Trifluoperazine-Induced Suicidal Erythrocyte Death and S-Nitrosylation Inhibition, Reversed by the Nitric Oxide Donor Sodium Nitroprusside.

BACKGROUND AND PURPOSE: The high potency antipsychotic drug trifluoperazine (10-[3-(4-methyl-1-piperazinyl)-propyl]-2-(trifluoromethyl)-(10)H-phenothiazine dihydrochloride; TFP) may either counteract or promote suicidal cell death or apoptosis. Similar to apoptosis, erythrocytes may enter eryptosis, characterized by phosphatidylserine exposure at the cell surface and cell shrinkage. Eryptosis can be stimulated by an increase in cytoplasmic Ca2+ concentration ([Ca2+]i) and inhibited by nitric oxide (NO). We explored whether TFP treatment of erythrocytes induces phosphatidylserine exposure, cell shrinkage, and calcium influx, whether it impairs S-nitrosylation and whether these effects are inhibited by NO. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, and protein nitrosylation from fluorescence switch of the Bodipy-TMR/Sypro Ruby signal. RESULTS: Exposure of human erythrocytes to TFP significantly enhanced the percentage of annexin-V-binding cells, raised [Ca2+]i, and decreased S-nitrosylation. The effect of TFP on annexin-V-binding was not affected by removal of extracellular Ca2+ alone, but was significantly inhibited by pre-treatment with sodium nitroprusside (SNP), an effect significantly augmented by additional removal of extracellular Ca2+. A 3 hours treatment with 0.1 µM Ca2+ ionophore ionomycin triggered annexin-V-binding and cell shrinkage, effects fully reversed by removal of extracellular Ca2+. CONCLUSIONS: TFP induces eryptosis and decreases protein S-nitrosylation, effects blunted by nitroprusside. The effect of nitroprusside is attenuated in the presence of extracellular Ca2+.

Sub-chronic (13-week) oral toxicity study, preceded by an in utero exposure phase and genotoxicity studies with fish source phosphatidylserine in rats.

The safety of fish phosphatidylserine (PS) conjugated to DHA (InCog™) was examined in a series of toxicology studies as first step to support future use in infants and general population using in vitro genotoxicity tests and in a sub-chronic toxicity study with an in-utero exposure phase. PS is a major lipid in the cell membrane, active in various membrane-mediated processes. PS-DHA, present in human milk, has been suggested to be important for early brain development. Rats were exposed to diets containing 1.5%, 3% or 4.5% InCog or two control diets. Parental (F0) animals were fed throughout mating, gestation and lactation. Subsequently, a subchronic, 13-week study was conducted on the F1 animals followed by 4 weeks of recovery. The genotoxicity tests showed no mutagenicity potential. No significant toxicological findings were found in the F0 rats or the F1 pups. In the 13-weeks study, an increase in the presence of renal minimal-mild multifocal corticomedullary mineralization was noted in nine females of the high-dose group. This change was not associated with any inflammatory or degenerative changes in the kidneys. The no-observed-adverse-effect level (NOAEL) in the present study was placed at 3% in the diet (mid-dose group), equivalent to an overall intake of at least 2.1 g InCog/kg bw/day in the F1 generation.

Natural antioxidants improve red blood cell "survival" in non-leukoreduced blood samples.

BACKGROUND: Blood collected in an anticoagulant can be kept refrigerated in an unmodified state within 5 - 6 weeks. Oxidative damage is considered to be a one of the major factors contributing to the development of storage lesions. Lipid and membrane proteins oxidation results in changes in cation gradients that affect the cell survival. AIM: In the present study we used the natural antioxidants and ion channels blockers (L-carnosine, spermine, phloretin and their mixtures) to prolong "survival" of red blood cells (RBCs), measured as the lack of PS exposure and cell hemolysis, in the Alsever's preservative solution upon hypothermic storage. RESULTS: We show that the mixture of carnosine (20 mM), spermine (20 µM) and phloretin (100 µM) effectively blunted phosphatidylserine (PS) exposure, Ca(2+) accumulation and RBCs hemolysis in non-leukoreduced low (∼ 2%) hematocrit samples after 36 days of storage as well as after 1 day of post-storage incubation of the stored cells in physiological saline solution. In addition, a slight but significant decrease in PS exposure was observed in non-leukoreduced high (∼ 20%) hematocrit samples after 36 days of storage with the mixture of substances. CONCLUSION: We conclude that the use of the mixture of natural antioxidants (carnosine, spermine, and phloretin) as an additive to blood preservative solution provides better RBCs storage and "survival".

Lonomia obliqua (Lepidoptera, Saturniidae) caterpillar bristle extract induces direct lysis by cleaving erythrocyte membrane glycoproteins.

Lonomia obliqua caterpillar bristle extract induces hemolysis in vitro on washed human and rat erythrocytes, in either the absence or presence of exogenous lecithin. In the former condition, phospholipases A(2) are key enzymes involved in hemolysis. However, the mechanism whereby this extract causes direct hemolysis is not known. Thus, the aim of this study was to investigate the hemolytic mechanism of the crude extract of the caterpillar L. obliqua on human erythrocytes in the absence of lecithin. The extract significantly increased the erythrocyte osmotic fragility and promoted the removal of glycophorins A and C, and band 3 from the erythrocyte membrane. The use of Ca2+ and Mg2+ ions significantly potentiated glycoprotein removal, remarkably of erythrocyte band 3. The composition of fatty acids was analyzed by HPLC in both L. obliqua caterpillar bristle extract and human erythrocyte membranes incubated with the extract. The levels of unsaturated fatty acids were remarkably augmented in erythrocytes incubated with the extract than in control erythrocytes, modifying thereby the saturated/unsaturated fatty acid ratio. Altogether, evidence is provided here that the interplay of at least three mechanisms of action accounts for the direct activity of the bristle extract on erythrocyte membrane, leading to hemolysis: the removal of glycoproteins and band 3; the insertion of fatty acids; and the action of phospholipases. Such mechanisms might affect erythrocyte flexibility and deformability, which may induce hemolysis by increasing erythrocyte fragility. However, whether the direct hemolytic activity of L. obliqua caterpillar is the major cause of intravascular hemolysis during envenomation still needs further investigation.

Thymoquinone-induced suicidal erythrocyte death.

Thymoquinone is a nutrient with anticarcinogenic activity by stimulating suicidal death of tumor cells. Similar to nucleated cells, erythrocytes may experience suicidal death or eryptosis, characterized by exposure of phosphatidylserine at the erythrocyte surface and by cell shrinkage. Triggers and signaling of eryptosis include increase in cytosolic Ca(2+)activity, ceramide formation, and stimulation of protein kinase C. The present experiments explored, whether thymoquinone influences eryptosis. According to annexin V-binding, thymoquinone (3 microM) increased the percentage of phosphatidylserine-exposing erythrocytes. According to forward scatter in FACS analysis, thymoquinone (10 microM) led to cell shrinkage. The effect of thymoquinone was not paralleled by appreciable ceramide formation (immunofluorescent antibody) or hemolysis (hemoglobin release). It was not significantly blunted in the nominal absence of extracellular Ca(2+) but was inhibited by staurosporine (500 nM). In conclusion, thymoquinone triggers suicidal erythrocyte death, an effect paralleling the apoptotic effect on nucleated cells.

Gold stimulates Ca2+ entry into and subsequent suicidal death of erythrocytes.

The suicidal death of erythrocytes, eryptosis, is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine exposure at the erythrocyte surface. Erythrocyte cell membrane scrambling is stimulated by increase of cytosolic Ca2+ concentration ([Ca2+](i)) and formation of ceramide. Phosphatidylserine (PS) exposing cells are rapidly cleared from circulating blood. Ca2+ entry and/or ceramide formation and thus eryptosis are triggered by lead, mercury, aluminium, and copper ions. The present study explored whether eryptosis could be similarly triggered by exposure to gold. To this end, erythrocytes from healthy volunteers were exposed to AuCl and phosphatidylserine exposure (annexin V binding), cell volume (forward scatter), [Ca2+](i) (Fluo3-dependent fluorescence), and ceramide formation (anti-ceramide-FITC fluorescence) were determined by flow cytometry. Exposure of erythrocytes to low concentrations of AuCl (> or =0.75microg/ml) increased [Ca2+](i) but did not affect ceramide formation. AuCl at concentrations > or =0.5microg/ml significantly increased the number of PS exposing erythrocytes and decreased forward scatter at low concentrations of AuCl pointing to cell shrinkage. Aurothiomalate (> or =1microg/ml), a gold containing drug effective against rheumatoid arthritis, similarly triggered PS exposure of erythrocytes. The present observations disclose a novel action of gold, which may well contribute to side effects during treatment with gold preparations.

Cytotoxicity of submicron emulsions and solid lipid nanoparticles for dermal application.

The cytotoxicity and physical properties of various submicron O/W emulsions and solid lipid nanoparticles for dermal applications were investigated. Droplet size and zetapotential of submicron emulsions depended on the composition of the cosurfactant blend used. The viability of J774 macrophages, mouse 3T3 fibroblasts and HaCaT keratinocytes was significantly reduced in the presence of stearylamine. Nanoparticles consisting of stearic acid or different kinds of adeps solidus could be manufactured when formulated with lecithin, sodium taurocholate, polysorbate 80 and stearylamine. Survival of macrophages was highly affected by stearic acid and stearylamine. In general a viability of more than 90% was observed when semi-synthetic glycerides or hard fat was employed to formulate nanoparticles.

Activated protein C prevents development of phosphatidylserine-induced intrauterine growth restriction in mice.

We examined the effect of activated protein C (APC) on the development of intrauterine growth restriction (IUGR) in an experimental animal model we established. The IUGR in mice was induced by artificial phosphatidylserine (PS)/phosphatidylcholine (PC) microvesicles that represent procoagulant phospholipids derived from activated platelets. This model represents the placental insufficiency associated with the phospholipid-induced hypercoagulable state in placental circulation. APC prolonged the activated partial thromboplastin time (aPTT) using mouse plasma and dose dependently inhibited thrombin generation in a chromogenic assay in defibrinated plasma of mice. Administration of exogenous APC at concentrations that maximally inhibited thrombin generation in defibrinated plasma prevented a significant reduction in fetal body weight and induced marked histological changes including congestion and fibrin depositions in IUGR mouse placentas. These results suggest that the inhibition of thrombin generation in the placental circulation by APC prevents the development of IUGR that is dependent on coagulation associated with PS/PC from activated platelets.

The effect of factor Xa/phospholipid infusion on the acute phase response in baboons.

Disseminated intravascular coagulation (DIC) is a frequent complication of septicemia or tissue injury and may be accompanied by elevations of interleukin-6, a mediator of the acute phase response. It is not known whether thrombin or fibrin deposition may directly induce an acute phase response. To study this, we employed a baboon model of in vivo thrombin generation, induced by the administration of purified bovine Factor Xa and phospholipid vesicles. Two Xa/phospholipid dosages were used, a low dosage (2 animals) leading to a rapid 49% decrease in fibrinogen and a high dosage (two injections at 5h interval; 3 animals) leading to complete fibrinogen depletion. Thereafter, fibrinogen levels increased in both treatment groups, reached a maximum of 2.52 +/- 0.23 g/l (mean +/- SE, n = 5; p < 0.01 with respect to basal levels) at day 2, and returned to normal by day seven. In five control (injection of 0.15% NaCl) baboons no significant changes of fibrinogen were observed (maximal values: 1.88 +/- 0.12 g/l). Serum concentrations of C-reactive protein, an acute phase protein, increased from 3.7 +/- 0.4 mg/l to a maximum of 33.0 +/- 7.3 at day one, which was five-fold higher (p < 0.01) than in control animals at day one (6.2 +/- 0.5 mg/l). Transient increases were observed within 6h for interleukin-6 from basal values of 6.2 +/- 1.7 ng/l to peak plasma levels of 42.9 +/- 21.4 ng/l, a value three-fold higher (p = 0.07) than in control animals (14.8 +/- 4.0 ng/l). The preliminary results of this observational study suggest that factor Xa/phospholipid infusion is followed by an acute phase response, leading after one day to significant increases of fibrinogen and of C-reactive protein.

PC:PS liposomes induce a recruitment of neutrophils and the release of TNF alpha in the lungs of mice sensitized with Saccharopolyspora rectivirgula.

The aim of this study was to verify the effect of nasally instilled liposomes (L) or dexamethasone-containing L (Ldexa) on normal or inflamed lung tissue. Three groups of mice were studied. Group I was given saline instillations for 3 weeks prior to the instillation with liposomes. In groups II and III lung inflammation was induced by repeated instillations of Saccharopolyspora rectivirgula before the instillation of liposomes (group II) or liposomes containing dexamethasone (group III). Animals from all groups were killed at regular time intervals for up to 48 h after the instillation of liposomes. The total cell count in bronchoalveolar lavage fluid did not differ significantly between groups I and II. However, in group III it decreased rapidly from 6.2 to 2.8 x 10(5) cells mL-1 within 2 h. Differential counts did not change in group I, but in group II a transient neutrophilia was observed 180 min after the instillation of liposomes. In group III, the instillation of dexamethasone-containing liposomes depleted all neutrophils and lymphocytes after 4 h. No TNF alpha was found in samples of lavage fluid from any of the groups at time 0. In group I, liposomes induced the production of 0.03 ng mL-1 of TNF alpha in the 1 h sample only. In group II, TNF alpha peaked to 1 ng mL-1 at 1 h and had decreased to 0.35 ng mL-1 by 3 h. In group III, TNF alpha peaked at 1 h, but only reached a level of 0.1 ng mL-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Toxic factors in the red blood cell membrane.

The toxic effects of hemolysed RBCs have been studied for more than 100 years, but the specific factors involved have not been identified. This study focused on phosphatidylethanolamine (PE) and phosphatidylserine (PS), two aminophospholipids that normally reside on the cytoplasmic side of the red cell membrane. An in vitro experiment with murine peritoneal exudate macrophages showed that PE and PS: a) stimulated the production of H2O2, complement factor C3a, prostacyclin, and thromboxane at a dose of 5 micrograms/ml; b) produced cell injury, evidenced by release of lipid peroxides, LDH, and by morphologic changes on phase-contrast and electron microscopy at a dose of 50 micrograms/ml; and c) caused cell death in 50-66% of cells at a dose of 100 micrograms/ml. An in vivo experiment showed that PE and PS injected intravenously into various groups of rabbits: a) caused only transient hypotension at a dose of 0.05 mg/kg body weight; b) caused significant hypotension, cardiac arrhythmias, bronchospasm, activation of intravascular coagulation, complement, platelets, and leukocytes with release of histamine, serotonin, and thromboxane at a dose of 0.10 mg/kg; and c) caused cardiac arrest and death at a dose of 0.30 mg/kg. In contrast, the phospholipids of the outer cell membrane (phosphatidylcholine and phosphatidylinositol) caused minimal toxicity in vitro and none in vivo.

Toxicity of some charged lipids used in liposome preparations.

The inhibition of 3H-thymidine incorporation into DNA of L1210 cells in culture by liposomes was used as an index of liposome toxicity. Inhibition of 3H-thymidine incorporation appeared to be dose dependent for some lipid compositions tested. The commonly used neutral lipids, phosphatidylcholine and cholesterol did not appear to inhibit 3H-thymidine incorporation. Phosphatidic acid, an adjunct for preparing anionic liposomes, appeared to be non-toxic compared to phosphatidylserine and dicetylphosphate (alternative adjuncts for preparing anionic liposomes). Stearylamine, a synthetic lipid which continued to dominate the preparation of cationic liposome was the most toxic of the lipids tested.