Mechanisms of xenobiotic-sensitive apoptotic cell death of erythrocytes

Abstract

Erythrocytes are similar to nucleated cells in that they undergo suicidal death or eryptosis. Mechanism involved in eryptosis may depend on the activation of Ca2+-sensitive cation channels leading to increase intracellular Ca2+ activity. Enhanced cytosolic Ca2+ concentration stimulates phosphatidylserine exposure at the cell surface and cell shrinkage. Eryptosis is a physiological process which may have an important contribution in the limitation of erythrocyte survival. Excessive eryptosis due to stress, toxicity, diseases or defective compensatory mechanism may lead to anemia. The present observations determined the role of xenobiotics in the regulation of eryptosis as well as their toxic effects for erythrocytes. The first part of the study explored the mechanisms adopted by different foods born mycotoxins (enniatin A, ochratoxin A and zearalenone) in the triggering of eryptosis. Exposure of erythrocytes for 48 hours to enniatin A (≥2.5µM) significantly increased [Ca2+]i, decreased [ATP]i, decreased forward scatter, triggered annexin-V-binding and elicited hemolysis. Decreased [ATP]i by glucose depletion for 48 hours was similarly followed by increased [Ca2+]i, decreased forward scatter and annexin-V-binding. Annexin-V-binding was blunted by Ca2+-removal, by the cation channel inhibitor amiloride (1mM), by the protein kinase C inhibitor staurosporine (500nM) but not by the pancaspase inhibitor zVAD (10µM). A 48 hour treatment of erythrocytes with ochratoxin A was followed by significant increase of Fluo3-fluorescencei (≥ 2.5 µM), increase of ceramide abundance (10 µM), decrease of forward scatter (≥ 5 µM) and increase of annexin-V-binding (≥ 2.5 µM). Ochratoxin A exposure slightly but significantly enhanced hemolysis (10 µM). Ochratoxin (10 µM) enhanced erythrocyte adhesion to HUVEC. Removal of extracellular Ca2+ significantly blunted, but did not abrogate ochratoxin A-induced annexin V binding. Similar to enniatin A and ochratoxin A, a 48 h treatment of erythrocytes with zearalenone (≥ 25 µM) was resulted into significant increase of [Ca2+]i, significant decrease of forward scatter, and significant increase of annexin-V-binding. The effect on annexin V binding was significantly blunted in the nominal absence of extracellular Ca2+. Zearalenone stimulates the suicidal erythrocyte death, an effect at least partially due to stimulation of Ca2+ entry. The present findings show that, all three mycotoxin (enniatin A, ochratoxin A and zearalenone) which were previously reported to induce apoptotic cell death in nucleated cells, also under their in vivo plasma concentrations act as potent stimulators of suicidal death of erythrocytes despite the absence of gene expression and mitochondria. The second part of the study explored the mechanisms involved in the eryptosis induced by therapeutically important phytochemicals (withaferin A, oridonin and dicoumarol). For 48 hour, erythrocytes were exposed to the three different phytochemicals with indicated concentrations leveling the range of their in vivo plasma levels. Withaferin A significantly decreased forward scatter (at ≥10 µM withaferin concentration) and increased [Ca2+]i (≥5 µM), ROS-formation (≥10 µM) ceramide-formation (≥10 µM) as well as annexin-V-binding (≥5 µM). Withaferin A treatment was followed by slight but significant increase of hemolysis. Extracellular Ca2+ removal, amiloride, and the antioxidant N-acetyl-L-cysteine significantly blunted withaferin A-triggered annexin-V-binding. Erythrocytes were exposed to oridonin. At concentration (≥25µM) oridonin significantly increased cytosolic Ca2+-concentration, increased ceramide formation, decreased forward scatter and triggered annexin V-binding (the latter in >20% of the erythrocytes). Oridonin did not decrease ATP concentration and hemolysed <5% of erythrocytes. The effects of oridonin on annexin V binding were partially reversed in the nominal absence of Ca2+ and by the addition of amiloride (1mM). Dicoumarol (≥10 µM) after incubation, significantly increased [Ca2+]i, enhanced cation channel activity, decreased forward scatter, triggered annexin-V-binding and elicited hemolysis. Following exposure to 30 µM dicoumarol, annexin-V-binding affected approximately 15%, and hemolysis 2% of treated erythrocytes. The stimulation of annexin-V-binding by dicoumarol was abrogated in the nominal absence of Ca2+. Collectively the data obtained from these studies reveal a completely novel effect of medicinally important phytochemicals (withaferin A, oridonin and dicoumarol) i.e the regulation of calcium-dependent suicidal death erythrocyte death.