Stimulation of suicidal erythrocyte death by benzethonium

Benzethonium, an antimicrobial surfactant widely used as preservative of pharmaceuticals, topical wound care product and oral disinfectant, triggers apoptosis of several cell types. The apoptosis is preceded and possibly triggered by mitochondrial depolarization. Even though lacking mitochondria, erythrocytes may similarly undergo suicidal cell death or eryptosis. Hallmarks of eryptosis include cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptosis may be triggered by energy depletion, which leads to increase of cytosolic Ca(2+)-activity with subsequent Ca(2+)-sensitive cell shrinkage and cell membrane scrambling. Ca(2+)-sensitivity is enhanced by ceramide. The present study explored the effect of benzethonium on eryptosis. Cell membrane scrambling was estimated from binding of fluorescent annexin V to phosphatidylserine, cell volume from forward scatter in FACS analysis, cytosolic Ca(2+)-concentration from Fluo3-fluorescence, hemolysis from hemoglobin release, lactate formation by colorimetry and ceramide utilizing fluorescent antibodies. A 48 hours exposure to benzethonium (=5μM) significantly increased cytosolic Ca(2+)-concentration, decreased forward scatter and triggered annexin V-binding affecting some 30% of the erythrocytes at 5 μM benzethonium. Only 5% of treated erythrocytes were hemolytic. The effects of benzethonium on annexin V binding were blunted in the nominal absence of Ca(2+) and in the presence of amiloride (1 mM) but not in the presence of the pancaspase inhibitor zVAD (10 μM). Benzethonium further significantly enhanced the effect of glucose depletion on cytosolic Ca(2+)-concentration and annexin V-binding, but significantly blunted the effect of glucose depletion on forward scatter. Benzethonium (5 μM) significantly enhanced lactic acid formation but not ceramide abundance. The present observations disclose a novel effect of benzethonium, i.e. triggering of suicidal death of erythrocytes.