Effect of Nitazoxanide on Erythrocytes

Nitazoxanide, a drug effective against a variety of pathogens, triggers apoptosis and is thus considered to be employed against malignancy. Similar to nucleated cells, erythrocytes may undergo an apoptosis-like suicidal cell death or eryptosis. Hallmarks of eryptosis include cell shrinkage and phospholipid scrambling of the cell membrane with translocation of phosphatidylserine to the erythrocyte surface. Stimulators of eryptosis include increase in cytosolic Ca2+ -activity ([Ca2+ ]i ). The Ca2+ -sensitivity of eryptosis is increased by ceramide. This study explored whether nitazoxanide triggers eryptosis. [Ca2+ ]i was estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin-V-binding, ceramide abundance utilizing fluorescent antibodies and haemolysis from haemoglobin release. A 48-hr exposure to nitazoxanide (1-50 μg/ml) did not significantly modify [Ca2+ ]i but significantly increased ceramide formation, decreased forward scatter (≥10 μg/ml), increased the percentage of annexin-V-binding erythrocytes (≥10 μg/ml) and, at higher concentrations (≥20 μg/ml), stimulated haemolysis. The stimulation of annexin-V-binding was significantly blunted in the absence of calcium. Nitazoxanide thus stimulates eryptosis, an effect in part due to ceramide formation.