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Introduction: Bivalve molluscs like Mytilus galloprovincialis are valuable bioindicators due to their filter-feeding lifestyle, wide distribution, and ability to concentrate xenobiotics. Studying the effects of pharmaceuticals on these molluscs is crucial given their presence in surface waters. This study investigated the response of M. galloprovincialis to chlorpromazine (Cpz), an antipsychotic with antiviral activity against influenza, HIV, and coronaviruses in human cells.Methods: In this study, we examined the 14-day impact of chlorpromazine (Cpz) on the model species M. galloprovincialis at two concentrations (Cpz 1: 12 ng L-1 or 37 pM; Cpz 2: 12 µg L-1 or 37 nM). To ensure controlled exposure, a stock solution of Cpz was prepared and introduced into the tanks to match the intended concentrations. Seawater and stock solutions were refreshed every 48 h. The primary focus of this study centered on evaluating cell viability, cell volume regulation, and oxidative stress indicators.Results: Although cell volume regulation, as assessed by decreasing regulatory volume Regulation volume decrease, did not show statistically significant changes during the experiment, digestive cell viability, on the other hand, showed a significant decrease (p < 0.01) in the Cpz 2 group, suggesting effects on the general health and survival of these cells. Biochemically, in both Cpz 1 and Cpz 2, superoxide dismutase activity increased, while catalase (CAT) decreased, causing an elevated lipid peroxidation thiobarbituric acid-reactive substances and protein carbonyls, particularly in the Cpz 2 group. The level of reduced glutathione (GSH) increased in both exposures, whereas the level of GSSG increased only in the Cpz 1 group. Consequently, the GSH/GSSG ratio was elevated in the Cpz 2 group only.Discussion: A comparison of the magnitudes of anti- and pro-oxidative manifestations indicated a pro-oxidative shift in both exposures. These findings show that Cpz induces non-specific symptoms of biochemical and cellular disturbances in M. galloprovincialis even at the low picomolar concentration.

Peracetic acid (PAA) is an oxidative disinfectant with a broad spectrum of antimicrobial activity and low environmental impact. In this study, we investigated the physiological impacts of PAA application in Atlantic salmon (Salmo salar) parr reared in freshwater recirculating aquaculture systems over a 4-week period. PAA at a target concentration of 1 mg/L was administered either in pulse (every 3 days) or continuous. The group that did not receive PAA served as a control. Fish tissue samples were collected for histology, gene expression, and biochemical analyses at day 0 and after 2 and 4 weeks of exposure. The expression of genes encoding for antioxidant defence in the olfactory organs, skin, and gills changed during the trial, but the temporal effects were more pronounced than inter-treatment impacts. The glutathione group of antioxidant genes was more responsive to PAA. In most cases, an upregulation was observed. Significantly lower levels of reactive oxygen species were identified in the plasma and skin mucus of the two PAA-exposed groups at week 4; nonetheless, significantly increased levels of total antioxidant capacity were only observed in the skin mucus of fish from the continuous treatment group. Additional markers of oxidative stress (i.e., 8-oxo-2′-deoxyguanosine and o,o'-dityrosine) were analysed in the skin, gills, liver, and dorsal fins. These markers were unaffected by the two PAA treatments. Sporadic reversible structural alterations were observed in the three mucosal organs; the changes were time-dependent, and the effects of PAA treatment were minimal. The number of mucous cells varied over time but not within treatments except in the skin of the pulse group at week 4 where a reduction was observed. The ratio of acidic and neutral mucous cells in the skin and gills were affected by PAA treatments especially in the pulse group. Overall, this study revealed that Atlantic salmon parr mobilised mucosal and systemic antioxidant defences against the oxidative disinfectant PAA, but it was evident that the mode of application did not impose a strong influence. The minimal effects of PAA application on the indicators of health and welfare underscore the potential use of PAA as a routine disinfectant in recirculating aquaculture systems.

Background/Aims: The thaliana phytoalexin Camalexin has been proposed for the treatment of malignancy. Camalexin counteracts tumor growth in part by stimulation of suicidal death or apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms contributing to the complex machinery executing eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, protein kinase C and caspases. The present study explored, whether Camalexin induces eryptosis and, if so, to shed light on mechanisms involved. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo-3 fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to Camalexin significantly increased the percentage of annexin-V-binding cells (≥ 10 µg/ml), significantly decreased forward scatter (≥ 5 µg/ml) and significantly increased Fluo-3-fluorescence (≥ 10 µg/ml), but did not significantly modify DCFDA fluorescence or ceramide abundance. The effect of Camalexin on annexin-V-binding was significantly blunted by removal of extracellular Ca2+, by kinase inhibitors staurosporine (1 µM) and chelerythrine (10 µM), as well as by caspase inhibitors zVAD (10 µM) and zIETD-fmk (50 µM). Conclusions: Camalexin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part depending on Ca2+ entry, as well as staurosporine and chelerythrine sensitive kinase(s) as well as zVAD and zIETD-fmk sensitive caspase(s).

Background/Aims: The atypical retinoid E23-(40-hydroxyl-30-adamantylbiphenyl-4-yl) acrylic acid (ST1926, adarotene) is used in the treatment of malignancy. The effect of ST1926 is at least in part due to stimulation of apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal death of erythrocytes. Hallmarks of eryptosis include cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the stimulation of eryptosis includes increase of cytosolic Ca2+ activity [Ca2+]i, oxidative stress and ceramide. The present study explored, whether adarotene induces eryptosis and, if so, to test for the involvement of Ca2+ entry, oxidative stress and ceramide. Methods: Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to adarotene (9 µM) significantly increased the percentage of annexin-V-binding cells, an effect paralleled by significant decrease of forward scatter, as well as significant increase of Fluo3-fluorescence, DCFDA fluorescence, and ceramide abundance. The effect of adarotene (9 µM) on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. Conclusions: Adarotene stimulates phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled by and at least in part due to Ca2+ entry, oxidative stress and ceramide.

Background/Aims: The analkaloid drug quinine is utilized mainly for the chemoprophylaxis of malaria. The multiple side effects of quinine include hemolytic anemia and hemolytic uremic syndrome, disorders involving suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling contributing to stimulation of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide and D4476 sensitive casein kinase. The present study explored the putative effect of quinine on eryptosis and elucidated cellular mechanisms involved. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCF dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to quinine (≥ 50 µM) significantly increased the percentage of annexin-V-binding cells without significantly affecting forward scatter. Quinine significantly increased Fluo3-fluorescence, DCF fluorescence and ceramide abundance. The effect of quinine on annexin-V-binding was significantly blunted by removal of extracellular Ca2+ and by addition of D4476 (10 µM). Conclusions: Quinine triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, oxidative stress, ceramide and D4476 sensitive casein kinase.

Background/Aims: The CDC25B inhibitor NSC-95397 triggers apoptosis of tumor cells and is thus considered for the treatment of malignancy. The substance is effective in part by modification of gene expression. Similar to apoptosis of nucleated cells erythrocytes may undergo eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, as well as activation of protein kinases. The present study explored, whether NSC-95397 induces eryptosis and, if so, to shed some light on the mechanisms involved. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to NSC-95397 significantly increased the percentage of annexin-V-binding cells (≥ 1 µM), significantly decreased forward scatter (≥ 2.5 µM), and significantly increased Fluo3-fluorescence (≥ 1 µM), DCFDA fluorescence (5 µM) and ceramide abundance (≥ 5 µM). The effect of NSC-95397 (5 µM) on annexin-V-binding was slightly, but significantly blunted by removal of extracellular Ca2+ and by addition of the protein kinase C inhibitor staurosporine (1 µM). Conclusions: NSC-95397 triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part requiring entry of Ca2+ and activation of staurosporine sensitive kinase(s).

Background/Aims: The natural anthraquinone derivative emodin (1,3,8-trihydroxy-6-methylanthraquinone) is a component of several Chinese medicinal herbal preparations utilized for more than 2000 years. The substance has been used against diverse disorders including malignancy, inflammation and microbial infection. The substance is effective in part by triggering suicidal death or apoptosis. Similar to apoptosis of nucleated cells erythrocytes may enter suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the triggering of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study aimed to test, whether emodin induces eryptosis and, if so, to elucidate underlying cellular mechanisms. Methods: Phosphatidylserine abundance at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: Exposure of human erythrocytes for 48 hours to emodin (≥ 10 µM) significantly increased the percentage of annexin-V-binding cells, and at higher concentrations (≥ 50 µM) significantly increased forward scatter. Emodin significantly increased Fluo3-fluorescence (≥ 10 µM), DCFDA fluorescence (75 µM) and ceramide abundance (75 µM). The effect of emodin on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. Conclusions: Emodin triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to stimulation of Ca2+ entry and paralleled by oxidative stress and ceramide appearance at the erythroctye surface.

Background/Aims: The phytochemical polyphenol rottlerin is a potent activator of diverse Ca2+ -sensitive K+ channels. Those channels play a decisive role in the execution of eryptosis, the suicidal death of erythrocytes, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the stimulation of eryptosis includes increase of cytosolic Ca2+ activity ([Ca2+]i) and ceramide. The present study explored, whether rottlerin induces eryptosis and, if so, to test for the involvement of Ca2+ entry and ceramide. Methods: Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified by determination of haemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to rottlerin (1 - 5 µM) significantly increased the percentage of annexin-V-binding cells, an effect paralleled by significant decrease of forward scatter. Up to 5 µM rottlerin failed to significantly increase average Fluo3-fluorescence. Rottlerin (5 µM) did, however, significantly increase the ceramide abundance. Rottlerin (5 µM) further significantly increased hemolysis. The effect of rottlerin (5 µM) on annexin-V-binding was virtually abolished by removal of extracellular Ca2+. Conclusions: Rottlerin stimulates eryptosis with erythrocyte shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled by and at least in part due to Ca2+ entry and ceramide.

Background/Aims: The serine/threonine protein phosphatase 1 and 2a inhibitor Calyculin A may trigger suicidal death or apoptosis of tumor cells. 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. Triggers of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+] i). Eryptosis is fostered by activation of staurosporine sensitive protein kinase C, SB203580 sensitive p38 kinase, and D4476 sensitive casein kinase. Eryptosis may further involve zVAD sensitive caspases. The present study explored, whether Calyculin A induces eryptosis and, if so, whether its effect requires Ca2+ entry, kinases and/or caspases Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, and [Ca2+] i from Fluo-3 fluorescence, as determined by flow cytometry. Results: A 48 hours exposure of human erythrocytes to Calyculin A (≥ 2.5 nM) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter and significantly increased Fluo-3 fluorescence. The effect of Calyculin A on annexin-V-binding was significantly blunted by removal of extracellular Ca2+, by staurosorine (1 µM), SB203580 (2 µM), D4476 (10 µM), and zVAD (10 µM). Conclusions: Calyculin A triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part requiring Ca2+ entry, kinase activity and caspase activation.

Background/Aims: The bis-indole alkaloid Fascaplysin is effective against malignancy, an effect at least partially due to stimulation of tumor cell apoptosis. Similar to apoptosis of nucleated cells, erythrocytes could enter suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explored, whether Fascaplysin induces eryptosis and, if so, to shed light on the cellular mechanisms involved. Methods: Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified from the hemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to Fascaplysin (≥ 5 µM) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, and significantly increased Fluo3-fluorescence, DCFDA fluorescence as well as ceramide abundance. The effect of Fascaplysin on annexin-V-binding and forward scatter was significantly blunted but not abolished by removal of extracellular Ca2+. Conclusions: Fascaplysin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, oxidative stress and ceramide.

Background/Aims: The bioactive steroid sapogenin diosgenin is considered for a wide variety of applications including treatment of malignancy. The substance counteracts tumor growth in part by stimulating apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the stimulation of eryptosis includes increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explored, whether diosgenin induces eryptosis and, if so, to decipher cellular mechanisms involved. Methods: Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCF dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified by determination of haemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to diosgenin significantly increased the percentage of annexin-V-binding cells (≥ 5 µM), significantly decreased forward scatter (15 µM), significantly increased Fluo3-fluorescence (≥ 10 µM), significantly increased DCF fluorescence (15 µM), significantly increased ceramide abundance (15 µM) and significantly increased hemolysis (15 µM). The effect of diosgenin (15 µM) on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. Conclusions: Diosgenin stimulates eryptosis with erythrocyte shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled by and at least in part due to Ca2+ entry, oxidative stress and ceramide.

Background: The microtubule assembly inhibitor nocodazole has been shown to trigger caspase-independent mitotic death and caspase dependent apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may undergo 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), oxidative stress and ceramide. The present study explored, whether and how nocodazole induces eryptosis. Methods: Flow cytometry was employed to determine phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, the abundance of reactive oxygen species (ROS) from 2′,7′-dichlorodihydrofluorescein (DCF) diacetate dependent fluorescence as well as ceramide surface abundance utilizing specific antibodies. Tubulin abundance was quantified by TubulinTracker™ Green reagent and visualized by confocal microscopy. Results: A 48 hours exposure of human erythrocytes to nocodazole (≥ 30 µg/ml) significantly increased the percentage of annexin-V-binding cells without significantly modifying average forward scatter. Nocodazole significantly increased Fluo3-fluorescence, significantly increased DCF fluorescence and significantly increased ceramide surface abundance. The effect of nocodazole on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+ and was not modified in the presence of Caspase 3 inhibitor zVAD (1 µM). Nocodazole treatment reduced the content of total tubulin. Conclusions: Nocodazole triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of Ca2+ entry, oxidative stress and ceramide.

Background/Aims: The alkylating drug oxaliplatin is widely used for chemotherapy of malignancy. Oxaliplatin is effective by inducing both, necrosis and apoptosis. Similar to necrosis or apoptosis of nucleated cells, erythrocytes may enter hemolysis, which is apparent from hemoglobin release or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include oxidative stress and/or Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether and how oxaliplatin induces eryptosis. Methods: Phosphatidylserine exposure at the cell surface was quantified utilizing annexin-V-binding, cell volume estimated from forward scatter, hemolysis deduced from hemoglobin release, [Ca2+]i determined utilizing Fluo-3 fluorescence, and reactive oxygen species (ROS) abundance visualized using 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) dependent fluorescence. Results: A 48 hours exposure of human erythrocytes to oxaliplatin (10 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, significantly increased Fluo-3 fluorescence, and significantly increased DCFDA fluorescence. The effect of oxaliplatin on annexin-V-binding and forward scatter was rather augmented by removal of extracellular Ca2+, but was significantly blunted in the presence of the antioxidant N-acetyl-cysteine (1 mM). Conclusions: Oxaliplatin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect partially dependent on ROS formation.

Background/Aims: Fucoxanthin, a carotenoid isolated from brown seaweeds, induces suicidal death or apoptosis of tumor cells and is thus considered for the treatment or prevention of malignancy. In analogy to apoptosis of nucleated cell, erythrocytes may enter eryptosis, the suicidal death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and activation of p38 kinase or protein kinase C. The present study explored, whether and how fucoxanthin induces eryptosis. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, hemolysis from hemoglobin release, [Ca2+]i from Fluo3-fluorescence, and abundance of reactive oxygen species (ROS) from DCFDA dependent fluorescence and lipid peroxidation using BODIPY fluoresence. Results: A 48 hours exposure of human erythrocytes to fucoxanthin significantly increased the percentage of annexin-V-binding cells (≥ 50 µM), significantly decreased average forward scatter (≥ 25 µM), significantly increased hemolysis (≥ 25 µM), significantly increased Fluo3-fluorescence (≥ 50 µM), significantly increased lipid peroxidation, but did not significantly modify DCFDA fluorescence. The effect of fucoxanthin on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+, and was insensitive to p38 kinase inhibitor skepinone (2 µM) and to protein kinase C inhibitor calphostin (100 nM). Conclusion: Fucoxanthin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of Ca2+ entry.

Background: The P-glycoprotein inhibitor zosuquidar (LY335979) is clinically used to augment the effect of cytostatic drugs on suicidal tumor cell death or apoptosis. The present study explored whether the substance is cytotoxic to erythrocytes. Upon injury, erythrocytes may undergo suicidal cell death or eryptosis, which is characterized by cell shrinkage and translocation of cell membrane phosphatidylserine to the erythrocyte surface. Signaling of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i), oxidative stress and activation of several kinases, such as p38 kinase and protein kinase C. Methods: Phosphatidylserine abundance at the erythrocyte surface was quantified from binding of FITC-labelled annexin-V, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, and reactive oxygen species (ROS) from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. Results: A 48 h treatment of human erythrocytes with zosuquidar significantly increased the percentage of annexin-V-binding cells (2 and 4 µg/ml), significantly decreased forward scatter (4 µg/ml), significantly increased [Ca2+]i (4 µg/ml), but did not significantly modify ROS. The up-regulation of annexin-V-binding following zosuquidar (4 µg/ml) treatment was significantly blunted by removal of extracellular Ca2+, by presence of p38 kinase inhibitor SB203580 (2 µM) and by presence of protein kinase C inhibitor calphostin (100 nM). Conclusions: Exposure of erythrocytes to zosuquidar triggers suicidal erythrocyte death with erythrocyte shrinkage and erythrocyte membrane scrambling, an effect involving Ca2+ entry and requiring activity of SB203580 and calphostin sensitive kinases.

Background/Aims: The anti-inflammatory, anti-autoimmune, antiparasitic, and anti-viral ether phospholipid edelfosine (1-O-octadecyl-2-O-methylglycero-3-phosphocholine) stimulates apoptosis of tumor cells and is thus considered for the treatment of malignancy. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and phospholipid scrambling of the cell membrane with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i) and oxidative stress. The present study explored, whether and how edelfosine induces eryptosis. Methods: Flow cytometry and photometry, respectively, were employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, hemolysis from hemoglobin release, [Ca2+]i from Fluo3-fluorescence, and abundance of reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. Results: A 6 hours exposure of human erythrocytes to edelfosine (5 µM) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, and significantly increased Fluo3-fluorescence, but did not significantly modify DCFDA fluorescence. The effect of edelfosine on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. Conclusions: Edelfosine triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of Ca2+ entry.

Background/Aims: The benzophenone garcinol from dried fruit rind of Garcinia indica counteracts malignancy, an effect at least in part due to stimulation of apoptosis. The proapototic effect of garcinol is attributed in part to inhibition of histone acetyltransferases and thus modification of gene expression. Moreover, garcinol triggers mitochondrial depolarisation. Erythrocytes lack gene expression and mitochondria but are nevertheless able to enter apoptosis-like suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include oxidative stress, energy depletion and Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether and how garcinol induces eryptosis. Methods: To this end, phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, hemolysis from hemoglobin release, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence and cytosolic ATP levels utilizing a luciferin-luciferase-based assay. Results: A 24 hours exposure of human erythrocytes to garcinol (2.5 or 5 µM) significantly increased the percentage of annexin-V-binding cells. Garcinol decreased (at 1 µM and 2.5 µM) or increased (at 5 µM) forward scatter. Garcinol (5 µM) further increased Fluo3-fluorescence, increased DCFDA fluorescence, and decreased cytosolic ATP levels. The effect of garcinol on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. Conclusions: Garcinol triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of ROS formation, energy depletion and Ca2+ entry.

Background/Aims: The JAK1/JAK2 tyrosine kinase inhibitor ruxolitinib is widely used for the treatment of myeloproliferative neoplasm-associated myelofibrosis and other malignancies. Most important side effects include anemia. A common cause of anemia is accelerated suicidal death of erythrocytes or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Mechanisms contributing to the triggering of eryptosis include oxidative stress, Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i), and activation of distinct kinases, such as p38 mitogen activated protein (MAP) kinase. The present study explored whether and how ruxolitinib induces eryptosis. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, hemolysis from hemoglobin release, [Ca2+]i from Fluo3-fluorescence, and ROS formation from DCFDA dependent fluorescence. Results: A 48 hours exposure of human erythrocytes to ruxolitinib (25 µM) significantly increased the percentage of annexin-V-binding cells and significantly decreased forward scatter. Ruxolitinib did not significantly modify Fluo3-fluorescence and DCFDA fluorescence and the effect of ruxolitinib on annexin-V-binding was not significantly modified by removal of extracellular Ca2+. The effect of ruxolitinib on annexin-V-binding was, however, significantly blunted by the p38 MAP kinase inhibitor SB203580 and virtually abolished by the p38 MAP kinase inhibitor skepinone. Conclusion: Ruxolitinib triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part requiring p38 MAP kinase activity.

Background/Aims: The antinflammatory natural product boswellic acid is effective against cancer at least in part by inducing tumor cell apoptosis. Similar to apoptosis of nucleated cells erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include oxidative stress, increase of cytosolic Ca2+-activity ([Ca2+]i), energy depletion, ceramide formation and p38 kinase activation. The present study tested, whether and how boswellic acid induces eryptosis. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, hemolysis from hemoglobin release, [Ca2+]i from Fluo3-fluorescence, ceramide abundance utilizing specific antibodies, reactive oxygen species (ROS) from 2′,7′-dichlorodihydrofuorescein diacetate (DCFDA) fluorescence, and cytosolic ATP concentration utilizing a luciferin-luciferase assay kit. Results: A 24 hours exposure of human erythrocytes to boswellic acid (5 µg/ml) significantly increased the percentage of annexin-V-binding cells (to 9.3 ±0.9 %) and significantly decreased forward scatter. Boswellic acid did not significantly modify [Ca2+]i, cytosolic ATP, ROS, or ceramide abundance. The effect of boswellic acid on annexin-V-binding was significantly blunted, but not abolished by p38 kinase inhibitors skepinone (2 µM) and SB203580 (2 µM). Conclusions: Boswellic acid stimulates cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part dependent on p38 protein kinase activity.

Background: The anticarcinogenic drug PRIMA-1 (p53 reactivation and induction of massive apoptosis 1) induces suicidal death of tumor cells, an effect in large part attributed to the up-regulation of the proapoptotic transcription factor p53. Erythrocytes are lacking gene transcription but are nevertheless able to enter eryptosis, a 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 formation. The present study tested whether PRIMA-1 stimulates eryptosis. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ceramide abundance from binding of specific antibodies, and ROS formation from DCFDA fluorescence. Results: A 48 h exposure of human erythrocytes to PRIMA-1 (25 µM) significantly increased the percentage of annexin-V-binding cells without significantly influencing [Ca2+]i or forward scatter. PRIMA-1 (100 µM) induced annexin-V-binding was not significantly blunted by removal of extracellular Ca2+ or by the caspase-3 inhibitor zVAD. PRIMA-1 (100 µM) further increased the ceramide abundance at the cell surface and ROS formation. Conclusions: PRIMA-1 stimulates phosphatidylserine translocation at the erythrocyte cell membrane, an effect at least partially due to up-regulation of ceramide abundance and ROS formation.

Background: The artemisinin derivative artesunate is effective in the treatment of severe malaria and is considered for the treatment of malignancy. Artesunate triggers tumor cell apoptosis, an effect at least in part mediated by mitochondria. Even though lacking mitochondria, erythrocytes may similarly enter suicidal death or eryptosis, which is characterized by cell shrinkage and breakdown of the phospholipid asymmetry of the cell membrane with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i), ceramide formation, and oxidative stress. The present study explored whether artesunate stimulates eryptosis. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ceramide abundance from binding of specific antibodies, and oxidative stress from 2′,7′-dichlorodihydrofluorescein-diacetate fluorescence. Results: A 48 h exposure of human erythrocytes to artesunate significantly increased the percentage of annexin-V-binding cells (≥ 9 µg/ml) without significantly influencing forward scatter. Artesunate significantly increased [Ca2+]i. The stimulation of annexin-V-binding by artesunate (15 µg/ml) was significantly blunted but not abolished by removal of extracellular Ca2+. Artesunate increased the ceramide abundance at the cell surface and the 2′,7′-dichlorodihydrofluorescein-diacetate fluorescence. Conclusions: Artesunate stimulates phosphatidylserine translocation at the erythrocyte cell membrane, an effect at least partially due to increase of [Ca2+]i, stimulation of ceramide formation and generation of oxidative stress.

Background/Aims: Dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity, are required for initiation of specific T cell-driven immune responses. Phosphoinositide-3-kinase (PI3K) suppresses proinflammatory cytokine production in DCs, which limits T helper (Th1) polarization. PI3K is in part effective by downregulation of transcription factor NF-κB. Downstream signaling elements of PI3K include serum- and glucocorticoid-inducible kinase 1 (SGK1) and its phosphorylation target N-myc downstream regulated gene 1 (NDRG1). The present study explored whether SGK1 and NDRG1 play a role in the regulation of NF-κB and DC-maturation. Methods: DCs were isolated from bone marrow (BMDCs) or spleen of mice lacking functional SGK1 (sgk1-/-) and corresponding wild type mice (sgk1+/+). Protein abundance was determined by Western blotting. Transcription was inhibited by siRNA. Abundance of maturation markers was quantified by flow cytometry. FITC-dextran uptake was determined to quantify phagocytosis. Results: NDRG1 was similarly expressed in sgk1+/+ and sgk1-/-BMDCs, but SGK1-dependent phosphorylation of NDRG-1 was decreased in sgk1-/-BMDCs. Silencing of NDRG1 in sgk1+/+BMDCs as compared to control empty vector-treated BMDCs enhanced nuclear abundance of NF-κB subunit p65. Moreover, the abundance of phosphorylated NF-κB inhibitor IκBa, of phosphorylated IκB kinase (IKKa/ß) and of nuclear p65 were significantly higher in sgk1-/-BMDCs than in sgk1+/+BMDCs. Expression of maturation markers, MHC II, and CD86, was significantly larger and phagocytic capacity was significantly lower in sgk1-/- than in sgk1+/+BMDCs. Expression of CD86 and MHCII was also significantly higher in DCs isolated from the spleen of sgk1-/- mice than those from sgk1+/+mice. Conclusion: SGK1 and NDRG1 participate in the regulation of NF-κB signaling in and maturation of DCs.

Background: Mitotane (1,1-dichloro-2-[o-chlorophenyl]-2-[p-chlorophenyl]ethane), a cytostatic drug used for the treatment of adrenocortical carcinomas, is effective by triggering tumor cell apoptosis. In analogy to apoptosis of nucleated cells, eryptosis is the suicidal death of erythrocytes, which is typically paralleled by cell shrinkage and breakdown of cell membrane phosphatidylserine asymmetry with subsequent phosphatidylserine exposure at the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca2+ concentration ([Ca2+]i). The present study tested, whether treatment of human erythrocytes with mitotane is followed by eryptosis. Methods: [Ca2+]i was estimated from Fluo3 fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding, and hemolysis from hemoglobin release. Results: Exposure to mitotane (≥ 5 µg/ml ≈ 16 µM) significantly increased [Ca2+]i, increased annexin V binding and triggered hemolysis, but did not significantly modify forward scatter. The effect on annexin V binding was significantly blunted in the absence of extracellular Ca2+. Within 30 min Ca2+ ionophore ionomycin (1 µM) decreased forward scatter, an effect virtually abolished in the presence of mitotane (15 µg/ml). Conclusions: Mitotane increases [Ca2+]i with subsequent phosphatidylserine translocation. By the same token mitotane inhibits Ca2+ induced cell shrinkage.

Background/Aims: The protein kinase Akt2/PKBβ is a known regulator of macrophage and dendritic cell (DC) migration. The mechanisms linking Akt2 activity to migration remained, however, elusive. DC migration is governed by Ca2+ signaling. We thus explored whether Akt2 regulates DC Ca2+ signaling. Methods: DCs were derived from bone marrow of Akt2-deficient mice (akt2-/-) and their wild type littermates (akt2+/+). DC maturation was induced by lipopolysaccharides (LPS) and evaluated by flow cytometry. Cytosolic Ca2+ concentration was determined by Fura-2 fluorescence, channel activity by whole cell recording, transcript levels by RT-PCR, migration utilizing transwells. Results: Upon maturation, chemokine CCL21 stimulated migration of akt2+/+ but not akt2-/- DCs. CCL21-induced increase in cytosolic Ca2+ concentration, thapsigargin-induced release of Ca2+ from intracellular stores with subsequent store-operated Ca2+ entry (SOCE), ATP-induced inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ release as well as Ca2+ release-activated Ca2+ (CRAC) channel activity were all significantly lower in mature akt2-/- than in mature akt2+/+ DCs. Transcript levels of IP3 receptor IP3R2 and of IP3R2 regulating transcription factor ETS1 were significantly higher in akt2+/+ than in akt2-/- DCs prior to maturation and were upregulated by LPS stimulation (1h) in akt2+/+ and to a lower extent in akt2-/- DCs. Following maturation, protein abundance of IP3R2 and ETS1 were similarly higher in akt2+/+ than in akt2-/- DCs. The IP3R inhibitor Xestospongin C significantly decreased CCL21-induced migration of akt2+/+DCs and abrogated the differences between genotypes. Finally, knock-down of ETS1 with siRNA decreased IP3R2 mRNA abundance, thapsigargin- and ATP-induced Ca2+ release, SOCE and CRAC channel activation, as well as DC migration. Conclusion: Akt2 upregulates DC migration at least in part by ETS1-dependent stimulation of IP3R2 transcription.

Background: The nitrogen mustard derivative of estradiol-17β-phosphate estramustine is used for the treatment of prostate cancer. Estramustine may trigger suicidal death of cancer cells. Side effects of estramustine include anemia. At least in theory, estramustine could cause anemia by stimulation of eryptosis, the suicidal death of erythrocytes. Hallmarks of eryptosis include cell shrinkage, increased cytosolic Ca2+ activity ([Ca2+]), ceramide formation and phosphatidylserine translocation to the outer leaflet of the cell membrane with phosphatidylserine exposure at the erythrocyte surface. Eryptosis is stimulated by increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored whether estramustine triggers eryptosis. Methods: [Ca2+]i was estimated from Fluo3 fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding, and hemolysis from hemoglobin release. Results: A 24 h exposure to estramustine (≤ 100 µM) significantly increased [Ca2+]i, increased annexin V binding and increased hemoglobin release. The effect of estramustine on annexin V binding was significantly blunted by removal of extracellular Ca2+. Conclusions: Estramustine stimulates both, eryptosis and hemolysis. The estramustine induced translocation of phosphatidylserine to the cell surface is at least partially due to increase of cytosolic Ca2+ activity.

Background/Aims: Platelets are critically important for primary haemostasis and the major players in thrombotic vascular occlusion. Platelets are activated by agonists, such as thrombin and collagen-related peptide as well as second-wave mediators including thromboxane A2 via different intracellular signaling pathways resulting in degranulation, aggregation and thrombus formation. Platelet activation is paralleled by phosphorylation and activation of p38 MAPK. The limited specificity of hitherto known p38 MAPK inhibitors precluded safe conclusions on the precise role of p38 MAPK in the regulation of platelet function. The present study examined the impact of Skepinone-L, a novel and highly selective inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), on platelet activation and thrombus formation. Methods: Experiments were performed in freshly isolated human platelets. Protein phosphorylation was quantified by Western blotting, thromboxane B2 synthesis by enzyme immunoassay, ATP release by ChronoLume luciferin assay, cytosolic Ca2+ concentration by Fura-2 fluorescence-measurements, platelet aggregation by a light transmissions measurement and in vitro thrombus formation by a flow chamber. Results: Skepinone-L (1 μM) virtually abrogated the phosphorylation of platelet p38 MAPK substrate Hsp27 following stimulation with CRP (1 μg/ml), thrombin (5 mU/ml) or thromboxane A2 analogue U-46619 (1 μM). Furthermore, Skepinone-L significantly blunted activation-dependent platelet secretion and aggregation following threshold concentrations of CRP, thrombin and thromboxane A2 analogue U-46619. Skepinone-L did not impair platelet Ca2+ signaling but prevented agonist-induced thromboxane A2 synthesis through abrogation of p38 MAPK-dependent phosphorylation of platelet cytosolic phospholipase A2 (cPLA2). Skepinone-L further markedly blunted thrombus formation under low (500-s) and high (1700-s) arterial shear rates. Conclusions: The present study discloses a powerful inhibiting effect of p38 MAPK-blocker Skepinone-L on platelet activation and thrombus formation.

Several compounds from marine organisms have been studied for their potential use in aquaculture. Among the red algae, Asparagopsis taxiformis is considered one of the most promising species for the production of bioactive metabolites with numerous proposed applications. Here, the in vitro antibacterial activity, the easy handling and the absence of adverse effects on marine fish species are reported. Depending on the seasonal period of sampling, ethanol extracts of A. taxiformis exhibited significantly different inhibitory activity against fish pathogenic bacteria. The extract obtained in late spring showed strong antibacterial activity against Aeromonas salmonicida subsp. salmonicida, Vibrio alginolyticus, and V. vulnificus, and moderate activity against Photobacterium damselae subsp. damselae, P. damselae subsp. piscicida, V. harveyi and V. parahaemolyticus. Sea bass and gilthead sea bream were fed with pellets supplied with algae and algal extracts. The absence of undesired effects was demonstrated. Hematological and biochemical investigations allowed to confirm that the whole alga and its extracts could be proposed for a future application in aquaculture.