 45 min (Figure 2), which may represent the time threshold between the formation of two differently located band 3 aggregates – Triton-soluble, and, successively, cytoskeleton bound – we can postulate that the Tyr-phosphorylative process is a cellular defence against the incoming oxidative modifications induced by DDS-NHOH. In this process, introduction of negative charges, represented by phosphate groups, to band 3 protein would slow down its aggregation, at least up to the total arrest of the phosphorylative process. Subsequently, modifications would continue more profoundly, inducing not only more marked clustering of band 3 but also totally redistributing HMWA from soluble to insoluble (cytoskeleton) membrane fractions (Figure 4). This is further suggested by total rearrangement of band 3 HMWA at 0.6 mM DDS-NHOH: in these conditions, band 3 Tyr-P is very slight (Figures 1 and 2), and band 3 HMWA were located in the cytoskeleton even after 30 min incubation (data not shown).

This may fit the hypothesis that reactive radicals also generate a second species of radicals, probably thiyl radicals (McMillan et al., 2005), more reactive and efficacious in generating many and drastic alterations in membrane structure and composition.

This is in line with evidence from patients receiving dapsone therapy. In normal subjects, therapy leads to weakening of anti-oxidant defences (as indicated by decreased GSH content) and triggers membrane reorganization, as indicated by increased band 3 HMWA formation and higher sensitivity towards diamide-induced oxidative stress (Figure 8). When dapsone was administered to our G6PD deficient patient (P2), falls in both haemoglobin content and haematocrit were observed at P250, suggesting the onset of the haemolytic process. This cannot be explained by a simple fall in GSH content (see Table 2) since, even at 50 mg·day−1 dapsone (P250), almost one-third of glutathione was in reduced form, but incapable of preventing dapsone-induced erythrocyte modification. One explanation matches expectations with the in vitro effect of DDS-NHOH on membrane alterations: HMWA and IgG binding were clearly present (Figure 7), showing that DDS-NHOH induced membrane reorganization had occurred. However, band 3 Tyr-P was not detected, even in P250 erythrocytes, where the effects of DDS-NHOH were enhanced by the concomitant defect in anti-oxidant defences. During dapsone treatment, DDS-NHOH does not irreversibly inhibit Tyr-protein kinases and/or phosphatases, as indicated by the diamide-induced band 3 Tyr-P of patients' erythrocytes (especially in P2) (Figure 8). This was probably because the concentration of this effector is too low to have immediate effects on the enzymes, like those observed in our in vitro experiments, which would represent high, toxic levels of this metabolite.

DDS-NHOH cannot be directly detected in human plasma, as it is rapidly taken up by erythrocytes prior to its redox cycling with haemoglobin, forming methaemoglobin (Coleman and Jacobus, 1993). In any case, the major metabolic elimination of dapsone is by N-hydroxylation, which accounts for between 30% and 40% of an oral dapsone dose, and the efficiency of N-hydroxylation is related to dapsone clearance (May et al., 1990; 1992; Bluhm et al., 1999). Dapsone therapy includes a daily administration of 50–100 mg for leprosy and 100–300 mg for dermatitis herpetiformis (Leonard and Fry, 1991), leading to serum concentrations of 0.5–5 mg·L−1 (equivalent to 2–20 µM); therapeutic doses up to 400 mg have been reported (Elonen et al., 1979; Zuidema et al., 1986), as well as some cases of intoxication with dapsone, such as after an overdose with 10 g of dapsone, leading to serum concentrations of 120 mg·L−1 (about 0.5 mM, comparable to those used in our in vitro experiments). Another case of intoxication produced methaemoglobinemia at serum concentrations of 18.8 mg·L−1 (76 µM) (Woodhouse et al., 1983). The acetylation ratio (MADDS : DDS) shows a genetically determined bimodal distribution allowing the definition of ‘slow’ and ‘rapid’ acetylators (Zuidema et al., 1986); therefore the amount of dapsone available for N-hydroxylation can vary accordingly, and blood cell damage can be more prominent, notably in the presence of G6PD deficiency. Our study confirms previous reports, stressing that sensitiveness to the compound is clearly idiosyncratic and dependent on the patho/physiological patient status (May et al., 1990; 1992; Wertheim et al., 2006) who may also be at risk of the dapsone hypersensitivity syndrome (Sener et al., 2006) even at a dose of 100 mg·day−1. Alterations observed in the RBC membranes of P2 were very similar to those obtained in vitro with DDS-NHOH treatments, which were representative of a larger dapsone intake and may account for the toxic adverse effects reported after dapsone therapy. In patients receiving dapsone therapy, erythrocyte band 3 Tyr-P is not present in non-stimulated conditions, and this would be in agreement with the hypothesis that band 3 Tyr-P is related to the eryptosis (Bottini et al., 1997; Bordin et al., 2009), and, for this, is not compatible with patient tolerance. Diamide treatment can trigger this process to abnormally high levels, thus reflecting dapsone treatment-induced membrane alterations. Monitoring of band 3 alterations during dapsone intake in patients would be useful approach in preventing potential drug adverse effects due to incorrect dosage.

In conclusion, DDS-NHOH may be considered responsible for the adverse effects of dapsone on red blood cells. Net reorganization of membranes, leading to the autologous antibody binding seen following DDS-NHOH treatment of red blood cells in vitro, has also been demonstrated to characterize membranes from erythrocytes of patients receiving dapsone therapy. In addition, this alteration process is both concentration- and time-dependent, and involves oxidation of cytosol and membrane-bound proteins in a progressive mechanism which, in the end, causes shortening of the erythrocyte cell lifespan.

Acknowledgments

This work was supported by the Italian Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MURST).

The authors thank Claudio Bettella and Giancarlo Ruffato for supplying fresh blood from volunteers.

Glossary

Abbreviations

cdb3

cytoplasmic domain of band 3

DDS

dapsone

DDS-NHOH

dapsone hydroxylamine

G6PD

glucose 6 phosphate dehydrogenase

GSH

glutathione

GSSG:

oxidized glutathione

HMWA

high molecular weight aggregate

RBC

red blood cell

Tyr-P

tyrosine phosphorylation

Conflict of interest

The authors state no conflict of interest.

Supporting information

Additional Supporting Information may be found in the online version of this article:

Figure S1 Dose- and time-dependent effect of DDS-NHOH on Syk and SHP-2 recruitment to membrane.

bph0161-1186-SD1.tif (1.6MB, tif)
bph0161-1186-SD2.tif (1.7MB, tif)

Table S1 Test values.

bph0161-1186-SD3.doc (30.5KB, doc)

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

References

  1. Arese P, Turrini F, Schwarzer E. Band 3/complement-mediated recognition and removal of normally senescent and pathological human erythrocytes. Cell Physiol Biochem. 2005;16:133–146. doi: 10.1159/000089839. [DOI] [PubMed] [Google Scholar]
  2. Baggio B, Bordin L, Clari G, Gambero G, Moret V. Functional correlation between the Ser/Thr-phosphorylation of band 3 and band 3-mediated transmembrane anion transport in human erythrocytes. Biochim Biophys Acta. 1993a;1148:157–160. doi: 10.1016/0005-2736(93)90173-w. [DOI] [PubMed] [Google Scholar]
  3. Baggio B, Bordin L, Gambaro G, Piccoli A, Marzaro G, Clari G. Evidence of a link between band 3 phosphorylation and anion transport in patients with ‘idiopathic’ calcium oxalate nephrolithiasis. Miner Electrolyte Metab. 1993b;19:17–20. [PubMed] [Google Scholar]
  4. Bahadir S, Cobanoglu U, Cimsit G, Yayli S, Alpay K. Erythema dyschromicum perstans: response to dapsone therapy. Int J Dermatol. 2004;43:220–222. doi: 10.1111/j.1365-4632.2004.01984.x. [DOI] [PubMed] [Google Scholar]
  5. Bennett V, Stenbuck PJ. Association between ankyrin and the cytoplasmic domain of band 3 isolated from the human erythrocyte membrane. J Biol Chem. 1980;255:6424–6432. [PubMed] [Google Scholar]
  6. Betke K, Beutler E, Brewer GJ, Kirkman HN, Luzzato L, Motulsky AG, et al. Standardization of procedures for the study of glucose-6-phosphate dehydrogenase. 1967. Report of a WHO scientific group-WHO Technical Report-Serial 366.
  7. Bluhm RE, Adedoyin A, McCarver DG, Branch RA. Development of dapsone toxicity in patients with inflammatory dermatoses: activity of acetylation and hydroxylation of dapsone as risk factors. Clin Pharmacol Ther. 1999;65:598–605. doi: 10.1016/S0009-9236(99)90081-4. [DOI] [PubMed] [Google Scholar]
  8. Bordin L, Clari G, Moro I, Dalla Vecchia F, Moret V. Functional link between phosphorylation state of membrane proteins and morphological changes of human erythrocytes. Biochem Biophys Res Commun. 1995;213:249–257. doi: 10.1006/bbrc.1995.2123. [DOI] [PubMed] [Google Scholar]
  9. Bordin L, Brunati AM, Donella-Deana A, Baggio B, Toninello A, Clari G. Band 3 is a anchor protein and a target for SHP-2 tyrosine phosphatases in human erythrocytes. Blood. 2002;100:276–282. doi: 10.1182/blood.v100.1.276. [DOI] [PubMed] [Google Scholar]
  10. Bordin L, Ion-Popa F, Brunati AM, Clari G, Low PS. Effector-induced Syk-mediated phosphorylation in human erythrocytes. Biochim Biophys Acta. 2005a;1745:20–28. doi: 10.1016/j.bbamcr.2004.12.010. [DOI] [PubMed] [Google Scholar]
  11. Bordin L, Zen F, Ion-Popa F, Barbetta M, Baggio B, Clari G. Band 3 Tyr-P in normal and glucose-6-phosphate dehydrogenase-deficient human erythrocytes. Mol Membr Biol. 2005b;22:411–420. doi: 10.1080/09687860500233679. [DOI] [PubMed] [Google Scholar]
  12. Bordin L, Quartesan S, Zen F, Vianello F, Clari G. Band 3 Tyr-P in human erythrocytes from non-pregnant and pregnant women. Biochim Biophys Acta. 2006;1758:611–619. doi: 10.1016/j.bbamem.2006.03.009. [DOI] [PubMed] [Google Scholar]
  13. Bordin L, Fiore C, Bragadin M, Brunati AM, Clari G. Regulation of membrane band 3 Tyr-Pby proteolysis of p72syk and possible involvement in senescence process. Acta Biochim Biophys Sin. 2009;41:846–851. doi: 10.1093/abbs/gmp071. [DOI] [PubMed] [Google Scholar]
  14. Bordin L, Fiore C, Donà G, Andrisani A, Ambrosini G, Faggian D, et al. Evaluation of erythrocyte band 3 phosphotyrosine level, glutathione content, CA-125, and human epididymal secretory protein E4 as combined parameters in endometriosis. Fertil Steril. 2010 doi: 10.1016/j.fertnstert.2009.10.038. Epub ahead of print. [DOI] [PubMed] [Google Scholar]
  15. Bottini E, Bottini FG, Borgiani P, Businco L. Association between ACP1 and favism: a possible biochemical mechanism. Blood. 1997;89:2613–2615. [PubMed] [Google Scholar]
  16. Bradshaw TP, McMillan DC, Crouch RK, Jollow DJ. Formation of free radicals and protein mixed disulfides in rat red cells exposed to dapsone hydroxylamine. Free Radic Biol Med. 1997;22:1183–1193. doi: 10.1016/s0891-5849(96)00542-4. [DOI] [PubMed] [Google Scholar]
  17. Bratosin D, Mazurier J, Tissier JP, Estaquier J, Huart JJ, Ameisen JC, et al. Cellular and molecular mechanisms of senescent erythrocyte phagocytosis by macrophages. A review. Biochimie. 1998;80:173–195. doi: 10.1016/s0300-9084(98)80024-2. [DOI] [PubMed] [Google Scholar]
  18. Brunati AM, Bordin L, Clari G, James P, Quadroni M, Baritono E, et al. Sequential phosphorylation of protein band 3 by Syk and Lyn tyrosine kinases in intact human erythrocytes: identification of primary and secondary phosphorylation sites. Blood. 2000;96:1550–1557. [PubMed] [Google Scholar]
  19. Byrd SR, Gelber R. Effect of dapsone on haemoglobin concentration in patients with leprosy. Lep Rev. 1991;62:171–178. doi: 10.5935/0305-7518.19910020. [DOI] [PubMed] [Google Scholar]
  20. Ciccoli L, Rossi V, Leoncini S, Signorini C, Blanco-Garcia J, Aldinucci C, et al. Iron release, superoxide production and binding of autologous IgG to band 3 dimers in newborn and adult erythrocytes exposed to hypoxia and hypoxia-reoxygenation. Biochim Biophys Acta. 2004;1672:203–213. doi: 10.1016/j.bbagen.2004.04.003. [DOI] [PubMed] [Google Scholar]
  21. Coleman MD, Jacobus DP. Reduction of dapsone hydroxylamine to dapsone during methaemoglobin formation in human erythrocytes in vitro. Biochem Pharmacol. 1993;45:1027–1033. doi: 10.1016/0006-2952(93)90246-s. [DOI] [PubMed] [Google Scholar]
  22. Coleman MD, Breckenridge AM, Park BK. Bioactivation of dapsone to a cytotoxic metabolite by human hepatic microsomal enzymes. Br J Clin Pharm. 1989;28:389–395. doi: 10.1111/j.1365-2125.1989.tb03517.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Coleman MD, Tingle MD, Hussain F, Storr RC, Park BK. An investigation into the haematological toxicity of structural analogues of dapsone in vivo and in vitro. J Pharm Pharmacol. 1991;43:779–784. doi: 10.1111/j.2042-7158.1991.tb03481.x. [DOI] [PubMed] [Google Scholar]
  24. Cream JJ. Anaemia in dermatitis herpetiformis role of dapsone-induced haemolysis and malabsorption. Br J Dermatol. 1970;82:333–338. doi: 10.1111/j.1365-2133.1970.tb06829.x. [DOI] [PubMed] [Google Scholar]
  25. Elonen E, Neuvonen PJ, Halmekoski J, Mattila MJ. Acute dapsone intoxication: a case with prolonged symptoms. Clin Toxicol. 1979;14:79–85. doi: 10.3109/15563657909030116. [DOI] [PubMed] [Google Scholar]
  26. Fiore C, Bordin L, Pellati D, Armanini D, Clari G. Effect of glycyrrhetinic acid on membrane band 3 in human erythrocytes. Arch Biochem Biophys. 2008;479:46–51. doi: 10.1016/j.abb.2008.08.011. [DOI] [PubMed] [Google Scholar]
  27. Gonazales MA, Menendez C, Font F, Kahigwa E, Kimario J, Mshnda H, et al. Cost-effectiveness of iron supplementation and malaria chemoprophylaxis in the prevention of anaemia and malaria among Tanzanian infants. Bull WHO. 2000;78:97–107. [PMC free article] [PubMed] [Google Scholar]
  28. Grossman S, Budinsky R, Jollow D. Dapsone-induced hemolytic anemia: role of glucose-6-phosphate dehydrogenase in the hemolytic response of rat erythrocytes to N-hydroxydapsone. J Pharmacol Exp Ther. 1995;273:870–877. [PubMed] [Google Scholar]
  29. Hecht D, Zick Y. Selective inhibition of protein tyrosine phosphatase activities by H2O2 and vanadate in vitro. Biochem Biophys Res Commun. 1992;188:773–779. doi: 10.1016/0006-291x(92)91123-8. [DOI] [PubMed] [Google Scholar]
  30. Israili ZH, Cucinell SA, Vaught J, Davis B, Zesser JM, Dayton PG. Studies of the metabolism of DDS in man and experimental animals. Formation of N-hydroxy metabolites. J Pharmacol Exp Ther. 1973;187:138–151. [PubMed] [Google Scholar]
  31. Kay M. Immunoregulation of cellular life span. Ann N Y Acad Sci. 2005;1057:85–111. doi: 10.1196/annals.1356.005. [DOI] [PubMed] [Google Scholar]
  32. Leonard JN, Fry L. Treatment and management of dermatitis herpetiformis. Clin Dermatol. 1991;9:403–408. doi: 10.1016/0738-081x(91)90032-g. [DOI] [PubMed] [Google Scholar]
  33. Low PS, Rathinavelu P, Harrison ML. Regulation of glycolysis via reversible enzyme binding to the membrane protein band 3. J Biol Chem. 1993;268:14627–14631. [PubMed] [Google Scholar]
  34. Lutz HU, Bussolino F, Flepp R, Fasler S, Stammler P, Kazatchkine MD, et al. Naturally occurring anti-band-3 antibodies and complement together mediate phagocytosis of oxidatively stressed human erythrocytes. Proc Natl Acad Sci USA. 1987;84:7368–7376. doi: 10.1073/pnas.84.21.7368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. McMillan DC, Simson JV, Budinsky RA, Jollow DJ. Dapsone-induced hemolytic anemia: effect of dapsone hydroxylamine on sulfhydryl status, membrane skeletal proteins and morphology of human and rat erythrocytes. J Pharmacol Exp Ther. 1995;274:540–547. [PubMed] [Google Scholar]
  36. McMillan DC, Jensen CB, Jollow DJ. Role of lipid peroxidation in dapsone-induced hemolytic anemia. J Pharmacol Exp Ther. 1998;287:868–876. [PubMed] [Google Scholar]
  37. McMillan DC, Powell CL, Bowman ZS, Morrow JD, Jollow DJ. Lipid versus proteins as major targets of pro-oxidant, direct-acting hemolytic agents. Toxicol Sci. 2005;88:274–283. doi: 10.1093/toxsci/kfi290. [DOI] [PubMed] [Google Scholar]
  38. May DG, Porter JA, Uetrecht JP, Wilkinson GR, Branch RA. The contribution of N-hydroxylation and acetylation to dapsone pharmacokinetics in normal subjects. Clin Pharmacol Ther. 1990;48:619–627. doi: 10.1038/clpt.1990.204. [DOI] [PubMed] [Google Scholar]
  39. May DG, Arns PA, Richards WO, Porter J, Ryder D, Fleming CM, et al. The disposition of dapsone in cirrhosis. Clin Pharmacol Ther. 1992;51:689–700. doi: 10.1038/clpt.1992.81. [DOI] [PubMed] [Google Scholar]
  40. Musch MW, Hubert EM, Goldstein L. Volume expansion stimulates p72syk and p56lyn in skate erythrocytes. J Biol Chem. 1999;274:7923–7928. doi: 10.1074/jbc.274.12.7923. [DOI] [PubMed] [Google Scholar]
  41. Pantaleo A, Ferru E, Giribaldi G, Mannu F, Carta F, Matte A, et al. Oxidized and poorly glycosylated band 3 is selectively phosphorylated by Syk kinase to form large membrane clusters in normal and G6PD-deficient red blood cells. Biochem J. 2009;418:359–367. doi: 10.1042/BJ20081557. [DOI] [PubMed] [Google Scholar]
  42. Ruxton GD, Beauchamp G. Time for some a priori thinking about post hoc testing. Behav Ecol. 2008;19:690–693. [Google Scholar]
  43. Sangiolo D, Storer B, Nash R, Corey L, Davis C. Toxicity and efficacy of daily dapsone as Pneumocystis jiroveci prophylaxis after hematopoietic stem cell transplantation: a case-control study. Biol Blood Marrow Transplant. 2005;11:521–529. doi: 10.1016/j.bbmt.2005.04.011. [DOI] [PubMed] [Google Scholar]
  44. Schiff DE, Roberts WD, Sue YJ. Methaemoglobinemia associated with dapsone therapy in a child with pneumonia and chronic immune thrombocytopenic purpura. J Pediatr Hematol Oncol. 2006;28:395–398. doi: 10.1097/00043426-200606000-00015. [DOI] [PubMed] [Google Scholar]
  45. Sener O, Doganci L, Safali M, Besirbellioglu B, Bulucu F, Pahsa A. Severe dapsone hypersensitivity syndrome. J Investig Allergol Clin Immunol. 2006;16:268–270. [PubMed] [Google Scholar]
  46. Teare JP, Punchard NA, Powell JJ, Lumb PJ, Mitchell WD, Thompson RP. Automated spectrophotometric method for determining oxidized and reduced glutathione in liver. Clin Chem. 1993;39:686–689. [PubMed] [Google Scholar]
  47. Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem. 1969;27:502–522. doi: 10.1016/0003-2697(69)90064-5. [DOI] [PubMed] [Google Scholar]
  48. Ujiie H, Shimizu T, Ito M, Arita K, Shimizu H. Lupus erythematosus profundus successfully treated with dapsone: review of the literature. Arch Dermatol. 2006;142:399–401. doi: 10.1001/archderm.142.3.399. [DOI] [PubMed] [Google Scholar]
  49. Vyas PM, Roychowdhury S, Koukouritaki SB, Hines RN, Krueger SK, Williams DE, et al. Enzyme-mediated protein haptenation of dapsone and sulfamethoxazole in human keratinocytes: II. Expression and role of flavin-containing monooxygenases and peroxydases. J Pharmacol Exp Ther. 2006;319:497–505. doi: 10.1124/jpet.106.105874. [DOI] [PubMed] [Google Scholar]
  50. Wang DN. Band 3 protein: structure, flexibility and function. FEBS Lett. 1994;346:26–31. doi: 10.1016/0014-5793(94)00468-4. [DOI] [PubMed] [Google Scholar]
  51. Wertheim MS, Males JJ, Cook SD, Tole MD. Dapsone induced haemolytic anaemia in patients treated for ocular cicatricial pemphigoid. Br J Ophtalmol. 2006;90:516. doi: 10.1136/bjo.2005.085837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Winter HR, Wang Y, Unadkat JD. CYP2C8/9 mediate dapsone N-hydroxylation at clinical concentrations of dapsone. Drug Metab Dispos. 2000;28:865–868. [PubMed] [Google Scholar]
  53. Woodhouse KW, Henderson DB, Charlton B, Peaston RT, Rawlins MD. Acute dapsone poisoning: clinical features and pharmacokinetic studies. Hum Toxicol. 1983;2:507–510. doi: 10.1177/096032718300200306. [DOI] [PubMed] [Google Scholar]
  54. Zuidema J, Hilbers-Modderman ES, Merkus FW. Clinical pharmacokinetics of dapsone. Clin Pharmacokinet. 1986;11:299–315. doi: 10.2165/00003088-198611040-00003. [DOI] [PubMed] [Google Scholar]

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