Molecular Basis for the Selective Toxicity of Amphotericin B

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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1974, p. 377-382 Copyright 0 1974 American Society for Microbiology Vol. 5, No. 4 Printed in U.S.A. Molecular Basis for the Selective Toxicity of Amphotericin B for Yeast and Filipin for Animal Cells JANINA KOTLER-BRAJTBURG, H.

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1974, p Copyright American Society for Microbiology Vol. 5, No. 4 Printed in U.S.A. Molecular Basis for the Selective Toxicity of Amphotericin B for Yeast and Filipin for Animal Cells JANINA KOTLER-BRAJTBURG, H. D. PRICE, GERALD MEDOFF, DAVID SCHLESSINGER, AND GEORGE S. KOBAYASHI Departments of Medicine and Microbiology, Washington University School of Medicine, St. Louis, Missouri Received for publication 11 October 1973 Among the polyene antibiotics, many, like filipin, cannot be used clinically because they are toxic; amphotericin B, however, is useful in therapy of human fungal infections because it is less toxic. Both the toxicity of filipin and the therapeutic value of amphotericin B can be rationalized at the cellular and molecular level by the following observations: (i) these polyene antibiotics showed differential effects on cells; filipin was more potent in lysing human red blood cells, whereas amphotericin B was more potent in inhibiting yeast cell growth; and (ii) the effects of filipin were more efficiently inhibited by added cholesterol, the major membrane sterol in human cells, whereas the effects of amphotericin B were more efficiently inhibited by ergosterol, the major membrane sterol in yeast. The simplest inference is that the toxicity and effectiveness of polyenes are determined by their relative avidities for the predominant sterol in cell membranes. The effect of polyene antibiotics on cells is ergosterol in the fungal membrane than it does dependent upon the presence of sterols in the to the cholesterol in membranes of mammalian cell membrane (8, 10). At sufficiently high cells, whereas the opposite is true for filipin. concentrations, the polyenes cause a variety of Several previous studies support this notion toxic effects, including an increase in membrane permeability, leakage of cellular compo- show the expected differences (4, 5, 13). (14-16), but there are also others which do not nents, inhibition of macromolecular synthesis, To resolve these discrepancies, we performed and eventually death (see review in reference parallel studies using two polyene antibiotics, 6). Because all eukaryotic cells contain sterols, two cell types, and two sterols. The effects of the the therapeutic usefulness of the polyene antibiotics are limited by their toxicity to mam- and inhibition of yeast growth in the presence polyene antibiotics on lysis of red blood cells malian cells. Filipin, for example, is a polyene and absence of free cholesterol or ergosterol too toxic for clinical use. Amphotericin B were used as measures of the strength of sterolantibiotic binding. (AmB), however, is one of a few that is less toxic and is used clinically as an antifungal Our results indicate that the observed preferential toxicity of AmB for yeast cells compared agent in human infection. It is likely that the clinical efficacy of AmB is based on a relative with that observed for red blood cells is based on specificity for fungi. The following facts support this. (i) Yeast cells were found to be more membrane. In contrast. filipin is more toxic to greater avidity for the ergosterol in the fungal than 100 times as susceptible to AmB than red blood cells because of its greater avidity for were cultivated animal cells (9). (ii) AmB was cholesterol. found to be more potent against yeast cells (This paper was presented in part at the 73rd than filipin (15), whereas filipin was more Annual Meeting of the American Society for potent against red blood cells than was AmB Microbiology, 6-11 May 1973, Miami Beach, (7) Fla.) Ṡince fungal and mammalian cell membranes contain different sterols (1), a reasonable hypothesis to explain the different susceptibilities Chemicals. Filipin in the form of the unfrac- MATERIALS AND METHODS of fungal and animal cells to these two polyene tionated antibiotic complex, 86% pure, was donated antibiotics is that AmB binds more avidly to the by The Upjohn Co., Kalamazoo, Mich. AmB,

378 KOTLER-BRAJTBURG ET AL.,gg/mg, was donated by E. R. Squibb & Sons, Inc., New Brunswick, N.J. The polyenes were dissolved in dimethylformamide at a concentration of 1 mg/ml immediately before use.

2 378 KOTLER-BRAJTBURG ET AL.,gg/mg, was donated by E. R. Squibb & Sons, Inc., New Brunswick, N.J. The polyenes were dissolved in dimethylformamide at a concentration of 1 mg/ml immediately before use. To calculate the molarity of the polyenes, molecular weights of 654 and 924 were used for filipin and AmB, respectively. Cholesterol and ergosterol were purchased from Sigma Chemical Co., St. Louis, Mo. Stock solutions were prepared at a concentration of 1 mg per 2 ml of methanol and were stored at 4 C no longer than 5 days before use. Different amounts of this stock solution were transformed to aqueous assay solutions and dispersed on a Vortex mixer. To calculate the molarity of sterol in aqueous suspension, it was assumed that all of the sterol was suspended. Measurement of hemolysis. The effects of AmB and filipin were measured on red blood cells by determining the extent of hemolysis caused by the two polyenes in the presence and absence of the sterols. Ten milliliters of human blood obtained by venipuncture was immediately mixed with 0.2 ml of 0.2 M ethylenediaminetetraacetic acid in a conical tube and centrifuged (2,000 x g for 15 min). The pellet was resuspended and washed three times with 5 ml of cold isotonic saline buffered with 0.01 M sodium phosphate, ph 7.4. The red cells were then suspended in a volume of the buffer equal to the volume of the pellet, and the suspension was maintained at 4 C until used. Cells were always used on the day of preparation. The degree of hemolysis was determined by either of two methods. (i) Filipin-induced hemolysis as a function of time was determined by following the increase in transmittance at 675 nm in the assay tubes containing erythrocytes in saline buffer. (ii) Filipin- or AmB-induced hemolysis was monitored after removal of intact cells by centrifugation by measuring changes of absorbance caused by the hemoglobin released into solution. The transmittance and the absorbance were measured on a Coleman Junior spectrophotometer in cuvettes with a 1-cm pathlength. No lysis was observed in control tubes containing dimethylformamide and methanol alone at concentrations equivalent to those in the experimental tubes. The concentration of methanol in all tubes was 1%; the concentration of dimethylformamide was 1% or less. Tubes contained 2.5 ml of saline buffer, the polyene being tested with or without 12.8 IAM sterol. The exact number of red blood cells transferred from stock solution to the assay tubes was determined for each experiment as that amount which, after total hemolysis of samples of erythrocytes in 2.5 ml of hypotonic buffer (the isotonic buffer described above diluted 1:15 with water), gave an absorbance of 0.45 to Tubes were incubated for 30 min at 24 to 26 C before the erythrocytes were added. About 25 jsliters of erythrocyte stock suspension was used for the experiments with filipin, and 5 iliters was used for the experiments with AmB. For erythrocytes, incubations were carried out for 30 min at 24 to 26 C with filipin and for 1 h at 35 to 37 C with AmB. The tubes were then centrifuged at 2,000 x g for 15 min, and released hemoglobin was determined from the absorbance of the supematant solution. To ANTIMICROB. AG. CHEMOTHER. monitor the hemolysis conveniently for two different initial concentrations of erythrocytes, the absorbance was read at 520 nm for the filipin experiments and at 417 nm for the AmB experiments. These wavelengths were chosen to permit measurable hemolysis of two different initial concentrations of erythrocytes. The different conditions of the assays for filipin and AmB (amount of erythrocytes, time and temperature of incubation) were chosen to permit hemolysis by comparable concentrations of either polyene, which in turn permitted measurement of the inhibition of hemolysis by comparable concentrations of added sterols. Inhibition of growth of Saccharomyces cerevisiae. The effects of filipin and AmB on the growth of S. cerevisiae were measured by incubating the yeast with each of the polyenes in the presence or absence of one of the sterols. S. cerevisiae obtained from Hoffman-La Roche, Inc., Nutley, N.J., was maintained on Sabouraud dextrose agar and transferred weekly. An inoculum from the culture was transferred to 40 ml of CNB medium containing 0.16% yeast nitrogen base without amino acids, 0.5% (NHj),SO4, 1.0%/1 glucose, and 0.2% Casamino Acids. After 16 to 18 h of incubation at 28 C with shaking, 10 ml of the culture was diluted with 30 ml of fresh CNB medium and incubated for an additional 2 to 3 h. Hemocytometer and colony counts were done to determine the number of cells to be transferred to assay tubes. The counts presented in the results are hemocytometer counts, which were two to three times higher than the results obtained by colony counts. The growth experiments were performed in culture tubes (18 by 150 mm) capped with foam stoppers. The complete assay system contained 5 ml of CNB medium, cells, and polyenes with or without sterols. The concentrations of methanol and dimethylformamide were 1% or less. The control tubes contained medium, cells, and the corresponding amount of dimethylformamide and methanol. The reference tube contained only medium. The tubes were incubated with shaking at 28 C, and growth was quantitated by determination of the absorbancy at 450 nm in the Coleman Junior spectrophotometer. A special adapter made it possible to perform these measurements directly in the reaction tubes. The results were not corrected for nonlinearity of the measured turbidity versus cell number, and are expressed as percentage of growth inhibition. Full growth (100%) was defined as the difference in reading between a control culture grown in the absence of antibiotics and the same culture at zero times; thus, 100% growth inhibition represents no increase in turbidity from zero time readings. RESULTS Hemolytic action of filipin and amphotericin B and its inhibition by sterols. Polyene antibiotics induce the rapid lysis of mammalian erythrocytes in the presence of isotonic saline (7). By varying the cell number and the time and temperature of incubation, it was possible for each polyene to select conditions for which the range of effective concentra-

VOL. 5, 1974 tion of both were about the same, and for which lysis was abrupt above a relatively sharp threshold concentration. Under these conditions, hemolysis did not occur at less than 1.0 i 0.

3 VOL. 5, 1974 tion of both were about the same, and for which lysis was abrupt above a relatively sharp threshold concentration. Under these conditions, hemolysis did not occur at less than 1.0 i 0.3 ;M for filipin or less than MAM for AmB. Concentrations sufficient to give total lysis were 2.3 ± 0.4,uM for filipin and 3.2 ± 0.5 MM for AmB (represent mean ± SE, n = 3). Addition of appropriate concentrations of sterols to this assay system resulted in inhibition of hemolysis. The extent of protection depended on the exact experimental procedure. For example, protection was greater when polyenes were preincubated with sterol than it was when polyenes were added to a mixture of erythrocytes and sterols (Table 1). The hemolysis of erythrocytes induced by filipin was time dependent (Fig. 1), and cholesterol decreased the extent of hemolysis more effectively than did ergosterol. At higher concentrations of filipin, the inhibition was less pronounced but was still greater with cholesterol than ergosterol. The relative inhibition of hemolysis by sterols as a function of filipin concentration is shown in Fig. 2; the analogous experiment for AmB is shown in Fig. 3. At low concentrations of antibiotic, both sterols gave about the same protection; at concentrations of polyenes greater than twice that causing complete lysis, neither sterol was protective. However, at intermediate polyene concentrations, a difference in protection by the two sterols was clear. Cholesterol was more effective in inhibiting filipin-induced hemolysis than was ergosterol, whereas the reverse was true for AmB-induced hemolysis. In some experiments, only cholesterol could block the action of filipin, and only ergosterol could block the action of AmB. In others, the differences were not as pronounced, but were always present. TOXICITIES OF AMPHOTERICIN B AND FILIPIN 379 ci -I E c o 0 00D Time, minutes FIG. 1. Filipin-induced hemolysis as a function of time. (0) Filipin alone; (0) filipin preincubated with 12.8 MM cholesterol; (A) filipin preincubated with 12.8 gm ergosterol; (0) red blood cells alone. Each tube contained filipin at a final concentration 1.4 (A) or 2.8,uM (B). Erythrocytes were added at zero time, and the transmittance after complete hemolysis was 90%. 55 / / ergosterol 60 E sterol 20C ' /cholesterol -C O I Filipin concentration, (,um) FIG. 2. Inhibition of filipin-induced hemolysis by cholesterol and ergosterol as a function of filipin concentration. Erythrocytes were added to the saline buffer containing various concentrations of: untreated filipin (0); filipin preincubated with 12.8 MM cholesterol (0); filipin preincubated with 12.8 MMergosterol (A). After addition of the erythrocytes, the tubes were incubated for 30 min at 24 to 26 C and hemolysis was determined. TABLE 1. Filipin Sterol inhibition of filipin-induced hemolysis o stel Percent hemolysis Cholesterol Ergosterol Aa Bb Aa Bb a Sterol and filipin incubated for 30 min at room temperature before the addition of erythrocytes. b Erythrocytes and sterol incubated before the addition of filipin. Antifungal effects of the polyenes and the protection by sterol. Experiments were carried out with an initial inoculum of 3.2 x 106 to 3.6 x 101 cells of S. cerevisiae per ml in exponential growth. During 20 h of incubation, AmB at concentrations of 1.0 MuM and above gave complete inhibition of growth, whereas 2.8 gm filipin was required for the same effect. As in the hemolysis experiments, the effects of the polyene antibiotics were prevented by the presence of sterols. The results of a typical experiment are shown in Fig. 4. It is seen that cholesterol was more potent in preventing the effects of filipin, whereas ergosterol was more

380 KOTLER-BRAJTBURG ET AL. Amphotericin B concentration, (jum) FMa. 3. Inhibition of AmB-induced hemolysis by cholesterol and ergosterol as a function of AmB concentration.

4 380 KOTLER-BRAJTBURG ET AL. Amphotericin B concentration, (jum) FMa. 3. Inhibition of AmB-induced hemolysis by cholesterol and ergosterol as a function of AmB concentration. Conditions were 8imilar to those described in Fig. 2. The incubation after the addition of the erythrocytes was I h at 35 to 37 C. AmB (0); AmB preincubated with 12.8 pm cholesterol (0); AmB preincubated with 12.8 #M ergosterol (A). ANmwmtoB. Ar.. CmmoTHm. MM, and normal growth resulted. When the concentration of filipin was raised to 5.6 pm, cholesterol at 6.4 gm was protective, but ergosterol did not protect even at 12.8 pm. At 8.4 pm filipin, only cholesterol at a concentration of 12.8 pm protected. At 11.2 MM filipin, neither sterol was protective. AmB inhibited growth at 1.0 pm, and at this concentration its effect could be eliminated by both sterols at 6.4 MM. When the concentration of AmB was increased to 2.1 MM, only ergosterol protected. At a concentration of 4.2 MM, neither sterol protected. After 44 h, the inhibitory effects of the polyene were less pronounced and the organism grew to 80 and 60% of the control in the presence of 2.8 MM filipin and 1.0MuM AmB, respectively. The protective action of the sterols now extended to higher concentrations of the polyenes, but the selectivity remained unchanged. Filipin concentration, (um) Amphotericin B concentration, (pm) FIG. 4. Protective effects of cholesterol and ergosterol against the growth inhibition caused by different concentrations of filipin (A) and AmB (B). The initial inoculum of S. cerevisiae was 3.4 x 10' cells/ml, and the percentage of growth inhibition was calculated from the readings of the turbidity after 20h ofgrowth. Symbols: (0) no sterol; (O) 6.4 pm cholesterol; (U) 12.8 pm cholesterol; (A) 6.4 gm ergosterol; and (A) 12.8 um ergosterol. effective in antagonizing the effects of AmB. The action of 2.8MpM filipin was inhibited by the addition of either sterol at a concentration of 6.4 DISCUSSION The interaction of polyene antibiotics with sterol has been clearly demonstrated to be responsible for their effects. However, in spite of extensive work, there has been no attempt to compare the different toxicities of polyenes for various cell types and correlate this with their relative avidities for different sterols. By using artificial membrane preparations or yeast, some authors have concluded that filipin was always more potent than nystatin (4) and that the polyene antibiotics showed no detectable preference for ergosterol or cholesterol (13). Others have shown that membranes containing ergosterol responded to nystatin at much lower concentrations than did those containing cholesterol (3). Fluorometric studies have shown a preference of filipin for cholesterol as compared with ergosterol (14), but no marked preference of AmB for either sterol could be detected (2). Several biological studies which have used sterols to interfere with antifungal and hemolytic activities of polyene antibiotics also have had conflicting results. For example, Ghosh and Ghosh did not find any difference between ergosterol and cholesterol in protecting yeast against AmB and nystatin (5). Others (16) found that cholesterol more effectively protected against filipin than did ergosterol, whereas the effects of AmB and nystatin were better prevented by ergosterol. In the latter study, cholesterol was completely inactive against nystatin and only slightly active against

5 VOL. 5, 1974 AmB. Similar results of the sterols in preventing the antifungal effects of AmB were shown by Rebell (12), but in his study the difference in the protection given by cholesterol and ergosterol against AmB-induced hemolysis was insignificant. Our results can be summarized as follows. (i) We have confirmed that for both antibiotics filipin was more potent against red blood cells (7), and AmB was more potent against yeast cells (15). (ii) We have shown that the hemolytic and antifungal effects of filipin were more effectively blocked by ergosterol, whereas both effects of AmB were more effectively blocked by ergosterol. The specificity of the polyene antibiotics is obviously not complete. For example, the differential protective effects of the sterols were clearest at limiting concentrations of polyenes, whereas at the highest levels of polyenes, where unbound polyene would be in excess, neither sterol protected in the range tested. It is noteworthy that a higher molar ratio of sterol to polyene was required to protect erythrocytes as compared with yeast. This may only reflect the different experimental conditions of the two assays or the general strength of the two membranes; but it is also possible that the sterol in the erythrocyte membrane is more accessible to the polyene. The observation that the protective action of the added sterols was enhanced when polyenes and sterols were preincubated before the addition of red blood cells (Table 1) is consistent with the notion of complex formation between free sterols and antibiotics. The simplest interpretation of our results is that AmB-ergosterol interactions are stronger than those of AmB and cholesterol and that filipin-cholesterol interactions are stronger than those of filipin and ergosterol. This provides a rationalization for the toxicity of filipin and for the therapeutic value of AmB; the molecular basis for the cellular selectivity of the polyene antibiotics could reside in selectivity at the molecular level for the sterols. We do not believe that there are any strong conflicts between our results and those of others. However, in some cases, authors have used ranges of antibiotic concentrations which could have obscured differences, whereas in other cases only one system was used, so that correlation with relative biological effects was missed. Frequently the disagreements in interpretation in the literature are based on the use of different single systems rather than merely differences in experimental conditions. TOXICITIES OF AMPHOTERICIN B AND FILIPIN 381 In the clinical situation, two types of cells are treated with polyene antibiotics-the fungus and host cells. Thus, to understand relative toxicities of different polyenes and their basis in molecular interactions, at least two relevant cell types and at least two polyenes of different toxicity must be studied and compared. Our findings show that when this is done, an overall rationalization of relative toxicities and binding affinities becomes clear. ACKNOWLEDGMENTS This work was supported by a grant from the John A. Hartford Foundation, Inc. and Public Health Service grants AI and AI from the National Institute of Allergy and Infectious Diseases, and AM from the National Institute of Arthritis, Metabolism, and Digestive Diseases. LITERATURE CITED 1. Bishop, D. G The distribution and function of lipids in cells, p In A. R. Jonson and J. B. Davenport (ed.), Biochemistry and methodology of lipids. Wiley-Interscience, New York. 2. Bittman, R., and S. A. Fischkoff Fluorescence studies of the binding of the polyene antibiotics filipin III, amphotericin B, nystatin and lagosin to cholesterol. Proc. Nat. Acad. Sci. U.S.A. 69: Cass, A., A. Finkelstein, and V. Krespi The ion permeability induced in thin lipid membrane by the polyene antibiotics nystatin and amphotericin B. J. Gen. Physiol. 56: Demel, R. A., L. L. M. van Deenen, and S. C. Kinsky Penetration of lipid monolayers by polyene antibiotics. Correlation with selective toxicity and mode of action. J. Biol. Chem. 240: Ghosh, A., and J. J. Ghosh Effect of nystatin and amphotericin B on the growth of Candida albicans. Ann. Biochem. Exp. Med. 23: Hamilton-Miller, J. M. T Chemistry and biology of the polyene macrolide antibiotics. Bacteriol. Rev. 37: Kinsky, S. C Comparative responses of mammalian erythrocytes and microbial protoplast to polyene antibiotics and vitamin A. Arch. Biochem. Biophys. 102: Kinsky, S. C Membrane sterols and the selective toxicity of polyene antifungal antibiotics. Antimicrob. Ag. Chemother. 1963, p American Society for Microbiology, Ann Arbor, Michigan. 9. Kwan, C. N., G. Medoff, G. S. Kobayashi, D. Schlessinger, and H. J. Raskas Potentiation of antifungal effects of antibiotics by amphotericin B. Antimicrob. Ag. Chemother. 2: Lampen, J Amphotericin B and other polyenic antifungal antibiotics. Amer. J. Clin. Pathol. 52: Lampen, J. O., P. M. Arnow, Z. Borowska, and A. I. Laskin Location and role of sterol at nystatinbinding sites. J. Bacteriol. 84: Rebell, G Effect of lipoproteins on hemolytic and antifungal activity of amphotericin B and other polyene antibiotics. Antimicrob. Ag. Chemother. 1966, p American Society for Microbiology, Ann Arbor, Michigan. 13. Schlosser, E., and D. Gottlieb Sterols and the sensitivity of Pythium species to filipin. J. Bacteriol.

382 KOTLER-BRAJTBURG ET AL. 91:1080-1084. 14. Schroeder, F., J. F. Holland, and L. L. Bieber. 1972. Fluorometric investigation of the interaction of polyene antibiotics with sterols.

6 382 KOTLER-BRAJTBURG ET AL. 91: Schroeder, F., J. F. Holland, and L. L. Bieber Fluorometric investigation of the interaction of polyene antibiotics with sterols. Biochemistry 11: Zygmunt, W. A Intracellular loss of potassium in ANMMICROB. AG. CHEMOTHER. Candida albicans after exposure to polyene antifungal antibiotics. Appl. Microbiol. 14: Zygmunt, W. A., and P. A. Tavormina Steroid interference with antifungal activity of polyene antibiotics. Appl. Microbiol. 14:

Permeabilizing and Hemolytic Action of Large and Small

ANTIMICROBLAL AGENTS AND CHEMOTHERAPY, OCt. 198, p. 586-592 66-484/8/1-586/7$2./ Vol. 18, No. 4 Permeabilizing and Hemolytic Action of Large and Small Polyene Antibiotics on Human Erythrocytes JANINA BRAJTBURG,*