Mechanisms of xenobiotic-sensitive apoptotic cell death of erythrocytes

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

Growth and yield enhancement of carrot through integration of NPK and organic manures

A pot experiment was conducted at Horticulture Experimental Area, Gomal University, Dera Ismail Khan, Pakistan to investigate the combined effects of NPK and organic manures on growth and yield of carrot, for two consecutive years. The experiment was laid out in CRD with six treatments and four replications. Five different organic manures such as poultry manure (PM), sewage sludge (SS), farmyard manure (FYM), press mud (PrM) and goat manure (GM) were applied in combination with NPK, each at recommended levels for two successive years. A fertilizer check (control) was also included as treatment where no fertilizer and manure were used. The study revealed significant improvements in almost all growth and yield attributes by combined application of NPK and organic manures. Among different combinations, NPK + PM surpassed all other treatments by giving maximum leaves per plant (8.73 and 8.13), leaf length (38.17 and 36.77cm), root length (29.30 and 24.83cm), root diameter (3.10 and 3.27cm), root weight per plant (142.40 and 142.00g), total biomass per plant (169.33 and 166.67g) and root yield (56.67 and 56.83 t/ha), during both the experimental years. Similarly, NPK combination with green manure and sewage sludge also produced better results pertaining to carrot growth and production for two consecutive years. It was also observed during the study that control treatment showed poorest findings and placed at lowest levels

Geldanamycin-Induced Phosphatidylserine Translocation in the Erythrocyte Membrane

Background/aims: Geldanamycin, a benzoquinone ansamycin antibiotic, and its analogues induce apoptosis of tumor cells and are thus considered for the treatment of cancer. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine-exposure at the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+-concentration ([Ca2+]i) and formation of ceramide. The present study explored, whether geldanamycin modifies [Ca2+]i, ceramide formation, cell volume and phosphatidylserine abundance at the erythrocyte surface. Methods: Erythrocyte volume was estimated from forward scatter, phosphatidylserine-abundance from annexin V binding, hemolysis from hemoglobin release, ceramide formation from binding of fluorescent antibodies and [Ca2+]i from Fluo3-fluorescence. Results: A 48 hours exposure to geldanamycin significantly decreased forward scatter (≥ 5 µM), significantly increased annexin-V-binding (≥ 25 µM), but did not significantly modify Fluo3-fluorescence (up to 50 µM). The annexin-V-binding following geldanamycin treatment was not significantly modified by removal of extracellular Ca2+ but was paralleled by significantly increased ceramide formation (50 µM). Conclusions: Geldanamycin stinulated eryptosis, an effect at least partially due to ceramide formation

Triggering of erythrocyte membrane blebbing by ciprofloxacin

An extensively used fluoroquinolone antibiotic ciprofloxacin shows a broad-spectrum antibacterial activity against both gram-positive and gram-negative strains. It works mainly by the inhibition of DNA gyrase and topoisomerase IV which results in impaired DNA replication leading towards microbial cell death. Eryptosis is an alternative term used for suicidal erythrocyte death. In current study, eryptotic effect of ciprofloxacin was investigated by exposing erythrocytes for 48 hours to the different concentrations (45-90µM) of ciprofloxacin. The experimental work related to the investigation of eryptosis was done by cell size measurement and confirmation of calcium role in membrane blebbing. As a possible mechanism of eryptosis, oxidative stress induced by ciprofloxacin was determined by catalase, glutathion peroxidase and superoxide dismutase activities measurement. Similarly, necrotic effect of ciprofloxacin was also illustrated by hemolysis measurement. Results of our study revealed that the therapeutical doses of ciprofloxacin may induce oxidative stress by significant decrease in superoxide dismutase, catalase and glutathione peroxidase activities as well as induce eryptosis, featured by erythrocytes membrane blebbing and hemolysis

Patulin-Induced Suicidal Erythrocyte Death

Background: Patulin, the most common mycotoxin in apples and apple-derived products, triggers apoptosis and has thus been considered for the treatment of cancer. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by cell membrane scrambling leading to phosphatidylserine-exposure at the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i). The present study explored, whether exposure of human erythrocytes to patulin is followed by eryptosis. Methods: Forward scatter was measured to estimate cell volume, annexin V binding to detect phosphatidylserine-exposure, hemoglobin release to quantify hemolysis, and Fluo3-fuorescence to determine [Ca2+]i. Results: A 48 h exposure to patulin significantly increased [Ca2+]I (5 µM), significantly decreased forward scatter (5 µM) and significantly increased annexin-V-binding (2.5 µM). Patulin (10 µM) induced annexin-V-binding was virtually abrogated by removal of extracellular Ca2+. Conclusion: Patulin stimulates Ca2+ entry into erythrocytes, an effect triggering suicidal erythrocyte death or eryptosis

Triggering of Suicidal Erythrocyte Death by Celecoxib

toxin

Stimulation of Erythrocyte Death by Phloretin

Background: Phloretin, a natural component of apples, pears and strawberries, has previously been shown to stimulate apoptosis of nucleated cells. Erythrocytes may similarly enter suicidal death or eryptosis, which is characterized by cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i), ceramide, ATP depletion, and activation of protein kinase C (PKC) as well as p38 mitogen activated protein kinase (p38 kinase). Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, and ceramide abundance from binding of specific antibodies. Results: A 48 h exposure of human erythrocytes to phloretin significantly increased the percentage of annexin-V-binding cells (≥100 µM) without significantly influencing forward scatter. Phloretin did not significantly modify [Ca2+]i and the stimulation of annexin-V-binding by phloretin (300 µM) did not require presence of extracellular Ca2+. Phloretin did not significantly modify erythrocyte ATP levels, and the effect of phloretin on annexin-V-binding was not significantly altered by PKC inhibitor staurosporine (1 µM) or p38 kinase inhibitor SB2203580 (2 µM). However, phloretin significantly increased the ceramide abundance at the cell surface. Conclusions: Phloretin stimulates phospholipid scrambling of the erythrocyte cell membrane, an effect at least partially due to up-regulation of ceramide abundance

In Vitro Induction of Erythrocyte Phosphatidylserine Translocation by the Natural Naphthoquinone Shikonin

Shikonin, the most important component of Lithospermum erythrorhizon, has previously been shown to exert antioxidant, anti-inflammatory, antithrombotic, antiviral, antimicrobial and anticancer effects. The anticancer effect has been attributed to the stimulation of suicidal cell death or apoptosis. Similar to the apoptosis of nucleated cells, erythrocytes may experience eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and by phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include the increase of cytosolic Ca2+-activity ([Ca2+]i) and ceramide formation. The present study explored whether Shikonin stimulates eryptosis. To this end, Fluo 3 fluorescence was measured to quantify [Ca2+]i, forward scatter to estimate cell volume, annexin V binding to identify phosphatidylserine-exposing erythrocytes, hemoglobin release to determine hemolysis and antibodies to quantify ceramide abundance. As a result, a 48 h exposure of human erythrocytes to Shikonin (1 µM) significantly increased [Ca2+]i, increased ceramide abundance, decreased forward scatter and increased annexin V binding. The effect of Shikonin (1 µM) on annexin V binding was significantly blunted, but not abolished by the removal of extracellular Ca2+. In conclusion, Shikonin stimulates suicidal erythrocyte death or eryptosis, an effect at least partially due to the stimulation of Ca2+ entry and ceramide formation