Ilana Milo-Goldzweig

Trichothecene-induced hemolysis: I. The hemolytic activity of T-2 toxin

In the present investigation, the hemolytic properties of T-2 toxin were examined. Tests with different concentrations of T-2 toxin showed that essentially complete hemolysis of rat erythrocytes commenced after a lag period, the length of which depended on the concentration of toxin. Comparison of the characteristics of hemolysis caused by T-2 toxin, saponins, H2O2, and polyoxyethylene surfactants showed great similarity between T-2 toxin and the latter two which proceed by a free radical mechanism. The same mechanism was suggested for hemolysis caused by T-2 toxin on the basis of the additional following observations: (1) darkness inhibited hemolysis; (2) specific free radical scavengers, i.e., vitamin E, mannitol, and histidine, inhibited hemolysis caused by T-2 toxin.

On the mechanism of saponin hemolysis—I: Hydrolysis of the glycosidic bond

Abstract The mechanism of saponin induced hemolysis was investigated by extracting the active hemolysing factor from ghost cells of saponin hemolysed blood. The fact that only the corresponding aglycones could be extracted, shows that hydrolysis of the glycosidic bond precedes hemolysis. It is suggested that lack of hemolytic activity in a saponin is due either to its non adsorbability to the red cell or to the lack of a proper membrane glycosidase necessary for hydrolysing the glycosidic bond.

The susceptibility of cholesterol-depleted erythrocytes to saponin and sapogenin hemolysis.

The assumption that complex formation between erythrocyte membrane cholesterol and saponins or sapogenins is the cause for their hemolytic activity, was tested by measuring the susceptibility of cholesterol-depleted erythrocytes towards these hemolysins. For some of the hemolysins cholesterol depletion caused inhibition of hemolysis, for others an augmentation. The results suggest that cholesterol does not serve as a specific binding site for these hemolysins.

PADMA-28, a traditional tibetan herbal preparation inhibits the respiratory burst in human neutrophils, the killing of epithelial cells by mixtures of oxidants and pro-inflammatory agonists and peroxidation of lipids.

Both aqueous and methanolic fractions derived from the Tibetan preparation PADMA-28 (a mixture of 22 plants) used as an anti-atherosclerotic agent, and which is non-cytolytic to a variety of mammalian cells, were found to strongly inhibit (1) the killing of epithelial cells in culture induced by ‘cocktails’ comprising oxidants, membrane perforating agents and proteinases; (2) the generation of luminol-dependent chemiluminescence in human neutrophils stimulated by opsonized bacteria; (3) the peroxidation of intralipid (a preparation rich in phopholipids) induced in the presence of copper; and (4) the activity of neutrophil elastase. It is proposed that PADMA-28 might prove beneficial for the prevention of cell damage induced by synergism among pro-inflammatory agonists which is central in the initiation of tissue destruction in inflammatory and infectious conditions.

On the mechanism of saponin hemolysis. II. Inhibition of hemolysis by aldonolactones.

Abstract Saponin-induced hemolysis is inhibited by aldonolactones, which are specific glycosidase inhibitors. These results confirm our previous suggestion that hydrolysis of the glycosidic bonds of saponins is essential to their hemolytic activity. The aldonolactones tested exhibited little inhibitory specificity towards various saponins, suggesting the presence of non-specific glycosidases on the erythrocyte membrane.

Hemolysis caused by polyoxyethylene-derived surfactants. Evidence for peroxide participation.

The hemolytic properties of nonionic surfactants of the series CH3(CH2)15-17-O-(CH2CH2O)nCH2CH2OH were investigated and compared to those of saponins, sapogenins and H2O2. Antioxidants and anaerobic conditions were shown to inhibit the hemolysis, while glycyrrhizin was found to enhance it. Similar effects were obtained for H2O2 hemolysis, but not for saponin and sapogenin hemolysis. It is proposed that peroxides and free radicals are mainly responsible for the polyoxyethylene derived surfactants induced hemolysis.

Effect of ester groups on the haemolytic action of sapogenins—II: Esterification with bifunctional acids

Abstract The cause for the enhancement of haemolysing capacity of sapogenin esters was investigated. For this purpose the haemolysis induced by sapogenins esterified with an acid bearing an extra polar group (i.e. OH or COOH) was compared with that of the corresponding simple esters. It was shown that the presence of a free OH or COOH group in the acid part of the ester inhibits haemolysis. This inhibition may be overcome by acetylation or methylation of the OH or COOH group respectively. It appears that it is not the introduction of an ester group which enhances haemolysis but the removal of the polar group. Sapogenins bearing two polar groups, the esterification of one of them, no matter which, enhances haemolysis, whereas the blocking of the second group reduces this effect. It was further shown that neither the presence of an OH group nor that of any oxygen function at position 3 in the sapogenin is essential for haemolysis.

The hemolytic properties of non-ionic hemolysins

Abstract The hemolytic properties of various non-ionic hemolysins were examined. Equal hemolytic properties were observed for saponins, sapogenins, steroids, tri- and bicyclic compounds as well as for aliphatic compounds. All hemolysins proved to be irreversibly absorbed by the red cell membrane prior to hemolysis. It was underlined that while the structure of the carbon skeleton does not influence the hemolytic potency, the nature of the polar functions in the molecule is a decisive factor.

The hemolytic activity of citral: evidence for free radical participation

In the present investigation the hemolytic properties of citral were examined. Tests with different concentrations of citral showed that hemolysis of rat erythrocytes commenced after a lag period the length of which depended on the concentration of the hemolysin and tended to 100% hemolysis. Comparison of the characteristics of the hemolysis induced by high and low citral concentrations, indicated that two mechanisms are involved--a non specific steroid--terpenoid or glutathione depletion mechanism dominating at high citral concentrations and a free radical mechanism dominating at low citral concentrations. Experiments performed with various free radical scavengers indicate that 1O2 might be involved.

On the similarity of hemolysis induced by plant sapogenins and by neutral steroids

Abstract The hemolytic properties of a variety of neutral steroids has been investigated. Great similarity could be observed in their structural requirements as compared with those of the sapogenins. The following common factors were observed: esterification of an —OH group, or its oxidation to the respective ketone always enhances hemolysis. Hemolysis is only slightly influenced by either the A/B ring junction, or by the stereochemistry of the 3-substituent. Nevertheless the preferred structures for hemolysis are: 5α > 5β; 3α—OH > 3β—OH; 3β—acetoxy > 3α—acetoxy. It is suggested that sapogenin hemolysis should not be treated as a phenomenon specific to these compounds, since the same type of hemolysis is induced by a great variety of other compounds.