Boris Isomaa

Shape transformations induced by amphiphiles in erythrocytes

Shape alterations induced in human erythrocytes by cationic, anionic, zwitterionic and nonionic amphiphiles (C10-C16) at antihaemolytic concentrations (CAH50 and CAHmax) and at a slightly lytic concentration (2-10% haemolysis) were studied. Anionic (sodium alkyl sulphates) and zwitterionic amphiphiles (3-(alkyldimethylammonio)-1-propanesulfonates) proved to be potent echinocytogenic agents. Among the nonionic amphiphiles there were potent stomatocytogenicagents (octaethyleneglycol alkyl ethers, pentaethyleneglycol dodecyl ether), one potent echinocytogenic agent (dodecyl D-maltoside) and one weak echinocytogenic agent (decyl beta-D-glucopyranoside). Shape alterations induced by cationic amphiphiles (alkyltrimethylammonium bromides, cetylpyridinium chloride and dodecylamine hydrochloride) showed a strong time-dependence. These amphiphiles immediately induced strongly crenated erythrocytes which during incubation shifted to less crenated erythrocytes or to stomatocytes. All of the echinocytogenic amphiphiles induced echinocytes immediately, and there were only small alterations of the induced shape during incubation. Among the stomatocytogenic amphiphiles there were some that induced stomatocytes immediately or after a short lag time while others first passed the erythrocytes through echinocytic stages before stomatocytic shapes were attained. Erythrocytes treated with amphiphiles did not recover their normal discoid shape following repeated washing and reincubation for 1 h in amphiphile-free medium. Our study shows that shape alterations induced by amphiphiles in erythrocytes cannot be explained solely by assuming a selective intercalation of differently charged amphiphiles into the monolayers of the lipid bilayer as suggested in the bilayer couple hypothesis (Sheetz, M.P. and Singer, S.J. (1976) J. Cell Biol. 70, 247-251). We suggest that amphiphiles, when intercalated into the lipid bilayer, trigger a rapid formation of intrabilayer non-bilayer phases which protect the bilayer against a collapse and bring about a transbilayer redistribution of intercalated amphiphiles as well as of bilayer lipids.

Permeability alterations and antihaemolysis induced by amphiphiles in human erythrocytes

In an attempt to define the parameters in amphiphilic molecules important for their interaction with the erythrocyte membrane, the effects of cationic, anionic, zwitterionic and nonionic amphiphilic agents (C10-C16) on osmotic fragility and transport of potassium and phosphate in human erythrocytes were studied. All the amphiphiles protected the erythrocytes against hypotonic haemolysis. Half-maximum protection occurred at a concentration which was about 15% of that inducing 50% haemolysis. The concentrations of amphiphiles required to induce protection or haemolysis were related to the length of the alkyl chain in a way indicating that a membrane/aqueous phase partition is the mechanism whereby the amphiphile monomers intercalate into the membrane. At antihaemolytic concentrations all the amphiphiles increased potassium efflux and passive potassium influx. The increase in the fluxes was about the same in both directions through the membrane and there were no clear differences in the effects of the different amphiphilic derivatives at equi-protecting concentrations. Active potassium influx was decreased by cationic, zwitterionic and non-ionic amphiphiles. The ability of the amphiphiles to inhibit the influx was not related to the length of the alkyl chain. Anionic amphiphiles had no or only a weak stimulatory effect on the influx. Phosphate efflux was reduced by all the amphiphiles. The inhibitory potency of the different amphiphiles decreased in the following order; anionic greater than zwitterionic, non-ionic greater than cationic. Short-chained amphiphiles were more potent inhibitors than long-chained. The possible participation of non-bilayer phases (mixed inverted micelles) in the intercalation of amphiphiles into the membrane is discussed.

The acute toxicity of surfactants on fish cells, Daphnia magna and fish-a comparative study.

There is a need to replace acute toxicity tests on fish (LC(50)) with more cost effective assays. The main objective of this study was to explore whether gill epithelial cells, hepatocytes and Daphnia magna could be used to predict acute toxicity of surfactants on fish. The acute toxicity of 10 synthetic surfactants (anionic, cationic, nonionic and zwitterionic) and two resin acids were determined on hepatocytes and gill epithelial cells from rainbow trout (Oncorhynchus mykiss), on Daphnia magna and on fish. Cell viability was measured with the fluorescent viability probe calcein-AM, immobilization was determined for Daphnia and 24-hr LC(50) for rainbow trout. The EC(50) values for the cellular tests were clearly higher than the corresponding values for Daphnia and fish, indicating that the cellular tests with the endpoint used are less sensitive than whole organisms. A combination of the EC(50) values for Daphnia and freshly isolated gill epithelial cells in suspension showed, however, a good correlation with acute toxicity on fish (r(2)=0.91 and slope=1.09). The combination seems to be a promising in vitro alternative to acute toxicity tests on fish (LC(50)), but a more exhaustive comparison, including a broad spectrum of chemicals should be made before the predictive value of the combined in vitro test can be evaluated.

A comparison of the toxicity of 50 reference chemicals to freshly isolated rainbow trout hepatocytes and Daphnia magna

There is a need for rapid and cost-effective in vitro tests or test batteries in aquatic toxicology which could be used as tools in evaluating the toxicity of chemicals. In the present study the toxicity of 50 reference chemicals was evaluated by determining immobility in Daphnia magna (24 h incubation) and 86Rb-leakage in freshly isolated rainbow trout hepatocytes (3 h incubation). Regression analysis of the data on the EC50 for Daphnia and EC50 for the rubidium leakage in hepatocytes showed a correlation of 0.71 (p < 0.0001). The slope of the regression line (0.68) differed markedly from a 1:1 correlation. Daphnia was more sensitive to the chemicals than hepatocytes and the discrepancy between the two tests increased with increasing toxicity of the chemicals. A comparison of the data from the hepatocyte test with published data on the toxicity of the reference chemicals to cultured mammalian hepatocytes (24 h incubation), indicated that the lower sensitivity of the fish hepatocytes compared to Daphnia is not due to the shorter incubation time used in the hepatocyte test. A comparison of the data from the Daphnia test with published data on the toxicity of the reference chemicals to various mammalian cells, showed a similar discrepancy between the Daphnia test and the cellular tests as in the case of the comparison between the Daphnia test and the rainbow trout hepatocyte test, indicating that the Daphnia test in general is more sensitive than conventional cellular cytotoxicity tests. It is concluded that further studies, using more sensitive endpoints than rubidium leakage, are needed before it can be decided whether or not freshly isolated rainbow trout hepatocytes are suitable in routine toxicity testing.

Vesiculation induced by amphiphiles in erythrocytes

The ability of shape-transforming cationic, anionic, zwitterionic, and nonionic amphiphiles to induce vesiculation in human erythrocytes was studied. At concentrations where they exhibit maximum protection against hypotonic haemolysis (CAHmax) echinocytogenic amphiphiles induced a rapid release of exovesicles. Following 5 min of incubation, the vesicle release (acetylcholinesterase release) amounted from 4% (sodium alkyl sulphates) to 13% (zwittergents) of the total acetylcholinesterase activity of the erythrocytes. At concentrations corresponding to CAH50 the vesicle release was less than 15% of that released at CAHmax. The size and the appearance of the vesicles varied with the type of amphiphile. Stomatocytogenic amphiphiles which do not pass the erythrocytes through echinocytic stages, did not induce release of exovesicles. Electron and fluorescence microscopic observations of erythrocytes treated with stomatocytogenic amphiphiles strongly indicated that an endovesiculation had occurred. Amphiphiles which pass the erythrocytes through echinocytic stages before stomatocytic shapes are attained, induced a release of both exo- and endovesicles.

A comparison of the toxicity of 30 reference chemicals to Daphnia magna and Daphnia pulex

To determine whether significant differences exist in the sensitivity of different Daphnia species to toxicants, the acute toxicity of the first 30 MEIC (multicenter evaluation of in vitro cytotoxicity) reference chemicals was determined in two species of Daphnia: D. magna and D. pulex. Correlation and regression analysis of the EC50 data for immobilization showed a very good concordance (r = 0.97, slope = 1.02). A comparison between the EC50 data obtained for D. magna by two laboratories independently for the 50 MEIC chemicals also showed a good concordance (r = 0.93, slope = 0.91). In both comparisons the regression line did not differ significantly from the regression line for a 1:1 regression. The authors conclude that their study, including a set of reference chemicals, indicates that is no difference in the overall sensitivity of the two Daphnia species and the two clones of D. magna.

Morphological characterization of exovesicles and endovesicles released from human erythrocytes following treatment with amphiphiles

In order to morphologically characterize exo- and endovesicles released during treatment of erythrocytes with amphiphiles and to look for possible amphiphile-specific effects on the vesiculation pattern, human erythrocytes were treated at 37 degrees C with amphiphiles at concentrations where they exhibit maximum protection against hypotonic haemolysis (cAHmax). Released exo-and endovesicles and treated cells were studied by means of transmission (TEM) and scanning (SEM) electron microscopy. All sphero-echinocytogenic amphiphiles induced a release of both spherical and tubular exovesicles. Dodecyl maltoside, a nonionic amphiphile with a bulky polar head, induced a release of predominantly tubular exovesicles, while all other sphero-echinocytogenic amphiphiles induced a release of predominantly spherical exovesicles. Some branched tubular exovesicles were released by a double-chained cationic amphiphile. Tail- and tongue-like structures were often seen on the exovesicles. Spherical exovesicles were frequently invaginated. Stomatocytogenic amphiphiles induced endovesiculation. In erythrocytes treated with most of the stomatocytogenic amphiphiles the endovesicles were clustered, but with some amphiphiles the endovesicles were randomly distributed. Large ringformed endovesicles (octaethyleneglycol alkyl ethers) and endovesicles in chains (octyl and decyl glucopyranoside) also occurred. The endovesicle membrane was often budding onto the lumen of the vesicle and in some cases this could ultimately lead to a vesicle inside the endovesicle. We conclude that amphiphiles do not only trigger vesiculation, but may also specifically affect the vesiculation processes.

Cell selective cytotoxicity of a peptide toxin from the cyanobacterium Microcystis aeruginosa

The effects of a cyclic peptide toxin, isolated from the cyanobacterium Microcystis aeruginosa, on cell morphology and ion transport in human erythrocytes, isolated rat hepatocytes and mouse fibroblasts (3T3) were studied. Neither in erythrocytes nor in fibroblasts did the toxin cause morphological alterations. In hepatocytes the toxin induced marked morphological alterations at a concentration of about 50 nM. In erythrocytes and fibroblasts no effects on ion transport were observed. In hepatocytes the toxin induced a significant increase in both phosphate and potassium efflux at concentrations far below the concentration causing morphological alterations (0.1 and 1 nM, respectively). It is suggested that the cytotoxicity of the toxin is not due to a non-specific interaction with the plasma membrane and that the effects of the toxin in hepatocytes are probably due to an interaction of the toxin with cytoskeletal elements.

Amphiphile-induced antihaemolysis is not causally related to shape changes and vesiculation

A wide variety of structurally different antihaemolytic amphiphiles were tested for their ability to induce exovesiculation (acetylcholinesterase (AChE) release, transmission electron microscopic (TEM) studies), endovesiculation (fluorescein isothiocyanate conjugated dextran (FITC-dextran) internalization, TEM studies) and shape changes in human erythrocytes at concentrations where they exert maximum protection against hypotonic haemolysis. The results show that vesiculation is a common phenomenon induced by amphiphiles in erythrocytes. Sphero-echinocytogenic amphiphiles induced exovesiculation, whereas stomatocytogenic amphiphiles induced endovesiculation. The antihaemolytic potency of the amphiphiles was not related to their ability to induce exo- or endovesiculation, or to the type or extent of shape changes induced, and it could not be ascribed to any molecular feature of the amphiphiles or to their charge. It is proposed that amphiphiles, when intercalated into the lipid bilayer of the membrane, rapidly induce rearrangements within the bilayer and that these rearrangements are associated with an increase in the permeability of the membrane; it is suggested that a rapid efflux of ions decreases the difference in osmotic pressure between cell interior and hypotonic buffer, thereby protecting cells from being lysed.

Lipid and protein composition of exovesicles released from human erythrocytes following treatment with amphiphiles

Human erythrocytes were treated with different water-soluble amphiphiles (detergents) at sublytic concentrations, whereafter released exovesicles and treated cells were isolated. Lipid analyses showed that exovesicles had a lower cholesterol/phospholipid ratio and a higher phosphatidylserine/phospholipid ratio compared to parent cells. Protein analyses revealed that exovesicles were, relative to their total protein content, depleted in spectrin, actin and band 6 protein and enriched in band 3 protein and acetylcholinesterase. Exovesicles contained all major glycoproteins. By using a radiolabeled amphiphile ([14C]cetyltrimethylammonium bromide) it was shown that the amphiphile/phospholipid ratio was similar in the vesicle membrane and in the parent cell membrane. This indicates that no significant segregation of the intercalated amphiphile between the exovesicle membrane and the parent cell membrane occurs during the vesiculation process. It is suggested that the redistributions of membrane lipids and proteins during the vesiculation process are secondary to the detachment of the cytoskeletal network from the membrane.