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  • A model substance often used in


    A model substance often used in the studies of oxidative processes in erythrocytes is tert-butyl hydroperoxide (t-BHP), which, being lipophilic, passes easily across the erythrocyte membrane [9]. In erythrocytes treated with t-BHP (0.1–3mM) for less than 1h, a dramatic decline in the reduced glutathione (GSH) content is observed, followed by hemoglobin (Hb) oxidation, changes in cell morphology and in the ion permeability of cell membranes [10], [11], [12], [13], [14], [15]. Erythrocytes long exposed to t-BHP at various concentrations may undergo oxidative isotonic lysis [16], [17], [18]. As we showed earlier [19], incubation of erythrocytes with 1–3mM t-BHP in a Ca2+-containing medium for 20–30min resulted in a dose-dependent cell swelling. If the incubation medium was Ca2+-free, high in K+, and/or supplemented with 10μM clotrimazole (CLT), an inhibitor of Ca2+-activated K+ channels (Gardos channels), the extent of swelling was significantly greater. This result led us to conclude that t-BHP acted by activating Gardos channels of erythrocytes suspended in a Ca2+-containing medium, thereby raising K+ efflux and moderating cell swelling. This study addressed the effect of CLT on the swelling and lysis of normal human erythrocytes induced by t-BHP. For the first time, it was shown that 10μM CLT significantly enhanced the lytic effect of t-BHP on erythrocytes, even in a Ca2+-free medium. To elucidate the mechanism of action of CLT on the erythrocyte membrane, we investigated how CLT affected the rate of ROS generation and how GSH, methemoglobin (MetHb), and thiobarbituric nos inhibitor reactive substances (TBARS) varied with time in human erythrocytes treated with t-BHP. The results tempt us to speculate that t-BHP-induced swelling and eventually hemolysis may be due to membrane destabilization produced by hemin, which is released during oxidation of erythrocytes. Forming a complex with hemin, CLT further enhances the effect of hemin on the membrane.
    Materials and methods
    Discussion Interaction with the organic hydroperoxide t-BHP offers a useful model for studying oxidative damage to erythrocytes. Over decades, the cytotoxic effect of t-BHP and the mechanisms involved have been a subject of extensive studies [9], [10], [11], [12], [13], [14], [15], [16], [28], [29], [30], [31]. When t-BHP is added to an erythrocyte suspension, intracellular GSH and oxyHb oxidation and membrane lipid peroxidation are observed [10], [11], [28], [30], which result in oxidative damage to erythrocytes that includes impaired ability of the cells to maintain cation gradients [15], [25], [32], a decrease in the protein thiol content, rearrangement in the cytoskeleton structure, morphological changes, and eventually hemolysis [9], [18], [33]. Hb oxidation leads to the formation of denatured hemoglobin monomers (irreversible hemichromes), which bind to the cell membrane and release hydrophobic heme (hemin) [14], [30], [34]. Chiu [35] reported the results of direct measurements of the rate of hemin release from hemoglobin oxidized with H2O2. There are studies in which free hemin content was determined in the membranes of erythrocytes treated with phenylhydrazine [34] and menadione [36], [37]. Exogenous hemin (Ferriprotoporphyrin IX) is known to readily incorporate into the membrane, causing its destabilization and hemolysis [36], [38], [39], [40]. CLT is an imidazole derivative efficacious as an antifungal agent. CLT is also known to be a Gardos channel inhibitor, which can be used perorally in therapy for severe sickle-cell anemia and thalassemia [41], [42]. CLT inhibits in vitro growth of Plasmodium falciparum and may prove efficacious for treating malaria [43]. Moreover, CLT also possesses antitumor properties related to its ability to inhibit Ca2+-ATPase of the sarcoplasmic reticulum [44]. Recent data provide evidence that CLT forms a complex with heme (hemin) that produces a more potent lytic effect than heme alone [45].