Caterina Faggio

Stimulation of Erythrocyte Cell Membrane Scrambling by Mitotane

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.

Importance of prebiotics in aquaculture as immunostimulants. Effects on immune system of Sparus aurata and Dicentrarchus labrax

Infectious diseases in fish represent a major problem for the aquaculture field as they produce extensive damages and loss. Over the last few years, with increased development of the aquaculture industry, different methods have been used to contrast these pathologies. Common interest has led to the use of components (as additives in diets) that could contrast diseases without causing any negative impact on the environment. These components are represented by prebiotics, probiotics, and plant extracts. In this review, the effects of prebiotics are described. Prebiotics are indigestible fibres fermented by gut enzymes and commensal bacteria, whose beneficial effects are due to the by-products generated from fermentation. The influence of pre-biotics on the immune system of fish is called immunosaccharides. Mannanoligosaccharides (MOS), Fructooligosaccharides (FOS) and Inulin act at different levels in the innate immune response. For example, through phagocytosis, lysozyme activity, and the complement system activity, an increase in fish growth and an amelioration of their health status is brought about. In this review, the use of prebiotics in aquaculture, such as immunostimulants, has been highlighted: particularly in two teleost fish species, Sparus aurata and Dicentrarchus labrax. The results demonstrate that the road is still long and further studies are required, but the use of prebiotics, individually or coupled together, can open the doors to pioneering a new model of alternative components to antimicrobial agents.

Stimulation of Suicidal Erythrocyte Death by Artesunate

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.

Effect of nitazoxanide on erythrocytes.

Nitazoxanide, a drug effective against a variety of pathogens, triggers apoptosis and is thus considered to be employed against malignancy. Similar to nucleated cells, erythrocytes may undergo an apoptosis‐like suicidal cell death or eryptosis. Hallmarks of eryptosis include cell shrinkage and phospholipid scrambling of the cell membrane with translocation of phosphatidylserine to the erythrocyte surface. Stimulators of eryptosis include increase in cytosolic Ca2+‐activity ([Ca2+]i). The Ca2+‐sensitivity of eryptosis is increased by ceramide. This study explored whether nitazoxanide triggers eryptosis. [Ca2+]i was estimated from Fluo3‐fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin‐V‐binding, ceramide abundance utilizing fluorescent antibodies and haemolysis from haemoglobin release. A 48‐hr exposure to nitazoxanide (1–50 μg/ml) did not significantly modify [Ca2+]i but significantly increased ceramide formation, decreased forward scatter (≥10 μg/ml), increased the percentage of annexin‐V‐binding erythrocytes (≥10 μg/ml) and, at higher concentrations (≥20 μg/ml), stimulated haemolysis. The stimulation of annexin‐V‐binding was significantly blunted in the absence of calcium. Nitazoxanide thus stimulates eryptosis, an effect in part due to ceramide formation.

Stimulation of Suicidal Erythrocyte Death by PRIMA-1

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.

Estramustine-Induced Suicidal Erythrocyte Death

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.

Tanshinone IIA stimulates erythrocyte phosphatidylserine exposure

Tanshinone IIA, an antimicrobial, antioxidant, antianaphylactic, antifibrotic, vasodilating, antiatherosclerotic, organo-protective and antineoplastic component from the rhizome of Salvia miltiorrhiza, is known to trigger apoptosis of tumor cells. Tanshinone IIA is effective in part through mitochondrial depolarization and altered gene expression. Erythrocytes lack mitochondria and nuclei but may undergo eryptosis, an apoptosis-like suicidal cell death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptosis is triggered by increase of cytosolic Ca2+ activity, ATP depletion and ceramide formation. The present study explored, whether tanshinone IIA elicits eryptosis. Cytosolic Ca2+-concentration was determined from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from binding of fluorescent annexin V, hemolysis from hemoglobin concentration in the supernatant, ATP concentration utilizing luciferin–luciferase and ceramide formation utilizing fluorescent anticeramide antibodies. Clearance of circulating erythrocytes was estimated by CFSE-labeling. A 48 h exposure to tanshinone IIA (≥10 µM) significantly increased cytosolic Ca2+-concentration, decreased ATP concentration (25 µM), increased lactate concentration (25 µM), increased ceramide formation (25 µM), decreased forward scatter, increased annexin-V-binding and increased (albeit to a much smaller extent) hemolysis. The effect of 25 µM tanshinone IIA on annexin-V binding was partially reversed in the nominal absence of Ca2+. Labelled tanshinone IIA-treated erythrocytes were more rapidly cleared from the circulating blood in comparison to untreated erythrocytes. The present observations reveal a completely novel effect of tanshinone IIA, i.e. triggering of Ca2+ entry, ATP depletion and ceramide formation in erythrocytes, events eventually leading to eryptosis with cell shrinkage and cell membrane scrambling.

FTY720-Induced Suicidal Erythrocyte Death

FTY720 is a potent anti-inflammatory drug known to trigger suicidal death or apoptosis of a variety of nucleated cells. Erythrocytes may similarly undergo suicidal erythrocyte death or eryptosis. Hallmarks of eryptosis include cell membrane scrambling and cell shrinkage, which are triggered by increase in cytosolic Ca2+ concentration and ceramide. The present study explored whether FTY720 stimulates eryptosis. Cell membrane scrambling was determined from annexin V-binding, cell shrinkage from forward scatter in FACS analysis, cytosolic Ca2+ concentration from Fluo3 fluorescence, ceramide formation from fluorescence-labeled antibody binding and hemolysis from the hemoglobin concentration in the supernatant. Within 48 hours exposure to FTY720 (10 µM) significantly increased annexin V-binding, decreased forward scatter and increased cytosolic Ca2+ concentration but did not significantly modify ceramide formation. The effects of FTY720 were significantly blunted in the nominal absence of extracelluar Ca2+. In conclusion, at toxic concentrations, FTY720 stimulates suicidal cell death, an effect at least partially due to stimulation of Ca2+ entry.

Cytotoxicity, haemolymphatic parameters, and oxidative stress following exposure to sub-lethal concentrations of quaternium-15 in Mytilus galloprovincialis

The presence of a xenobiotic in the environment can often represent a risk for living organisms. Quaternium-15, a preservative, is one of the most used substances and is added to several cosmetics and other industrial products. For this reason,kwowing the bio-indicator of the marine environment, the toxicological effects potentially elicited by this preservative on the marine invertebrate Mytilus galloprovincialis were studied. The results of this work confirm that quaternium-15, used at 0.1 and 1mg/l concentrations, while metabolized in M. galloprovincialis, causes a decrease in cellular viability, and remarkable changes to the defense and antioxidant system. In fact, haemocyte viability is dramatically reduced, and haemolymphatic parameter measurements indicate a stress on the animal. Moreover, an increase in radical species production, in Thiobarbituric Acid Reactive Species (TBARS) concentration, and in the Heat Shock Protein 70 amount, were observed in hepatopancreas. These changes suggest that the antioxidant systems are activated to overwhelm the oxidative damage induced by quaternium-15. Quaternium-15 jeopardizes both the defense and antioxidant systems. These results provide essential information with the biological fate of quaternium-15 in aquatic organisms, and confirm that biomarkers represent an important tool for modern environmental assessments as they can help with the prediction of pollutants involved in the monitoring program.

Effect of sodium dodecyl sulfate (SDS) on stress response in the Mediterranean mussel (Mytilus Galloprovincialis): regulatory volume decrease (Rvd) and modulation of biochemical markers related to oxidative stress.

In this study the effects of an anionic surfactant, sodium dodecyl sulfate (SDS), are assessed on the Mediterranean mussel (Mytilus galloprovincialis), exposed for 18 days at a concentration ranging from 0.1 mg/l to 1 mg/l. The effects are monitored using biomarkers related to stress response, such as regulatory volume decrease (RVD), and to oxidative stress, such as reactive oxygen species (ROS), endogenous antioxidant systems and Hsp70 levels. The results demonstrate that cells from the digestive gland of M. galloprovincialis, exposed to SDS were not able to perform the RVD owing to osmotic stress. Further, SDS causes oxidative stress in treated organisms, as demonstrated by the increased ROS production, in comparison to the controls (p<0.05). Consequently, two enzymes involved in ROS scavenging, superoxide dismutase (SOD) and catalase (CAT) have higher activities and the proportion of oxidized glutathione (GSSG) is higher in hepatopancreas and mantle of treated animals, compared to untreated animals (p<0.05). Furthermore Hsp70 demonstrates an up-regulation in all the analyzed tissues of exposed animals, attesting the stress status induced by the surfactant with respect to the unexposed animals. The results highlight that SDS, under the tested concentrations, exerts a toxic effect in mussels in which the disruption of the osmotic balance follows the induction of oxidative stress.