Nucleotides Through the Plasma Membrane of Human ... - Europe PMC

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Nov 5, 1974 - fibroblast membrane as evidenced by rapid leakage of both large and small ... ratorium, Stockholm, Sweden, Eagle medium was from Flow ...
Vol. 11, No. 4 Printed in U.S.A.

INFECTION AND IMMUNrrY, Apr. 1975, p. 640-648 Copyright i 1975 American Society for Microbiology

Determination of Toxin-Induced Leakage of Different-Size Nucleotides Through the Plasma Membrane of Human Diploid Fibroblasts MONICA THELESTAM* AND ROLAND MOLLBY Department of Bacteriology, Karolinska Institutet, S-104 01 Stockholm 60, Sweden Received for publication 5 November 1974

Human diploid lung fibroblasts were treated with cytolytic bacterial toxins and the nature of the membrane damage was investigated. [3H]uridine was used for differential labeling of cytoplasmic components of small or large molecular size. Two principal size categories were achieved by labeling the fibroblasts in either early growth phase or stationary phase, a high-molecular-weight ribonucleic acid label and a low-molecular-weight nucleotide label. The size of the labeled molecules was determined by perchloric acid precipitation and gel chromatography. Leakage of labeled molecules of different size indicated the size of the "functional pores" in the plasma membrane caused by the test substance. The nonionic detergent Triton X-100 produced large functional pores in the fibroblast membrane as evidenced by rapid leakage of both large and small labeled molecules. Theta-toxin from Clostridium perfringens and the polyene antibiotic filipin both gave rise to considerably smaller functional pores in the plasma membrane. Although small molecules easily passed the treated membrane, large molecules could not escape from the cells even after prolonged treatment with these substances or by increasing their concentration. By contrast, the leakage profiles obtained with melittin from bee venom or with delta-toxin from Staphylococcus aureus in each case suggested the formation initially of pores of intermediate size that increased upon prolonged incubation or when higher concentrations were used.

Cytotoxicity tests in general have suffered from the limitation of being only qualitative and based on subjective judgments concerning the degree of damage to the cell. Even more limited has been the possibility of estimating damage confined specifically to the cell cytoplasmic membrane. Conventional tests for in vitro cytotoxicity of most microbial protein toxins have involved microscopic observation of morphological changes in tissue culture cells. Furthermore, cell damage has been estimated by (i) measuring total protein, (ii) measuring the pH of the tissue culture medium, (iii) dye exclusion tests, and (iv) measuring certain enzyme activities (33, 40). In only a few studies with bacterial toxins has the leakage of isotopically labeled compounds through the cell membrane been followed (7, 42). In the field of immunology, however, more sophisticated methods have long been employed. Release of amino acids, protein, and nucleic acids after immune cytolysis was studied by Green et al. (10, 12). Leakage of 51Cr as a criterion of cell damage is now widely used in 64()

immunological studies (32, 47). Recently the release of low-molecular-weight substances such as adenosine 5'-triphosphate (16) and nicotinamide adenine dinucleotide (26) has been used as an index of plasma membrane damage. The principle that leakage of cytoplasmic material into the surrounding medium will indicate damage to the plasma membrane has been applied by us in two recent studies on the cytolytic effect of bacterial toxins and enzymes (28, 42). Release of [3H]uridine was used as a marker for membrane damage on human diploid fibroblasts. This technique provides information concerning permeability changes in the cell membrane under the influence of test substances, and it offers the following advantages: (i) high sensitivity and (ii) reproducible estimation of degree of the membrane damage. However, it gives little information about the nature of lesions leading to the permeability changes observed. The present investigation was undertaken to develop the assay system previously described.

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TOXIN-INDUCED NUCLEOTIDE LEAKAGE

The aim was to obtain further information on the kind of lesions caused by the cytolytic toxins tested by measuring the release of cytoplasmic constituents of different size. This paper presents a method for differential labeling of either

high-

or

low-molecular-weight (poly)nucleotides

in human diploid fibroblasts, and how release of these markers can be used to compare the effects caused by some cytolytic toxins. MATERLALS AND METHODS Chemicals. Hanks balanced salt solution and trypsin were obtained from Statens Bakteriologiska Laboratorium, Stockholm, Sweden, Eagle medium was from Flow Laboratories Ltd., Irvine, Scotland, [5'H Juridine (specific activity, 24 Ci/mmol) was from The Radiochemical Centre, Amersham, Bucks, England, and Aquasol TM Universal Cocktail was from New England Nuclear Chemicals GmbH, Frankfurt, West Germany. Sodium pyruvate, sodium borate, sodium chloride, lithium chloride, magnesium chloride, boric acid, trypan blue, uranyl acetate, perchloric acid, and tris(hydroxymethyl)aminomethane (Tris) were purchased from E. Merck, Darmstadt, West Germany. Transfer ribonucleic acid (RNA) (type IV), uridine, and the mono-, di-, and triphosphates of uridine were obtained from Sigma Chemical Co., St. Louis, Mo; yeast RNA (sodium salt, E2151) from Mann Research Laboratory, New York, N.Y.; dimethylsulfoxide from Mallinckrodt; Sephadex from Pharmacia, Uppsala, Sweden; and BioGel from BioRad Laboratory, Richmond, Calif. Unless otherwise stated all chemicals were of analytical grade. Toxic substances. Triton X-100 (technical grade) was purchased from Rohm and Haas Co., Philadelphia, Pa. Filipin complex was a kind gift from G. B. Whitfield, the Upjohn Co., Kalamazoo, Mich. (U-5956, ref 8393-DEG-11-18, crystalline complex 66% pure). Filipin was dissolved in ME2SO to 0.1 M stock solutions. Purified melittin, free from phospholipase activity, was kindly supplied by W. Vogt and P. G. Lankisch, Max-Planck Institute, Gottingen, Germany. Delta-toxin from Staphylococcus aureus was purified according to Kreger et al. (22). Theta-toxin from Clostridium perfringens was highly purified by methods similar to those reported for phospholipase C (29). The purity was confirmed by sodium dodecyl sulfate-polyacrylamide electrophoresis, crossed immunoelectrophoresis, and isoelectric focusing in gel (38; Mollby, unpublished data). Both toxins were shown to be devoid of lipase, phosphatase, protease, deoxyribonuclease, ribonuclease, hyaluronidase, glucosidase, and phospholipase activity when tested as described by Mollby et al. (27). Cultivation of cells. The basic methods have been described in detail by Thelestam et al. (42). Briefly, human diploid embryonic lung fibroblasts were grown in Eagle medium (8) supplemented with 10% calf serum, 5 mM glutamine, 1 mM sodium pyruvate and penicillin (100 IU/ml), and streptomycin (100 ,g/ml). Two cell strains were used with essentially similar results: WI-38 fibroblasts were obtained from L. Hayflick (14) and a similar strain was isolated and

641

described by Litwin (23). Monolayers were detached with 0.25% crude trypsin in Hanks balanced salt solution. Cells to be used for cytotoxicity tests were cultivated on polystyrene trays with a culture well area of 7 cm2 (FB-6TC, Linbro Chemicals Co., Inc., New Haven, Conn.). Seeding density was 90,000 cells per culture in 3 ml of medium. The cultures were incubated at 37 C in a humid atmosphere containing 5% CO2-95% air. After 5 to 7 days monolayers had developed. These contained 0.6 to 0.7 x 106 cells per culture well. Labeling of cells. (i) Nucleotide labeling. Confluent monolayer cultures (day 6 to 7 after seeding, i.e., in stationary growth phase) were labeled with [3H luridine (1 ACi/ml) in Eagle medium at 37 C. The labeling period of 2 h was followed by a 2-h chase with nonradioactive medium. Three rinses with Hanks balanced salt solution preceded the test. (ii) RNA labeling. Twenty-four hours after the cells had been seeded, cultures (in early growth phase) were labeled with [3H]uridine during the next 24 h (1 MCi/ml). After the labeling period incubation was continued in nonradioactive medium for 3 to 5 days until complete monolayers had developed. The cultures were rinsed three times with Hanks balanced salt solution before the test. Autoradiography according to Litwin and Thelestam (24) indicated that all the cells were uniformly labeled regardless of which method had been used. Likewise all cells appeared equally affected after incubation with active test substance, showing a uniform loss of their cytoplasmic grains. Maximal release. The maximal release of cytoplasmic radioactivity was determined as earlier described by Thelestam et al. (42). Briefly, the cells were lysed by disintegrating the cell membranes in a 0.06 M sodium borate buffer (pH 7.8) with a rubber policeman. Nuclei and cellular debris were removed by centrifugation (1,000 x g, 10 min, 4 C). The supernatant is referred to in the text as "cell lysate"; the radioactivity in the lysate was determined. This value was multiplied with a factor 1.25 to give the maximal release (25). Nuclear radioactivity was measured after dissolving the washed nuclear pellet in 1 M NaOH and neutralizing with concentrated HCl. Measurement of radioactivity. Samples of 0.1 ml were transferred to 10 ml of Aquasol in scintillation vials (Nuclear Chicago) and counted for 1 or 4 min in a Nuclear Chicago liquid scintillator. No significant quenching was observed in the solutions used. Estimation of size category of labeled material. Size category of labeled material in cell lysates was estimated by the following methods. (i) Perchloric acid precipitation. Cold perchloric acid (0.5 ml) 25% (wt/vol) containing 0.75% (wt/vol) uranylacetate, was added to a 1-ml sample in which 200 gg of carrier yeast RNA had been included (19). The precipitate was collected by centrifugation at 2,000 x g for 10 min at 4 C and hydrolyzed in boiling 25% perchloric acid for 30 min. Radioactivity in the hydrolyzed precipitate was considered as representing RNA. The nonprecipitable radioactivity represented acid-soluble nucleotides. (ii) Gel chromatography. Cell lysate or leaked

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INFECT. IMMUN.

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material was subjected to gel chromatography on Sephadex G-25 or BioGel P-60. The gels were equilibrated in 0.01 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, pH 7.6, containing 0.14 M LiCl and 1 mM MgCl2 (34). The Sephadex G-25 column was 2.5 by 50 cm, the flow rate was 2.7 ml/h x cm2, and 5-ml fractions were collected. The BioGel P-60 column was 2.5 by 120 cm, the flow rate was 3 ml/h x cm2, and 6-ml fractions were collected. Radioactivity was determined in 0.1-ml samples of each fraction. Toxin treatment of cells. Labeled cultures were incubated with test substance diluted in Tris-buffered saline. If not otherwise stated, incubation was performed at 37 C for 30 min. After this time the incubation mixture was carefully sucked off and centrifuged (1,000 x g, 10 min, 4 C). Radioactivity was measured in the supematant. Released radioactivity was expressed as percentage of maximal release, i.e., ([toxin-induced release - spontaneous release]/ [maximal release - spontaneous release]) x 100. The spontaneous release never exceeded 10% of the maximal release. The absolute value for maximal release was usually around 150,000 counts/min per culture. All tests were performed in duplicate or triplicate. The toxin-treated cultures were microscopically observed for visible signs of damage. Assay of hemolytic activity. Hemolytic activity was assayed by incubating washed erythrocytes (final concentration 0.5%, vol/vol) with twofold dilutions of the hemolysin in Tris-buffered saline. Hemolysis was estimated visually after 1 h at 37 C and finally after further incubation for 2 h at 4 C. The reciprocal of the dilution of the enzyme hemolyzing 50% of the erythrocytes indicated the number of hemolytic units (HU) per ml of undiluted sample. Delta-toxin was assayed on human erythrocytes (42) and theta-toxin with or without prior reduction on sheep erythrocytes (41).

RESULTS Optimization of labeling conditions. Labeling conditions either were designed so as to label (i) low-molecular-weight or (ii) high-molecularweight material with the use of [3H ]uridine. (i) To achieve a low-molecular-weight cytoplasmic label, the cells were labeled for 2 h in the stationary-growth phase. The leakage test was performed after a 2-h chase with nonradioactive medium. This procedure was chosen with regard to the following results (Fig. 1): the increase in absolute amount of incorporated activity per culture was parallel to the increase in cell number; and in the stationary-growth phase, the ratio of cytoplasmic to nuclear radioactivity reached a plateau, where the cytoplasmic radioactivity was about 1.5 times that of the nuclear. (ii) To achieve a high-molecular-weight cytoplasmic label, the cells were labeled for 24 h in early growth phase. The leakage test was performed after a nonradioactive chase for 3 to 5 days. This procedure was chosen with regard to

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FIG. 1. Optimization of nucleotide labeling. Cell cultures were grown in Eagle medium and each day the number of cells was estimated. At the same time a parallel set of cultures was labeled for2 h with 1 gCi of [3H]uridine per ml. Radioactive medium was replaced by fresh, nonradioactive medium and incubation was continued for another 2 h. After rinsing, the cells were lysed and the nuclei were separated by centrifugation. The pellet was washed once, dissolved in 1 M NaOH, and neutralized before scintillation counting. Radioactivity in the supernatant is referred to as cytoplasmic counts per minute and in the pellet as nuclear counts per minute. Symbols: 0, Number of cells; 0, cytoplasmic counts per minute per cell; 0, ratio of cytoplasmic counts per minute to nuclear counts per minute. TABLE 1. Perchloric acid precipitation of cell lysates Type of label

% radioactivity soluble in cold perchloric acid

% radioactivity insoluble in cold perchloric acid

Nucleotide RNA

90 15

10 85

the following results: analysis on gel chromatography (Sephadex G-25) showed that the greatest relative amount of high-molecular-weight label was obtained when the cells were incubated with [3H]uridine on the first day after seeding. A labeling time of 6, 12, 18, and 24 h was investigated. The absolute amount of radioactivity taken up and retained by the cells increased with increasing labeling time. Molecular size of labeled material. Less than 15% of the radioactivity in cell lysates from cells labeled according to (i) was precipitable by cold perchloric acid, whereas about 85% of the corresponding (ii) label was insoluble (Table 1). Cell lysates were chromatographed on a Sephadex G-25 column to resolve the labeled material into fractions of different molecular sizes. Material labeled in stationary phase was distributed into two peaks (Fig. 2). The first peak probably represents phosphorylation products of uridine and polymers of varying degree.

TOXIN-INDUCED NUCLEOTIDE LEAKAGE

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through the cytoplasmic membrane. The isotope release was identical for RNA- and nucleotide-labeled cells (Fig. 4). On BioGel P-60, the released material gave similar elution profiles as was obtained with lysates from RNA- or nucleo-

The second peak was shown to coincide with the elution profile of uridine. By contrast, the material labeled in early growth phase was eluted with the void volume (Fig. 2). Cell lysates were also fractionated on BioGel P-60 columns (Fig. 3A). The material labeled in stationary phase was still eluted as low-molecular-weight substances. Also, the material labeled in early growth phase seemed to contain small amounts of low-molecular-weight substances. However, the majority of the labeled material was found in two peaks. On further chromatography on BioGel P-200, the material in the first peak appeared to have a molecular weight of >200,000. This peak will be referred to as ribosomal RNA. The second peak possessed an identical elution profile to commercially available transfer RNA(molecular weight, 25,000). For all subsequent tests, labeling was used to incorporate radioactivity into low-molecularweight or high-molecular-weight material, respectively. Cells labeled, as stated above, in the following will be referred to as nucleotide or RNA labeled. Toxin-induced leakage; dose response curves. Labeled cultures were incubated with the nonionic detergent Triton X-100. This treatment caused a leakage of radioactive material

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FIG. 2. Sephadex G-25 chromatography of cell lysates. Cell cultures were labeled by incubation with [3H]uridine (1 ACi/ml) for 24 h in early growth phase. This was followed by nonradioactive medium for 3 to 5 days. Parallel cultures were labeled as described for Fig. 1 on the day the experiment was performed. Cell lysates were chromatographed on Sephadex G-25. Radioactivity was measured in each fraction, background activity was subtracted from each value, and data were expressed as percentage of total recovered activity. Symbols: 0, Nucleotide label; A RNA label. C

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Kav x 100 FIG. 3. BioGel P-60 chromatography of cell lysates and material released with toxic substances. Cell lysates (A), prepared as for Fig. 2, were chromatographed on BioGel P-60. RNA- and nucleotide-labeled cultures were treated with Triton X-100 (0.05% vol/vol) (B), delta-toxin (1 HU/ml) (C), or theta-toxin (2 HU/ml) (D), respectively. After a 30-min incubation period at 37 C, the incubation media were removed, centrifuged, and chromatographed on BioGel P-60. Results were expressed as for Fig. 2. Symbols: A, RNA label; 0, nucleotide

label.

644

INFECT. IMMUN.

THELESTAM AND MOLLBY

Toxin-induced leakage; time-course relationships. The detergent Triton X-100 caused the RNA label to leak out as rapidly as m the nucleotide label (Fig. 7). E The release of RNA label caused by melittin and delta-toxin increased more slowly than the E nucleotide release, which was very rapid (Fig. 8). However, after 2 h the two curves representc ing release of RNA and nucleotide label approach each other. This may indicate either (i) that initially small lesions were enlarged during incubation or (ii) that upon prolonged incubation the high-molecular-weight material leaked Concentration (%, V/v) out slowly through lesions of constant size. FIG. 4. Dose-response curve with Triton X-100. Filipin and theta-toxin caused rapid release Release of radioactive substances from RNA- and of nucleotide label (Fig. 9). By contrast the nucleotide-labeled cultures treated with Triton X-100 release of RNA label increased only slowly, at the concentrations indicated. Arrow indicates when morphological changes, as seen in the light micro- reaching at most 40 to 50% of maximal release after incubation for 3 h. The material released scope, first appeared. Symbols: A, RNA label; 0, from RNA-labeled cells after incubation with nucleotide label. theta-toxin for 30 and 180 min, respectively, u

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tide-labeled cells (Fig. 3A and B). Consequently, the detergent did not degrade the RNA label before or after its release from the cells and this high-molecular-weight material passed the damaged membrane as easily as the labeled nucleotides. Treatment of labeled cultures with melittin and delta-toxin caused a lower relative release of RNA label than of nucleotide label in the concentrations used (Fig. 5), although the differences were greater at lower concentrations. BioGel P-60 chromatography of material released after delta-toxin treatment (Fig. 3C) indicated that only a small amount of the high-molecular-weight fraction of the RNA label was released from the cells. By contrast, filipin and theta-toxin induced a leakage of significantly lower amounts of RNA label than of nucleotide label, even at high concentrations (Fig. 6). The RNA label released with a high concentration of theta-toxin (2 HU/ml) was shown to consist of only transfer RNA and nucleotides when chromatographed on BioGel P-60 (Fig. 3D). The results imply that the substances tested gave rise to membrane lesions of different types. It is evident that theta-toxin and filipin produced lesions, which permitted leakage of only small molecules, as compared to the leakage of both small and large molecules after treatment with Triton X-100. The functional pores caused by Triton X-100 were thus of significantly greater dimensions than the ones caused by theta-toxin and filipin. Delta-toxin and melittin, on the other hand, appeared to produce lesions of an intermediate type.

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FIG. 6. Dose response curves with filipin and theta-toxin. Release of radioactive substances from RNA- and nucleotide-labeled cultures treated with filipin (A) and theta-toxin (B) at the concentrations indicated. Arrow indicates when morphological changes, as seen in the light microscope, first appeared. Symbols: A, RNA label; 0, nucleotide label.

TOXIN-INDUCED NUCLEOTIDE LEAKAGE

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rounded and transparent with extremely prominent nucleoli. In spite of these rather severe effects the cell monolayers were not broken and the typical macroscopical array pattern was preserved. were

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DISCUSSION The technique presented in this study is based on the assumption that leakage of cytoplasmic material can be taken as a criterion of plasma membrane damage. It is furthermore assumed that the molecular weight (i.e., size) of (min) the released material may be regarded as indicincuFIG. 7. Effect of Triton X-100 in relation bation time. Release of radioactive substances from ative of the relative size of the membrane E

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180

time

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RNA- and nucleotide-labeled cultures treated with Triton X-100 (0.025% vol/vol) for the time periods indicated. Arrow indicates when morphological changes, as seen in the light microscope, first appeared. Symbols: A, RNA label; 0, nucleotide label.

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FIG. 10. BioGel P-60 chromatography of material released with theta-toxin after incubation for 30 and 180 min, respectively. RNA-labeled cultures were incubated with theta-toxin (1 HU/ml) for (A) 30 and (B) 180 min, respectively. The incubation media were removed, centrifuged, and chromatographed on BioGel P-60. hfesults were expressed as for Fig. 3.

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THELESTAM AND MOLLBY

lesions produced. The same basic assumptions underlie several other studies using cytoplasmic leakage as an indicator of cytotoxicity. Such investigations were carried out by Green et al. (10, 12), who studied complement-induced immune lysis of Krebs ascites tumor cells. Cell membrane lesions were characterized on basis of the molecular size of the released material. In 1961, Goodman (11) introduced the use of 5'Cr as a cytoplasmic marker for mune lysis of Ehrlich ascites

the study of imtumor cells. Wig-

zell (46) later used 5'Cr to standardize a cytotoxicity test, which has been used and further developed in several studies (32). However, as 5'Cr becomes nonspecifically bound to intracellular proteins of different sizes, the release of this label gives little information on the degree of membrane damage (4, 16). Recent studies on mouse and chicken embryo fibroblasts have indicated a reduced rate of uridine transport into density-inhibited cells (45). In the present study, however, uptake of labeled uridine into the cytoplasm of fibroblasts in stationary phase was as efficient as uptake in log phase (Fig. 1). However, its incorporation into macromolecules was substantially slowed down under the conditions used. For this reason, nucleotide labeling resulted in only a low-molecular-weight cytoplasmic label with uridine (molecular weight, 244) as the smallest component (Fig. 2). Thus, measurement of membrane damage by release of nucleotide label should be at least as sensitive a method as the release of adenosine 5'-triphosphate or nicotinamide adenine dinucleotide (16, 26). Other sensitive methods have been obtained by measuring release of potassium or rubidium ions (7, 9) or release of non-metabolizable amino acids (Thelestam, unpublished data). Ribosomal RNA turns over in the growing fibroblast considerably more slowly than the other RNA species (1, 5). This fact constituted the basic principle for using the same precursor to label big molecules by incorporating [3H ]uridine in early growth phase. Perchloric acid precipitation and gel chromatography showed that this hypothesis was indeed valid.Figure 3 shows that the RNA label consisted of both ribosomal RNA (molecular weight, > 200,000) and transfer RNA (molecular weight, 25,000). Triton X-100 rapidly gave rise to large membrane lesions permitting the high-molecularweight RNA label to escape from the cells as easily as the nucleotide label. The result is in accordance with the current concept of the mechanism of Triton X-100 solubilization of biological membranes. Simons et al. (37) showed that this detergent interacts with the

INFECT. IMMUN.

hydrophobic lipid-protein bonds in the plasma membrane forming soluble protein-detergent complexes. It is noteworthy, however, that the cells were not solubilized, although 90% of the radioactivity had leaked out. They were highly granular but still remained attached to the plastic surface and retained the macroscopic array pattern typical for fibroblasts. In a previous investigation using nucleotidelabeled cells (42), the cytotoxic effects of deltatoxin closely resembled the effects obtained with Triton X-100. Both substances caused a rapid and temperature-independent nucleotide release. It was thus suggested that delta-toxin had a detergent-like effect, which was in agreement with earlier investigations on the surfactant properties of this toxin (2, 3, 20, 22). However, the refined technique presented here allowed us to distinguish the effect of the detergent from that of the toxin. The latter caused a slower release of the large molecules, whereas the lesions produced by Triton X-100 permitted immediate leakage of the RNA label. However, prolonged incubation or increased concentrations of the toxin resulted in the same final effects as found with Triton X-100. It was of interest to compare the effects of Triton X-100 and delta-toxin with those of the better characterized polypeptide melittin, the principal toxin in bee venom. Melittin has been shown to be surface active (13, 35) and to interfere with the nonpolar fatty acids of the membrane phospholipids (48). As seen from Fig. 5 and 8, the lesions obtained with melittin are of the same size category as those obtained with delta-toxin, i.e., initially smaller than the ones produced by Triton X-100. Also, a recent study on spin-labeled membranes showed that the influence of this polypeptide on the physical state of the membranes investigated was not comparable with that of Triton X-100 (15). The leakage profiles produced with filipin or theta-toxin were clearly different from those caused by Triton X-100. They also differed from those of delta-toxin and melittin, as the functional pores obtained with filipin and thetatoxin did not grow larger with prolonged incubation or increased concentrations. Nevertheless the morphological effects with these substances were as severe as those found with Triton X-100. Filipin is the best characterized member of the group of polyene antibiotics. Its effects on a variety of different natural and artificial membranes has been studied (21, 30, 31, 43, 44). It is now well established that filipin (as probably all other polyene antibiotics) reacts with membrane sterols in such a way that the interaction between the sterol and lecithin is weakened (30,

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31). In this way functional pores are created, permitting leakage of cytoplasmic constituents through the otherwise undamaged membrane. In preliminary studies with amphotericin B and nystatin (unpublished data), we found that these polyenes also gave rise to the same kind of leakage pattern as was seen after filipin treatment of human diploid cells. Theta-toxin belongs to the group of oxygenlabile hemolysins, of which streptolysin 0 is best characterized (2, 3, 38). The latter is known to exert its cytolytic effect by interaction with membrane cholesterol in a manner similar to the action of filipin (3, 36). Also, theta-toxin has recently been shown to react with cholesterol resulting in the formation of arc-shaped structures (39). The present findings give further support of the interaction of theta-toxin with membrane cholesterol, as the leakage profiles of theta-toxin and filipin are very similar. Furthermore, cereolysin, another member of this group, was shown to affect the cells in an identical way (Thelestam, unpublished data). The fibroblast plasma membrane could thus be damaged to different degrees by different cytotoxic agents. The detergent Triton X-100, which acts by solubilizing the membrane components (primarily the proteins), gives rise to large functional pores. By contrast the agents, which interact specifically with the hydrophobic phospholipid-cholesterol bindings in the membrane, cause considerably smaller pores. Surface active proteins, interacting with the hydrophobic regions of the membrane, finally result in the same type of lesions as a detergent. Ringlike structures, "holes," of different sizes have been observed in the electron microscope after diverse treatments with cytolytic agents, in particular after complement induced lysis of erythrocytes (6, 17, 39). Several investigators claim that the observed ringlike structures are not transverse holes through the membrane but aggregates of the toxic agent and membrane components (18, 43, 44). Since the Stoke's radii of the nucleotide or the RNA label have not been determined in this investigation the actual diameter of the toxin-induced functional holes can not be compared to the holes described by other authors. Complement-induced lysis was not studied in the present system. It is more difficult to induce lysis by antibody-complement reaction on live fibroblasts than on erythrocytes or artificial membranes. This may be due to the fluidity and repair activities in the plasma membrane or the distribution of the surface antigens. A convenient method to characterize plasma membrane damage caused by cytolytic agents

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has been presented. Three different types of cytotoxic effects were distinguished with regard to their ability to induce membrane lesions of different sizes. Comparative studies of this method in relation to other known cytotoxicity tests will be published separately. ACKNOWLEDGMENTS We wish to thank T. Wadstrom and C. Smyth for their continuous interest in this work. The skillful technical assistance of G. Blomquist, M. Kjellgren, and L. Norenius is gratefully acknowledged. We are also greatly indebted to A. W. Bernheimer, A. Ehrnst, M. Gill, J. Litwin, E. Norrby, and A. Zetterberg for stimulating discussions and valuable criticism. This work was supported by the Swedish Medical Research Council (grant no. 16X-2562) and Karolinska Institutets Fonder. LITERATURE CITED 1. Abelson, H. T., L. F. Johnson, S. Penman, and H. Green. 1974. Changes in RNA in relation to growth of the fibroblast. II. The lifetime of mRNA, rRNA and tRNA in resting and growing cells. Cell 1:161-165. 2. Bernheimer, A. W. 1970. Cytolytic toxins of bacteria, p. 183-212. In S. J. Ajl, S. Kadis, and T. C. Montie (ed.), Microbial toxins, vol. 1. Academic Press Inc., New York. 3. Bernheimer, A. W. 1974. Interactions between membranes and cytolytic bacterial toxins. Biochim. Biophys. Acta 344:27-50. 4. Bunting, W. L., J. M. Kiely, and C. A. Owen. 1963. Radiochromium-labeled lymphocytes in the rat. Proc. Soc. Exp. Biol. 113:370-374. 5. Darnell, J. E. 1968. Ribonucleic acids from animal cells. Bacteriol. Rev. 32:262-290. 6. Dourmashkin, R. R., R. Hesketh, J. H. Humphrey, F. Medhurst, and S. N. Payne. 1972. Electron microscopic studies of the lesions in cell membranes caused by complement, p. 89-95. In D. G. Ingram (ed.), Biological activities of complement. S. Karger, Basel. 7. Duncan, J. L. 1974. Characteristics of streptolysin 0 hemolysis: kinetics of hemoglobin and '6rubidium release. Infect. Immun. 9:1022-1027. 8. Eagle, H. 1959. Amino acid metabolism in mammalian cell cultures. Science 130:432-437. 9. Gale, E. F. 1974. The release of potassium ions from Candida albicans in the presence of polyene antibiotics. J. Gen. Microbiol. 80:451-465. 10. Goldberg, B., and H. Green. 1960. Immune cytolysis. I. The release of ribonucleoprotein particles. II. Membrane-bounded structures arising during cell fragmentation. J. Biophys. Biochem. Cytol. 7:645-650. 11. Goodman, H. S. 1961. A general method for the quantitation of immune lysis. Nature (London) 190:269-270. 12. Green, H., R. A. Fleischer, P. Barrow, and B. Goldberg. 1959. The cytotoxic action of immune gamma globulin and complement on Krebs ascites tumor cells. J. Exp. Med. 109:511-521. 13. Habermann, E., and J. Jentsch. 1967. Sequenzanalyse des Melittins aus den tryptischen und peptischen Spaltstiicken. Hoppe-Seyler's Z. Physiol. Chem. 348:37-50. 14. Hayflick, L. 1965. The limited in vitro lifetime of human diploid cell strains. Exp. Cell Res. 37:614-636. 15. Hegner, D., U. Schummer, and G. H. Schnepel. 1973. The interaction of a lytic peptide, mellittin, with spin-labeled membranes. Biochim. Biophys. Acta 291:15-22. 16. Henney, C. S. 1973. Studies on the mechanism of

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