Search

Lantana (Lantana camara Linn.) is a noxious weed to which certain medicinal properties have been attributed, but its ingestion has been reported to be highly toxic to animals and humans, especially in the liver. The main hepatotoxin in lantana leaves is believed to be the pentacyclic triterpenoid lantadene A (LA), but the precise mechanism by which it induces hepatotoxicity has not yet been established. This work addressed the action of LA and its reduced derivative (RLA) on mitochondrial bioenergetics. At the concentration range tested (5-25 microM), RLA stimulated state-4 respiration, inhibited state-3 respiration, circumvented oligomycin-inhibited state-3 respiration, dissipated membrane potential and depleted ATP in a concentration-dependent manner. However, LA did not stimulate state-4 respiration, nor did it affect the other mitochondrial parameters to the extent of its reduced derivative. The lantadenes didn't inhibit the CCCP-uncoupled respiration but increased the ATPase activity of intact coupled mitochondria. The ATPase activity of intact uncoupled or disrupted mitochondria was not affected by the compounds. We propose, therefore, that RLA acts as a mitochondrial uncoupler of oxidative phosphorylation, a property that arises from the biotransformation (reduction) of LA, and LA acts in other mitochondrial membrane components rather than the ATP synthase affecting the mitochondrial bioenergetics. Such effects may account for the well-documented hepatoxicity of lantana., (2010 Elsevier Ltd. All rights reserved.)

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., (2010 Elsevier Ltd. All rights reserved.)

To approach the real haemolytic process of jellyfish toxins, both in vitro and in vivo haemolysis of tentacle-only extract (TOE) from jellyfish Cyanea capillata has been studied. Dose-response curves of the haemolytic activity of TOE in vitro were sigmoid shaped in both erythrocyte suspension and diluted whole blood, with the former more sensitive to TOE. The in vivo haemolysis increased sharply in the first 10 min and was followed by a gradual increase in the following 3h, with increasing blood potassium and lactic acid accordingly. SC5b-9 complexes were significantly up-regulated in vitro, but not in vivo. These results showed that the haemolysis of TOE in diluted whole blood and in vivo is not totally consistent with that in the erythrocyte suspension, and blood plasma might play a protective role against haemolysis. Thus we suggested that erythrocyte suspension can be used to test the damage of toxin on erythrocyte membrane, while the diluted whole blood may be more suitable to test the haemolysis of toxins., (Copyright 2010 Elsevier Ltd. All rights reserved.)

This study investigated the hemolytic and genotoxic effect of different organoselenium and organotellurium compounds in human blood cells, as simple tests for screening the toxicity of organochalcogenides. For osmotic fragility (OF) test, samples of total blood were incubated with the organochalcogens at 4, 8, 50, 75 and 100 microM or vehicle (DMSO) for 90 min at 37 degrees C. The EC(50) values for hemolysis were significantly increased in erythrocytes exposed to diphenyl selenide (II), diphenyl diselenide (III), diphenyl telluride (IV), diphenyl ditelluride (V), (S)-2-amino-1-diselenide-3-methylpropanyl (IX), butyl(styryl)telluride (XIII) and 2-(butyltellurium)furan (XIV) at higher concentrations tested. The exposure of erythrocytes to organochalcogens diphenyl diselenide (II) and butyl(styryl)telluride (XIII), which had greater hemolytic effect, did not modify catalase activity, reactive oxygen species (ROS) production and -SH content. On the other hand, Na(+)/K(+) ATPase activity of erythrocyte ghosts was significantly inhibited by the compounds diphenyl diselenide (II) and butyl(styryl)telluride (XIII) (P<0.05) in a concentration-dependent manner. The inhibition of Na(+)/K(+) ATPase activity was completely reversed by dithiothreitol (DTT); indicating reaction of these organochalcogens with thiol groups of the enzyme. The thiol oxidase activity of the compounds II and XIII was supported by the fact that the rate of DTT oxidation was increased significantly by both chalcogens. In the higher concentrations, the compounds (II) and (XIII) were strongly genotoxic and cytotoxic to human leukocytes cells, as verified by the DNA damage and cell viability evaluation. Our results suggest that at relatively high concentration organochalcogenides exhibit hemolytic and genotoxic action in human blood cells, which are probably linked to their thiol oxidase activity and preferential interaction with sulfhydryl groups critical to enzymes.

This work analyzed the effects of the aqueous crude extracts of Artemisia absinthium L., Lippia sp., Bryophyllum sp., Solidago microglossa DC, Cymbopogon citratus DC and Mentha x villosa HUDSON on the osmotic stability of human erythrocytes. Hemolysis was monitored by measurement of absorbance at 540 nm following addition of erythrocytes to NaCl solutions of varying concentration. Absorbance was fitted to sigmoid regression curves given by the Boltzmann equation, and hemolysis was characterized by the NaCl concentration leading to lysis of 50% of cells (H(50)), and by the intensity (H) and the amplitude (dS) of the lysis effect. The parameters were determined in the absence and presence of the crude extracts. The extracts of Artemisia absinthium, Lippia sp., C. citratus and M. villosa protected human erythrocytes against hypotonic shock, as evidenced by a decrease in the values of H and H(50) compared to the control solution (p<0.05). The extracts of Bryophyllum sp. and S. microglossa enhanced hemolysis, since their H(50) values were higher than in the control group (p<0.05), but they also showed protective effects, as evidenced by a decrease in H and an increase in dS.

Hemostatic disturbances are frequent findings in human accidents caused by Lonomia obliqua caterpillars in the southern region of Brazil. In severe envenomation, patients may present life-threatening bleedings. Such disturbances may be mimicked in rats, which also develop intravascular hemolysis. The scope of this study was to investigate the time-course and intensity of intravascular hemolysis induced by i.d. injection of 750 microg/kg crude L. obliqua bristle extract in rats. Total blood cell count, reticulocyte count, plasma hemoglobin and haptoglobin assays were performed in control and envenomed rats at 1, 6, 24 and 48 h after envenomation. Rats presented a drastic drop of haptoglobin levels at 1 and 6h with increased plasma hemoglobin levels, a decrease in packed cell volume values at 6, 24 and 48 h, and increased reticulocyte counts throughout after envenomation. Such observations indicated that intravascular hemolysis occurred as early as 1h following envenomation, and lasted for more than 6h. Intravascular hemolysis is probably induced by phospholipase A(2) and other proteins with direct hemolytic activity present in crude caterpillar bristle extract.

4-Hydroxytamoxifen (OHTAM) is the most active metabolite of the widely prescribed anticancer drug tamoxifen (TAM) used in breast cancer therapy. This work describes the effects of OHTAM on isolated human erythrocytes, using standardized test conditions, to check for a putative contribution to the TAM-induced hemolysis and to study basic mechanisms involved in the interaction of OHTAM with cell membranes. Incubation of isolated human erythrocytes with relatively high concentrations of OHTAM results in a concentration-dependent hemolysis, its hemolytic effect being about one-third of that induced by TAM. OHTAM-induced hemolysis is prevented by either alpha-tocopherol (alpha-T) or alpha-tocopherol acetate (alpha-TAc) and it occurs in the absence of oxygen consumption and hemoglobin oxidation, ruling out the oxidative damage of erythrocytes. However, OHTAM remarkably increases the osmotic fragility of erythrocytes, increasing the susceptibility of erythrocytes to hypotonic lysis. Additionally, the hemoglobin release induced by OHTAM is preceded by a rapid efflux of intracellular K(+). Therefore, our data suggest that OHTAM-induced hemolysis does not contribute to TAM-induced hemolytic anemia and it is a much weaker toxic drug as compared with TAM. Moreover, at variance with the membrane disrupting effects of TAM, OHTAM promotes perturbation of the membrane's backbone region due to its strong binding to proteins with consequent formation of membrane paths of permeability to small solutes and retention of large solutes like hemoglobin, followed by osmotic swelling and cell lysis. The prevention of OHTAM-induced hemolysis by alpha-T and alpha-TAc is probably committed to the permeability sealing resulting from structural stabilization of membrane.

1. Oxidative stress by phenazine methosulfate stimulated proteolysis in erythrocytes. 2. Gamma-irradiation of erythrocytes in the range of 50-1000 Gy also resulted in the induction of proteolysis. 3. Though it has been suggested that hyperthermia imposes an oxidative stress on a cell, hyperthermic exposure of erythrocytes (30 min, 39-49 degrees C) did not stimulate proteolysis during subsequent incubation of whole cells or hemolysates. 4. Proteolytic degradation of spectrin was accelerated during incubation of membranes isolated from cells heated above 45 degrees C but this effect seems to be due rather to thermal denaturation of spectrin than to oxidative modification of cellular proteins by hyperthermia.

Fish oil concentrates (Max EPA) were given without other diet modification for eight weeks to five insulin-dependent diabetics and five healthy volunteers, in order to determine their effect on possible in vitro indices of thrombosis. Cholesterol, HDL, LDL, fasting blood sugar, hemoglobin A1c, platelet count, and the osmotic fragility of red blood cells were not significantly changed from baseline values after eight weeks of fish oil consumption. Serum triglyceride levels were lowered by the fish oil (diabetics 130 +/- 23 to 89 +/- 26 mg/dl: normals 107 +/- 16 to 57 +/- 5 mg/dl). Nine out of ten subjects required more arachidonic acid to aggregate their platelets, and six out of ten required more collagen. Whole blood viscosity at low shear rates was increased in diabetics before the fish oil ingestion, and was reduced both in normals and in diabetics after eight weeks of treatment. Before fish oil administration, the diabetics had higher levels of von Willebrand Factor (vWF) (208 +/- 31%) than did controls (117 +/- 26%). There was a statistically significant decrease of serum von Willebrand Factor both in diabetics (p less than 0.01) and in normals (p less than 0.05) after six weeks of treatment. Analysis of the multimeric composition of the vWF indicated that the vWF molecule was not altered. Addition of eicosapentaenoic acid (EPA) or crude fish oil to human umbilical cord endothelial cell cultures did not change vWF levels in the supernatant. Whether these changes in platelet aggregation, whole blood viscosity and vWF can actually be translated into an in vivo amelioration of the vascular complications in diabetes remains to be determined in a carefully controlled clinical trial.

P. haemolytica leukotoxin caused rapid leakage of intracellular K+ (greater than 90% in 30 sec) from and cell swelling (approximately 100% increase in 15 min) of isolated bovine neutrophils. Incubation media made hypertonic by the addition of raffinose, dextran or inulin (carbohydrates with mol. wts of greater than or equal to 505) prevented leukotoxin-induced cell swelling, but not K+ leakage. Assuming that leukotoxin acts as a transmembrane molecular sieve, then the leukotoxin-induced functional transmembrane pore size in bovine neutrophil plasma membranes is slightly smaller than the molecular size of raffinose, i.e. 1.2 nm. Morphologically, leukotoxin caused bovine neutrophils to swell, lose their membrane ruffling, develop a finely porous surface, and form large plasma membrane defects. Exposure of neutrophils to leukotoxin caused slower (5-50 min) leakage of 80% of the cellular L-lactate: NAD oxidoreductase (E.C. 1.1.27, lactate dehydrogenase, LDH). Leukotoxin-induced K+ leakage and cell swelling developed in Ca2+-free medium, whereas leakage of lactate dehydrogenase develop only in medium containing Ca2+ and was inhibited by the addition of ethylene glycol-bis(B-aminoethyl ether)N,N,N1,N1-tetraacetic acid (EGTA). This sequence of leukotoxin-induced changes in neutrophils is compatible with the mechanism of action of pore-forming cytolysins.

We have previously shown that lysis of a nucleated mammalian cell requires several complement channels unlike lysis of erythrocytes and that this difference is due primarily to rapid elimination of channels from the plasma membrane. We have now investigated this problem further by studying the rate of channel elimination at low temp, the osmotic fragility of the cells, and the effectiveness of the membrane-associated ion pumps. When complement channels were formed for 3 min at 37 degrees C, followed by prolonged incubation at 2 degrees C, the C6 lytic dose-response curves indicated that a single channel was required for lysis of a cell, whereas multiple channels were required when the entire process was carried out at 30 degrees C. The shift from multi- to one-hit lytic behavior can be explained by the drastic reduction in the rate of channel elimination at low temp. C6 lytic dose-response curves with puromycin-treated cells were also found to display one-hit behavior, but, in this case, the rate of channel elimination was reduced only about 35-40% (which would not suffice to explain the one-hit lytic characteristics). However, cell death was more extensive for puromycin-treated cells than normal cells after incubation in buffers of low ionic strength, suggesting that an increase in osmotic fragility may be a contributing factor in the shift from multi- to one-hit behavior. Blocking of the membrane-associated Na+/K+-ATPases with ouabain did not affect the multi-channel requirement. Presumably, this means that the ion pumping rate does not significantly influence the number of channels required for lysis.