Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina 29425
Received April 3, 2001; accepted May 17, 2001
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ABSTRACT |
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Key Words: hemolytic anemia; favism; divicine; rat; glucose-6-phosphate dehydrogenase deficiency; erythrocytes; glutathione.
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INTRODUCTION |
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The mechanism underlying the onset of favism is not yet understood. However, it has been postulated that both pyrimidine aglycones, liberated upon digestion of their parent glucosides (Hegazy and Marquardt, 1984), are absorbed into the blood and induce oxidative damage within erythrocytes as a consequence of their redox activity (Chevion et al., 1982
; Winterbourn and Munday, 1990
). Studies of red cells withdrawn from patients during early and late stages of favic crises have indicated that GSH depletion and HMP shunt stimulation are key events that precede red cell loss (Gaetani et al., 1979
), and these biochemical responses are reported to be accompanied by alterations in the morphological appearance of the erythrocytes when viewed under light microscopy (Fischer et al., 1985
).
We have shown recently that divicine can induce a favic-like response when administered to G6PD-normal rats, and that divicine is directly hemotoxic to rat red cells (McMillan and Jollow, 1999). That is, when incubated with 51Cr-labeled red cells in vitro, divicine induces alterations in the cells such that when they are returned to the circulation of isologous rats, the treated cells are rapidly sequestered into the spleen. In view of the importance of sulfhydryl status in the progression of favism and the role of membrane skeletal proteins in the maintenance of normal red cell shape (Mohandas et al., 1983
), the present studies were undertaken to examine the sulfhydryl status, HMP shunt activity, morphology, and membrane skeletal proteins of rat erythrocytes exposed in vitro to hemotoxic concentrations of divicine. We report that divicine rapidly stimulates HMP shunt activity and depletes GSH in rat erythrocytes with concomitant formation of glutathione-protein mixed-disulfides. These biochemical alterations are associated with profound membrane skeletal protein damage and transformation of the cells to an extreme echinocytic morphology.
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MATERIALS AND METHODS |
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Animals.
Male Sprague-Dawley rats (130150 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.
Red cell incubation conditions.
Red cells 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). The red cells were resuspended to 40% hematocrit and used the same day they were collected. After a 5-min preincubation at 37°C, various concentrations of divicine dissolved in PBSG were added to red cell suspensions (2 ml) and allowed to incubate aerobically for up to 2 h at 37°C.
Analysis of red cell HMP shunt activity and sulfhydryl status.
The evolution of 14CO2 from 14C-1-glucose was used to estimate HMP shunt activity in rat red cell suspensions as described previously (Grossman et al., 1995). For determination of sulfhydryl status, aliquots (200 µl) of the red cell incubation mixtures were removed at various intervals after the addition of divicine and assayed for GSH, GSSG, and protein-SSG concentration by HPLC with electrochemical detection as described previously (Jensen et al., 1986
). The amount of sulfhydryl present in the sample was estimated by comparison of peak height to standards prepared identically to the samples.
Morphological examination of red cells.
Control and divicine-treated red cells were prepared for scanning electron microscopy in a manner similar to that described by Dewar (1982). After incubation, the red cells were washed once in PBSG, and fixed and dehydrated as described previously (Grossman et al., 1992). The cells were then cast with a thin coating of carbon and gold, and examined in a JEOL JSM-5410LV scanning electron microscope operating at 10 kV accelerating voltage.
Electrophoretic analysis of membrane skeletal proteins.
Red cell ghosts (unsealed membrane vesicles) were prepared from control and divicine-treated red cells by hypotonic lysis as described previously (Grossman et al., 1992). The ghosts were washed repetitively with buffer and solubilized with SDS. Electrophoretic and immunoblotting analysis of the solubilized membrane ghosts was carried out as described previously (McMillan et al., 1995
). The proteins were resolved on nonreducing, continuous gels (5% monomer and 1.5% bis-acrylamide crosslinker), and the skeletal protein bands were identified according to their migration distance (Fairbanks et al., 1971
).
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RESULTS |
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In view of the observed capacity of divicine to induce the formation of disulfide bonds between GSH and sulfhydryl groups of soluble protein in the red cell (Fig. 1), we examined the involvement of intermolecular disulfide bond formation in the alteration of the membrane skeletal proteins. Red cells were incubated with divicine (1.5 mM) for 1 h and then washed and exposed to DTT (5 mM) for an additional 1 h prior to lysis and solubilization. As shown in Fig. 5A
, exposure of untreated red cells to DTT alone (lane 2) had no effect on the normal electrophoretic pattern. However, post-treatment of divicine-treated red cells with DTT (lane 4) reversed the changes in the major structural proteins and decreased the level of membrane-bound hemoglobin (Fig. 5B
). Similar results were obtained when cysteamine was substituted for DTT, and when the divicine-treated cells were lysed and solubilized with SDS prior to addition of DTT (data not shown). These data indicate that the skeletal protein alterations induced by divicine were due to the formation of intermolecular disulfide bonds.
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DISCUSSION |
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We have used a direct synthetic method (Bailey et al., 1982) to prepare a stable hemisulfate salt of divicine (McMillan et al., 1993
). This compound was able to provoke a favic-like response in G6PD-normal rats. That is, divicine administration reduced the survival of previously infused 51Cr-labeled red cells and decreased the hematocrit in a dose-dependent manner (McMillan and Jollow, 1999
). The decrease in hematocrit was matched by an increase in spleen weight, reflective of the rapid uptake of divicine-damaged red cells. Of importance for the present studies, divicine hemotoxicity could be reproduced in vitro by direct exposure of 51Cr-labeled red cells to divicine before cells were returned to the circulation of isologous rats. Survival of these cells was also reduced in a concentration-dependent manner, and these cells were also found to be removed preferentially by the spleen. These observations indicate that the G6PD-normal rat red cell may be used as a model to examine the relevance of divicine-induced cellular alterations to the hemotoxic response.
The present studies demonstrate that divicine hemotoxicity in the rat is associated with the development of an oxidative stress within the red cell. This oxidative stress response was manifested by depletion of GSH with concomitant formation of glutathione-protein mixed-disulfides (Fig. 1), and by stimulation of HMP shunt activity (Fig. 2
). Under these experimental conditions, alterations in the electrophoretic pattern of membrane skeletal proteins and the appearance of membrane-bound hemoglobin were observed (Fig. 4
), and the red cells were transformed to an extreme echinocytic morphology (Fig. 3
). As previously reported, when rat red cells treated in this manner are returned to isologous rats, the cells are rapidly and selectively removed by splenic sequestration (McMillan and Jollow, 1999
).
The mechanism by which damaged or senescent red cells are removed by the spleen remains poorly understood. The exposure of protein, carbohydrate, and/or lipid epitopes on the external cell surface leading to recognition and ingestion by splenic macrophages have all been proposed to account for the removal of abnormal red cells from the circulation (Bratosin et al., 1998). Although the intracellular signal that provokes the appearance of an epitope on the external cell surface is also not known, it has long been postulated that alterations in the skeletal protein assembly that lies on the inner surface of the cell membrane may be responsible for initiating this process. In this regard, a number of studies have suggested a key role for the binding of hemoglobin to cytoskeletal protein in the mechanism underlying normal red cell senescence. For example, Low and colleagues (Turrini et al., 1991
) showed that hemoglobin binding to the membrane of red cells stimulated the deposition of autologous antibodies and complement, and that these cells were ingested by cultured monocytes.
As noted above, examination of skeletal proteins from divicine-treated red cells revealed marked changes in the electrophoretic pattern. Two major features of this response were the loss of membrane protein bands 1, 2.1, and 4.2, and the appearance of membrane-associated aggregates of hemoglobin monomers. The reversal of these changes by treatment of the cells with DTT (Fig. 5) with the liberation of hemoglobin monomer indicated that the hemoglobin addition reactions had occurred as a result of disulfide bond formation. Although the significance of these protein changes is not yet known, they are quite similar to those observed previously in ghosts obtained from rat red cells exposed to dapsone hydroxylamine (Grossman et al., 1992
), which is the hemolytic metabolite of the primary arylamine drug, dapsone.
The origin of the oxidant stress that converts the sulfhydryl groups of GSH, hemoglobin, and skeletal protein to mixed disulfides in these divicine-treated red cells is not yet known. Reversible oxidation of divicine to a quinone at the expense of molecular oxygen may generate reactive oxygen species (Chevion et al., 1982), which have the capacity to react with cellular thiol groups to form reactive thiyl radicals (Bradshaw et al., 1997
; Wefers and Sies, 1983
). Hemoglobin thiyl radicals could then attack the membrane proteins, resulting in the formation of hemoglobin-skeletal protein adducts and HMWP aggregates. Autooxidation of divicine in aerobic solution has been shown to generate hydrogen peroxide, and in the presence of GSH, the accumulation of GSSG has been observed (Arese, 1982
). In our studies, however, GSSG was not detected in divicine-treated red cells, suggesting that the loss of GSH was not due to detoxification of peroxide by GSH peroxidase. Alternatively, mixed disulfide formation could occur as a result of attack by a compound-centered free radical (Winterbourn, 1993
). In support of this concept, semiquinone radical intermediates of divicine have been detected in acellular systems by EPR spectroscopy (Albano et al., 1984
; Pedersen et al., 1988
). Whether or not these species are formed in red cells is not yet clear and warrants further investigation.
Several reports of divicine-induced morphological alterations have appeared in the literature (Beutler, 1978; Fischer et al., 1985
; Weed and Reed, 1966
). Fischer et al. (1985) described the appearance of a phenomenon referred to as plasma membrane "crossbonding." This altered morphology could be observed in red cells withdrawn from G6PD-deficient individuals during the early stages of a favic crisis, and is characterized by fusion of opposing faces of the internal surface of the plasma membrane, creating 2 compartments, one containing hemoglobin, and one apparently devoid of cellular material. Efforts to reproduce this response in vitro by exposure of rat red cells to divicine was achieved only when the cells were placed in a hypertonic medium (400 milliosmolar) to affect shrinkage of the cells. In the present in vitro incubation studies, intact treated cells with varying degrees of echinocytosis were evident but changes suggestive of crossbonding and cell fragmentation were not observed. Of interest, examination of red cells taken from rats 1 h after administration of a hemotoxic dose of divicine revealed a high proportion of knizocytes (pinched cells; triconcave disc) and occasional echinocytes (McMillan and Jollow, unpublished observations); however, crossbonded red cells were not evident under these in vivo conditions.
In summary, the data presented in this report demonstrate that an oxidative stress response is provoked in rat red cells exposed to concentrations of divicine that commit the cells to premature splenic sequestration. Under these experimental conditions, divicine hemotoxicity in the rat is associated with severe damage to the membrane skeleton, which is in turn manifested by alterations in red cell morphology.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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