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The Cr ion doping effect on various properties of Cr doped BaCrxFe12-xO19 nanoparticles was investigated, which were synthesized via a facile microemulsion approach and properties were studied using XRD, SEM, FTIR, Raman, photoluminescence and UV–visible techniques along with dielectric, optical and ferroelectric properties. The BaCrxFe12-xO19 structure was hexagonal involving P63/mmc space group with average crystalline size of 9–18 nm. The NPs exhibited agglomerated platelet heterogeneous morphology. The presence of the Ba-O-Fe functional group was also confirmed by FTIR analysis. The PL analysis revealed that the doping reduced the recombination rate and charge (e−-h+) separation is facilitated. The coercivity (Hc) and saturation polarization (Ps) increased with doping content and dielectric loss reduces with frequency and dopant concentration. The dopant contents also increased the AC conductivity and the optical bandgap found in 1.75–2.83 (eV) range. The BaCrxFe12-xO19 exhibited a significantly higher photocatalytic efficiency versus BaFe12O19, and 91 % CV dye was degraded in 90 min under visible light irradiation. Additionally, a recycling experiment was conducted to confirm the stability of the prepared photocatalyst and Cr doped BaCrxFe12-xO19 exhibited excellent stability and reusability. The Cr doping affected the dielectric, optical and ferroelectric properties and based on photocatalytic properties of BaCrxFe12-xO19, it has potential applications for the destruction of dyes in wastewater under visible light exposure, which will make the process highly feasible for photocatalytic applications.

Quorum sensing (QS) is a major controller of virulence and biofilm formation in pathogenic bacteria. The aim of the research was to screen novel synthetic compounds (18) from 2 series (Pyrazole and Diene dione) for quorum sensing and biofilm inhibitory potential against resistant pathogens isolated from patients with chronic sinusitis. Most of the compounds have documented zone of inhibition against Gram positive strains Staphylococcus aureus , Enterococcus faecalis and moderate activity against Gram negative Klebseilla pneumoniae and Proteus mirabilis in comparison with standard antibiotic. Compounds Q1 and Q7 have given the maximum zone of inhibition 18 and 20 mm with MICs 0.312 mg/mL and .156 mg/mL against S aureus and E faecalis, respectively. Some compounds were equally potent at inhibiting the formation of biofilm which later established by phase contrast microscopy. Regarding quorum sensing inhibition, the tested concentration of synthetic compound UA3 0.313 mg/mL inhibited violacein production without decreasing Chromobacterium pseudoviolaceum count which was significantly lower than determined MIC’s. It was depicted from the results that selected compounds exhibited low level of cytotoxicity toward human red blood cells. Hence, these findings revealed that most novel compounds were effective antibacterial, whereas compound UA3 has shared significant anti-quorum sensing potential against Chromobacterium pseudoviolaceum.

Methotrexate (MTX) is a common chemotherapeutical agent and folate antagonist with reported apoptotic activity in nucleated cells. The presented research work was planned to investigate the eryptotic effects of methotrexate after the exposure of erythrocytes to therapeutical doses (10–15 μM) of methotrexate. Eryptosis and the role of calcium in the stimulation of membrane blebbing were evaluated through the determination of mean cell volume. Oxidative stress induced by methotrexate (10–15 μM) was determined by antioxidative enzyme activities. Cytotoxic activity against human erythrocytes was examined through hemolysis assay. Exposure of erythrocytes to methotrexate results in significant reduction of superoxide dismutase, catalase, and superoxide dismutase activities at 10 and 15 μM in comparison to the untreated cells. Erythrocytes mean cell volume (MCV) was increased after 48 hours exposure of erythrocytes to methotrexate (10 μM). Significantly increased hemolysis percentage was observed at 10 μM after 48 hours incubation of erythrocytes with methotrexate. The results of the study suggested that the therapeutical doses (10–15 μM) of methotrexate may lead to increase in eryptotic and hemolytic activity of erythrocytes through free radical generation and subsequent calcium entry.

Background Bifenthrin is an insecticide and anti-estrogenic compound primarily used to control residential pests by depolarizing sodium gated voltage channels in the nervous system. Eryptosis, the suicidal death of erythrocytes, featured by PS exposure, membrane blebbing and cell shrinkage. Anemia is an outcome of uncontrolled eryptosis. Research Design In this study, erythrocytes were treated with different concentrations (.5-1-1.5 μM) of bifenthrin over a period of 48 hours. In order to investigate the oxidative stress induced by bifenthrin, catalase, superoxide dismutase, and glutathione peroxidase activities were investigated. Results Obtained data indicated the decrease in the enzymes (superoxide dismutase, glutathione peroxidase, and catalase) activities in bifenthrin treated cells at 1 μM concentration. In addition, measurement of cell size and confirmation of the role of calcium in the stimulation of the eryptotic activity of bifenthrin were performed. A significant increase in mean cell volume was found in the presence of bifenthrin and a decrease in mean cell volume in the presence of calcium channel blocker was observed. Similarly, there was also a significant increase in the percentage of hemolysis indicating the necrotic activity of bifenthrin. Conclusions It is concluded that the indicated doses of bifenthrin triggered oxidative stress which may lead to early cell death by eryptosis and hemolysis.

Iron oxide nanoparticles (Fe2O3 NPs) were fabricated through green route using pomegranate (Punica granatum) seeds extract. The Fe2O3 NPs were characterized by UV–vis, XRD, EDX, SEM and AFM techniques. The adopted green rout furnished semi spherical Fe2O3 NPs, uniformly distributed and particle size in the range of 25–55 nm. The LCMS/MS was performed for the identification of biomolecule present in the extract of pomegranate seeds and p-hydroxy benzoic acid, gallic acid, methyl gallate, catechin, kaempferol-3-O-sophoroside, 3-deoxyflavonoids, magnolol, ferulic acid, vanillic acid and pinocembrin along with other minor constituents were detected in the extracts using for Fe2O3 NPs. The synthesized Fe2O3 NPs showed excellent photocatalytic activity against reactive blue under UV light irradiation and maximum degradation of 95.08% was achieved with 56 min of reaction time. In view of promising activity, the Fe2O3 NPs could be used photocatalyst for the degradation of dyes in wastewater and pomegranate seeds extract can be applied as eco-benign and cost effective approach for Fe2O3 NPs synthesis. Keywords: Punica granatum, Green synthesis, Iron oxide nanoparticles, Photocatalysis, Dye degradation

La1-xBixCo1-yFeyO3 perovskite was prepared by micro-emulsion method and effect of the Fe and Bi doping on the properties of perovskite was investigated. As-prepared perovskite was characterized by X-ray diffraction (XRD), Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM), Energy-dispersive X-ray (EDX) and atomic force microscopy (AFM) techniques. La1-xBixCo1-yFeyO3 perovskite showed distorted rhombohedral structure and particle size was in the range of 33.05–57.41 nm. Doping of perovskite with Bi and Fe enhanced the direct current (DC) resistivity. Dielectric parameters were studied in the range of 20 to 20 MHz and vibrating magnetometery revealed that the Mr and Ms values were higher for La0.75Bi0.25Co0.75Fe0.25O3, whereas LaCoO3 showed higher value of Hc. Photocatalytic activity (PCA) was evaluated by degrading congo red dye and Bi and Fe doped perovskite showed significantly higher activity versus LaCoO3. Results revealed that the doping of LaCoO3 with Bi and Fe enhanced the magnetic, dielectric and catalytic properties of LaCoO3. Keywords: LaCoO3 perovskite, Bi and Fe doping, Characterization, Properties, Photocatalysis

Omeprazole, a proton pump inhibitor blocks the H + /K + -ATPase channels of gastric parietal cells. It is used for the treatment of peptic ulcer. Prolonged use of omeprazole may involve in inducing anemia. The key marker of eryptosis includes membrane blebbing, cell shrinkage and phosphatidylserine (PS) exposure at the cell surface. In current study, the eryptotic, oxidative as well as hemolytic effects of therapeutical doses (0.5, 1 and 1.5 µM) of omeprazole were investigated after exposing erythrocytes for 48 hours. Investigation of eryptosis was done by cell size measurement, PS exposure determination and calcium channel inhibition. As a possible mechanism of omeprazole induced eryptosis, oxidative stress was investigated by determining the catalase, glutathione peroxidase and superoxide dismutase activities. Similarly, necrotic effect of omeprazole on erythrocytes was also evaluated through hemolysis measurement. Results of our study illustrated that 1.5 µM of omeprazole may induce significant decrease in superoxide dismutase, glutathione peroxidase and catalase activities as well as triggered the erythrocytes shrinkage, PS exposure and hemolysis. Role of calcium was also confirmed in inducing erythrocyte shrinkage. It is concluded that the exposure of erythrocytes with 1.5 µM omeprazole may enhance the rate of eryptosis and hemolysis by inducing oxidative stress.

Naproxen sodium is a nonsteroidal anti-inflammatory drug (NSAID) having antipyretic and analgesic properties, mainly used for the treatment of rheumatoid arthritis and osteoarthritis. Eryptosis is an alternative term used for suicidal erythrocyte death. In the current study, eryptotic effect of naproxen sodium characterized by membrane blebbing was investigated in erythrocytes after 48 hours of treatment with different concentrations (1-25 µM). The experimental work related to investigation of eryptosis was done by cell size measurement and confirmation of calcium role in the induction of membrane blebbing. As a possible mechanism of eryptosis, oxidative stress induced by naproxen sodium was determined by catalase, glutathione peroxidase, and superoxide dismutase activities. Similarly, hemolytic effect of naproxen sodium was also determined by hemolysis measurement. Results of our study illustrated that the therapeutic doses (10-25 µM) of naproxen sodium induce oxidative stress, confirmed by significant decrease in superoxide dismutase, catalase, and glutathione peroxidase activities that lead to the triggering of cell death by eryptosis and hemolysis.

Atorvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzymeA reductase, is usually used for the treatment of hypercholesterolemia. Besides its pharmacological and side actions, its toxic effects on human nucleus devoid of erythrocytes are still unknown. Eryptosis is an alternative term used for suicidal erythrocyte death. Membrane blebbing is among the common markers of eryptosis. In this study, eryptotic effect of atorvastatin was investigated by exposing the erythrocytes for 48 hours to different concentrations (1-10 µM) of atorvastatin. The experimental work related to investigation of eryptosis was done by cell size measurement and calcium channel inhibition. As a possible mechanism of eryptosis, atorvastatin-induced oxidative stress was evaluated by determining catalase, glutathione peroxidase, and superoxide dismutase activities. Similarly, necrotic effect of atorvastatin was also determined by hemolytic assay. Results of our study illustrated that the tested doses of atorvastatin may induce oxidative stress as observed by significant reduction in superoxide dismutase, glutathione peroxidase, and catalase activities as well as induce eryptosis, featured by erythrocytes membrane blebbing. The study concluded that induction of oxidative stress by atorvastatin may lead to eryptosis.

Background/Aims: Epidemiological evidence suggests that vitamin D deficiency is associated with anemia. The potent metabolite 1,25(OH)2 vitamin D3 [1,25(OH)2D3] activates various signaling cascades regulating a myriad of cellular functions including suicidal cell death or apoptosis. Suicidal death of erythrocytes or eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine (PS) externalization. Stimulation of eryptosis may limit lifespan of circulating erythrocytes and thus cause anemia. In the present study, we explored the effect of a high vitamin D diet (10,000 I.U. vitamin D for 14 days) in mice on eryptosis. Methods: Plasma concentrations of erythropoietin were estimated using an immunoassay kit, blood count using an electronic hematology particle counter, relative reticulocyte numbers using Retic-COUNT® reagent, PS exposure at the cell surface from annexin V binding, cell volume from forward scatter, and cytosolic Ca2+ ([Ca2+]i) from Fluo3-fluorescence in FACS analysis. Results: Vitamin D treatment decreased mean corpuscular volume, reticulocyte count, and plasma erythropoietin levels. Vitamin D treatment slightly but significantly decreased forward scatter but did not significantly modify spontaneous PS exposure and [Ca2+]i of freshly drawn erythrocytes. Vitamin D treatment augmented the stimulation of PS exposure and cell shrinkage following exposure to hyperosmotic shock (addition of 550 mM sucrose) or energy depletion (glucose removal) without significantly modifying [Ca2+]i. Conclusions: The present observations point to a subtle effect of exogenous vitamin D supplementation on erythrocyte survival.

Background/Aims: Janus kinase-3 (JAK3) is activated during energy depletion. Energy-consuming pumps include the Na+/K+-ATPase. The present study explored whether JAK3 regulates Na+/K+-ATPase in dendritic cells (DCs). Methods: Ouabain (100 µM)-sensitive (Iouabain) and K+-induced (Ipump) outward currents were determined by utilizing whole cell patch-clamp, Na+/K+-ATPase α1-subunit mRNA levels by RT-PCR, Na+/K+-ATPase protein abundance by flow cytometry or immunofluorescence, and cellular ATP by luciferase-assay in DCs from bone marrow of JAK3-knockout (jak3-/-) or wild-type mice (jak3+/+). Ipump was further determined by voltage clamp in Xenopus oocytes expressing JAK3, active A568VJAK3 or inactive K851AJAK3. Results: Na+/K+-ATPase α1-subunit mRNA and protein levels, as well as Ipump and Iouabain were significantly higher in jak3-/-DCs than in jak3+/+DCs. Energy depletion by 4h pre-treatment with 2,4-dinitro-phenol significantly decreased Ipump in jak3+/+ DCs but not in jak3-/-DCs. Cellular ATP was significantly lower in jak3-/-DCs than in jak3+/+DCs and decreased in both genotypes by 2,4-dinitro-phenol, an effect significantly more pronounced in jak3-/-DCs than in jak3+/+DCs and strongly blunted by ouabain in both jak3+/+ and jak3-/-DCs. Ipump and Iouabain in oocytes were decreased by expression of JAK3 and of A568VJAK3 but not of K851AJAK3. JAK3 inhibitor WHI-P154 (4-[(3'-bromo-4'-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline, 22 μM) enhanced Ipump and Iouabain in JAK3 expressing oocytes. The difference between A568VJAK3 and K851AJAK3 expressing oocytes was virtually abrogated by actinomycin D (50 nM). Conclusions: JAK3 down-regulates Na+/K+-ATPase activity, an effect involving gene expression and profoundly curtailing ATP consumption.

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.

Background/Aims: Aristolochic Acid, a component of Aristolochia plants, has been shown to cause acute kidney injury, renal aristolochic acid nephropathy, Balkan endemic nephropathy, and urothelial carcinoma. Aristolochic acid nephropathy may be associated with severe anemia. The anemia could theoretically be due to stimulation of eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with translocation of phosphatidylserine to the erythrocyte cell membrane surface. Signalling involved in the stimulation of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i) and formation of ceramide. Methods: Cell volume was estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, [Ca2+]i from Fluo3 fluorescence, and ceramide abundance from binding of fluorescent antibodies in flow cytometry. Results: A 48 hours exposure to Aristolochic Acid (≥ 75 g/ml) was followed by a significant decrease of forward scatter and increase of annexin-V-binding. The effects were paralleled by a significant increase of [Ca2+]i and significantly blunted, but not abrogated by removal of extracellular Ca2+. Aristolochic Acid further significantly increased ceramide abundance. Conclusions: Aristolochic Acid triggers eryptosis, an effect at least in part due to entry of extracellular Ca2+ and ceramide formation. i 2014 S. Karger AG, Basel

Background: Piperlongumine, a component of Piper longum fruit, is considered as a treatment for malignancy. It is effective by inducing apoptosis. Mechanisms involved in the apoptotic action of piperlongumine include oxidative stress and activation of p38 kinase. In analogy to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine-exposure at the erythrocyte surface. Signaling involved in eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i), formation of ceramide, oxidative stress and activation of p38 kinase. Methods: Cell volume was estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, [Ca2+]i from Fluo3 fluorescence, reactive oxygen species from 2',7'-dichlorodihydrofluorescein-diacetate fluorescence, and ceramide abundance from binding of fluorescent antibodies in flow cytometry. Results: A 48 h exposure to piperlongumine (30 µM) was followed by significant decrease of forward scatter and increase of annexin-V-binding. Piperlongumine did not significantly modify [Ca2+]i and the effect was not dependent on presence of extracellular Ca2+. Piperlongumine significantly increased ROS formation and ceramide abundance. Conclusions: Piperlongumine triggers cell membrane scrambling, an effect independent from entry of extracellular Ca2+ but at least partially due to ROS and ceramide formation.

The polyphenolic flavonoid Baicalein has been shown to trigger suicidal death or apoptosis of tumor cells and is thus considered for the prevention and treatment of malignancy. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i) and ceramide. The present study explored whether Baicalein stimulates eryptosis. To this end, forward scatter was taken for measurement of cell volume, annexin-V-binding for phosphatidylserine-exposure, Fluo3 fluorescence for [Ca2+]i and fluorescent antibodies for ceramide abundance. As a result, a 48 h exposure of human erythrocytes to Baicalein was followed by significant decrease of forward scatter (≥10 µM), significant increase of the percentage of annexin-V-binding cells (≥25 µM), significant increase of [Ca2+]i (50 µM) and significant increase of ceramide abundance (50 µM). The effect of Baicalein (50 µM) on annexin-V-binding was significantly blunted but not abrogated by removal of extracellular Ca2+. In conclusion, at the concentrations employed, Baicalein stimulates suicidal erythrocyte death or eryptosis, an effect at least in part due to the combined effects of Ca2+ entry and ceramide formation.

Background/Aims: Cryptotanshinone, a component of Salvia miltiorrhiza Bunge roots, may trigger suicidal death or apoptosis of tumor cells and has thus been recommended for the prevention and treatment of malignancy. On the other hand, Cryptotanshinone has been shown to counteract apoptosis of neurons and hepatocytes. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and phosphatidylserine translocation to the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca2+-activity ([Ca2+]i). The present study explored whether Cryptotanshinone stimulates eryptosis. Methods: Forward scatter was taken as measure of cell volume, annexin V binding for identification of phosphatidylserine-exposing erythrocytes and Fluo3-fluorescence for determination of [Ca2+]i. Results: A 48 h exposure of human erythrocytes to Cryptotanshinone (10 µM) was followed by significant decrease of forward scatter, significant increase of the percentage annexin-V-binding cells and significant increase of [Ca2+]i. The effect of Cryptotanshinone (1 µM) on annexin-V-binding was virtually abrogated by removal of extracellular Ca2+. Conclusion: Cryptotanshinone is a powerful stimulator of suicidal erythrocyte death or eryptosis, which is effective mainly, if not exclusively, by stimulation of Ca2+ entry.

Background/Aims: Gedunin, an inhibitor of heat shock protein HSP90, triggers apoptosis of tumor cells and is thus effective against malignancy. Moreover, the drug has antimalarial potency. In analogy to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by phosphatidylserine translocation to the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca2+-activity ([Ca2+]i). The present study explored whether gedunin stimulates eryptosis. Methods: Forward scatter was determined to estimate cell volume, annexin V binding to identify phosphatidylserine-exposing erythrocytes, hemoglobin release to depict hemolysis, and Fluo3-fluorescence to quantify [Ca2+]i. Results: A 48 h exposure of human erythrocytes to gedunin significantly increased [Ca2+]i (12 µM), significantly decreased forward scatter (24 µM) and significantly increased annexin-V-binding (12 µM). The effect of gedunin (24 µM) on annexin-V-binding was virtually abrogated by removal of extracellular Ca2+. Conclusion: Gedunin stimulates suicidal erythrocyte death or eryptosis, an effect mainly if not exclusively due to stimulation of Ca2+ entry.

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.

Background: Novobiocin, an aminocoumarin antibiotic, interferes with heat shock protein 90 and hypoxia inducible factor dependent gene expression and thus compromises cell survival. Similar to survival of nucleated cells, erythrocyte survival could be disrupted by eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and by phospholipd scrambling of the cell membrane with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i). The Ca2+ sensitivity of phospholipid scrambling is enhanced by ceramide. The present study explored, whether novobiocin elicits eryptosis. Methods: [Ca2+]i was estimated from Fluo3-fluorescence, ceramide abundance utilizing fluorescent antibodies, cell volume from forward scatter, phosphatidylserine-exposure from annexin V binding. Results: A 48 hours exposure to novobiocin (500 µM) was followed by a significant increase of [Ca2+]i, decrease of forward scatter, increase of annexin-V-binding and enhanced ceramide formation. Removal of extracellular Ca2+ virtually abrogated the increase of annexin-V-binding following novobiocin exposure. Conclusions: Novobiocin stimulates eryptosis, an effect at least in part due to entry of extracellular Ca2+ and formation of ceramide.

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.

The selective cyclooxygenase-2 (COX-2) inhibitor celecoxib triggers apoptosis of tumor cells and is thus effective against malignancy. The substance is at least partially effective through mitochondrial depolarization. Even though lacking mitochondria, erythrocytes may enter apoptosis-like suicidal death or eryptosis, which is characterized by cell shrinkage and by phosphatidylserine translocation to the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca2+-activity ([Ca2+]i). The present study explored whether celecoxib stimulates eryptosis. Forward scatter was determined to estimate cell volume, annexin V binding to identify phosphatidylserine-exposing erythrocytes, hemoglobin release to depict hemolysis, and Fluo3-fluorescence to quantify [Ca2+]i. A 48 h exposure of human erythrocytes to celecoxib was followed by significant increase of [Ca2+]i (15 µM), significant decrease of forward scatter (15 µM) and significant increase of annexin-V-binding (10 µM). Celecoxib (15 µM) induced annexin-V-binding was blunted but not abrogated by removal of extracellular Ca2+. In conclusion, celecoxib stimulates suicidal erythrocyte death or eryptosis, an effect partially due to stimulation of Ca2+ entry.

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.

The antidepressant fluoxetine inhibits ceramide producing acid sphingomyelinase. Ceramide is in turn known to trigger eryptosis the suicidal death of erythrocytes characterized by cell shrinkage and exposure of phosphatidylserine at the erythrocyte surface. Ceramide is effective through sensitizing the erythrocytes to the pro-eryptotic effect of increased cytosolic Ca2+ activity ([Ca2+]i). In nucleated cells, fluoxetine could either inhibit or stimulate suicidal death or apoptosis. The present study tested whether fluoxetine influences eryptosis. To this end cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin release and [Ca2+]i from Fluo-3 fluorescence intensity. As a result, a 48 h exposure of erythrocytes to fluoxetine (≥25 µM) significantly decreased forward scatter, increased annexin V binding and enhanced [Ca2+]i. The effect on annexin V binding was significantly blunted, but not abolished, in the absence of extracellular Ca2+. In conclusion, fluoxetine stimulates eryptosis, an effect at least in part due to increase of cytosolic Ca2+ activity.

The nitrosourea alkylating agent, carmustine, is used as chemotherapeutic drug in several malignancies. The substance triggers tumor cell apoptosis. Side effects of carmustine include myelotoxicity with anemia. At least in theory, anemia could partly be due to stimulation of eryptosis, the suicidal death of erythrocytes, characterized by cell shrinkage and breakdown of phosphatidylserine asymmetry of the cell membrane with phosphatidylserine exposure at the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i). The present study tested whether carmustine triggers eryptosis. To this end [Ca2+]i was estimated from Fluo3 fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding, and hemolysis from hemoglobin release. As a result a 48 h exposure to carmustine (≥25 µM) significantly increased [Ca2+]i, decreased forward scatter and increased annexin V binding. The effect on annexin V binding was significantly blunted in the absence of extracellular Ca2+. In conclusion, carmustine stimulates eryptosis at least partially by increasing cytosolic Ca2+ activity.

Background: Levofloxacin, a fluoroquinolone, is an extensively used antibiotic effective against both positively and negatively staining bacteria. It works by inhibiting bacterial topoisomerase type II and topoisomerase type IV, resulting in impaired DNA synthesis and bacterial cell death. Eryptosis is another term for apoptotic cell death of erythrocyte marked by cell shrinkage, phosphatidylserine (PS) flipping, and membrane blebbing. Methods: The intent of the present research was to look at the eryptotic effect of levofloxacin by exposing erythrocytes to therapeutical doses (7, 14 µM) of levofloxacin for 48 hours. Cell size evaluation, PS subjection to outside, and calcium channel inhibition were carried out to investigate eryptosis. Oxidative stress generated by levofloxacin was measured as a putative mechanism of eryptosis using glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase activities. Similarly, hemolysis measurements demonstrated levofloxacin’s cytotoxic effect. Results: Our findings showed that therapeutic doses of levofloxacin can cause a considerable decline in antioxidant enzymes activities, as well as induce cell shrinkage, PS externalization, and hemolysis in erythrocytes. The role of calcium in triggering erythrocyte shrinkage was also confirmed. Conclusion: In conclusion, our findings showed that the indicated levofloxacin doses caused oxidative stress, which leads to erythrocyte death via eryptosis and hemolysis. These findings emphasize the importance of using levofloxacin with caution and the need for additional research to mitigate these side effects.