As we showed earlier a to min

As we showed earlier [19], a 20- to 30-min incubation with 2mM t-BHP resulted in erythrocyte swelling, which was less in a Ca2+-containing medium. Given that this effect was abolished in the presence of the Gardos channel inhibitor CLT, as well as in media high in K+, we suggested the contribution from the Gardos effect, that is, partial cell dehydration because of elevated intracellular Ca2+. The results of the present study confirmed this suggestion. However, CLT appeared to enhance t-BHP-induced erythrocyte swelling not only in a Ca2+-containing medium, but also in a Ca2+-free medium (Fig. 1), which means that, besides Gardos channel inhibition, there should be other, Ca2+-independent, mechanisms behind this effect. A longer incubation resulted in larger swelling (Fig. 2) and, at 2h, lysis began to occur. By 3h, hemolysis reached 30–40% at a t-BHP concentration of 2mM. Whatever the t-BHP concentration, the extent of hemolysis did not depend on the presence of Ca2+ in the medium (Fig. 3, panels A and B). One can see that CLT considerably and significantly enhanced hemolysis at all t-BHP concentrations in both Ca2+-containing and Ca2+-free media. Given that CLT produces little, if any, effect on the rate of free radical generation (Fig. 5), on the kinetics of GSH degradation (Fig. 6), methemoglobin formation (Fig. 7), and TBARS generation (Fig. 8), it is reasonable to conclude that 10μM CLT does not cause additional oxidative damage to Colistin Sulfate sale treated with t-BHP. Therefore, our results tempt us to speculate that CLT enhances t-BHP-induced changes in erythrocyte volume and lysis largely by forming a complex with hemin released during hemoglobin oxidation in erythrocytes, and thereby destabilizes the cell membrane more potently [45], [46]. Indirect evidence in support of this hypothesis was obtained in experiments where we resuspended t-BHP treated erythrocytes in a buffer containing albumin at different concentrations (Fig. 4). Albumin is known as an active chelator of free heme, contributing, along with hemopexin, into clearing free heme from cell membranes [40], [47], [48]. As can be seen in Fig. 4, the extent of hemolysis decreased with increasing albumin concentration. A possible explanation for these results is that albumin might remove hemin and its complex with CLT from the cell membranes. Heme/hemin is a functional group of various heme proteins, such as hemoglobin, myoglobin, cytochromes, catalase, and peroxidase. Heme release can occur in, for example, thalassemia, sickle cell anemia, glucose 6-phosphate dehydrogenase deficiency, hemorrhage, and muscle injury. In some pathological cases, heme is present in vivo at high micromolar concentrations [40]. Being a lipophilic molecule, heme is capable of intercalating into the cell membrane, causing cell injury. The heme-degrading enzyme heme oxygenase (HO) plays a significant role in protecting cells from toxic effects of free heme [40], [49]. This study is the first to show that CLT at a concentration of 10μM significantly increases erythrocyte swelling and nearly doubles lysis induced by the model oxidant t-BHP, which indicates that the membrane is damaged to a much greater extent in the presence of CLT. Therefore, it is tempting to speculate that CLT may act as a cytotoxic agent in pathologies associated with oxidative stress and increased hemoglobin release (e.g., intravascular hemolysis). The consequences of its clinical use may be positive and negative. For example, a concern arises that the use of CLT during painful crisis in sickle cell anemia [42] may have adverse effects on erythrocytes, thereby exacerbating the course of anemia. On the other hand, we assume that the effect of CLT on cell membrane damage during free radical oxidation may be employed to increase the efficacy of antitumor therapy, even more so that there are data demonstrating inhibition of the heme-degrading enzyme heme oxygenase (HO-1) in the presence of the drug [50].