S Elberg

Role of cell defense against oxidative damage in the resistance of Candida albicans to the killing effect of amphotericin B.

A laboratory-derived mutant of Candida albicans B311 (L) and a clinical isolate (C) of C. albicans, both lacking membrane ergosterol, were less susceptible to amphotericin B (AmB)-induced cell membrane permeability to K+ and lethality than was the wild-type laboratory strain (B311) which contained ergosterol. The resistance of L and C to AmB-induced killing was much greater than the level of resistance to AmB-induced cell membrane permeability. L and C were also less susceptible to killing by H2O2 than was B311, and when treated with menadione, they each produced less H2O2 than did B311. In addition, their levels of catalase activity were 3.8-fold (L) and 2-fold (C) higher than that of B311. The ergosterol deficiency in L and C probably impaired AmB binding to the cells, thereby lowering AmB effectiveness as measured by both cell membrane permeability and killing. Resistance of strains L and C to oxidation-dependent damage likely contributed to a diminished response to AmB-induced lethality.

Involvement of oxidative damage in erythrocyte lysis induced by amphotericin B.

Lysis of human erythrocytes induced by amphotericin B was retarded when the oxygen tension of the incubation mixture was reduced or when the antioxidant catalase was added; lysis was accelerated when cells were preincubated with the prooxidant ascorbate. In the atmosphere of reduced oxygen tension, the erythrocytes containing carboxyhemoglobin lysed at a slower rate than did the cells containing oxyhemoglobin. Consistent with a role for oxidative damage in lysis, the mixture of erythrocytes and amphotericin B showed an increase in malonyldialdehyde, the product of peroxidation, which paralleled the progression of hemolysis. In contrast, the permeabilizing effect of amphotericin B, measured as a decrease in intracellular K+, was not affected by changes in oxygen tension, catalase, or ascorbate treatment. These results imply that oxidant damage is involved in the lytic, but not in the permeabilizing, action of amphotericin B.

Effects of the detergent sucrose monolaurate on binding of amphotericin B to sterols and its toxicity for cells.

Amphotericin B (AmB) is a potent antifungal agent used to treat patients with systemic mycoses. The cytotoxicity of AmB is related to its binding to membrane sterols and its clinical usefulness is based on its greater affinity to ergosterol, the fungal sterol, compared to the mammalian cell sterol, cholesterol (1-3). Here we report that sucrose monolaurate (L.S.) decreased the binding of AmB to cholesterol without interfering with its binding to ergosterol. Furthermore, the toxicity of AmB for mouse erythrocytes (RBC) and cultured mouse fibroblasts, L-929, cells was significantly decreased by low concentrations of L.S., whereas under the same conditions, its toxicity for Candida albicans was unaffected. We observed a very good correlation between the spectroscopic and cell studies. The results reported here on the effects of L.S. on the selectivity of AmB toxicity for fungal cells compared to animal cells and the relative nontoxic nature of sugar esters suggest a potential for compounds of this type to enhance the therapeutic index of AmB.

Stimulatory, permeabilizing, and toxic effects of amphotericin B on L cells.

High concentrations of amphotericin B (AmB) killed mouse L cells, but low concentrations increased plating efficiency and stimulated the incorporation of labeled precursors into DNA and RNA. Thus, there were two disparate effects of AmB on L cells, stimulatory and toxic, and they occurred in distinct dose-related stages. AmB also affected the permeability of L cells. In dose-response studies, increases in cell membrane permeability, measured as the loss of K+ ions, occurred along with the stimulation of [3H]uridine incorporation into RNA. In contrast, stimulation of [3H]thymidine incorporation into DNA was only observed in cells recuperating from AmB-induced permeability changes. When the K+ concentration in the medium was lowered to 0.5 from 4.5 mM, or when 1 mM ouabain was added to the cultures, cell killing was potentiated, but the stimulatory and permeabilizing effects of subtoxic concentrations of AmB were unaffected. Furthermore, etruscomycin, a polyene antibiotic without any permeabilizing effects, nevertheless induced an enhancement of plating efficiency and of incorporation of [3H]uridine into RNA and [3H]thymidine into DNA. Our results suggest that the dose-related stimulatory, permeabilizing, and toxic effects of AmB most probably have distinct mechanisms of action and may be independent of one another.

Amphotericin B incorporated into egg lecithin-bile salt mixed micelles: molecular and cellular aspects relevant to therapeutic efficacy in experimental mycoses.

The cellular activities of amphotericin B (AmB) used as Fungizone were compared with those of AmB complexed to either egg lecithin and glycocholate (Egam) or egg lecithin and deoxycholate (Edam). Under conditions in which leakage of K+ from erythrocytes and cultured L cells treated with Fungizone was almost complete, Egam and Edam containing concentrations of AmB severalfold greater than the concentration of AmB in Fungizone had no effect but retained the ability to decrease the level of retention of K+ in fungal cells. Analysis by absorption and circular dichroism spectroscopy demonstrated that when these formulations containing AmB at concentrations of less than 10(-5) M were added to buffer, the AmB dissociated slowly as monomers from Egam or Edam and dissociated rapidly as a mixture of monomers and self-associated species from Fungizone. We propose that in Egam and Edam, the absence of free AmB in the self-associated form reduces the toxicity of AmB to mammalian cells, whereas the presence of monomeric AmB results in the retention of the antifungal activities of these complexes.

Increase in colony-forming units of Candida albicans after treatment with polyene antibiotics.

Polyene antibiotics, at concentrations which do not cause detectable toxic effect, induce an increase in the number of colon-forming units of yeast cells of Candida albicans. This effect, which we attribute to an increase in plating efficiency, is probably caused by binding of the polyenes to fatty acids in the cell wall of fungi.

Influence of extracellular K+ or Mg2+ on the stages of the antifungal effects of amphotericin B and filipin.

The macrolide heptaene amphotericin B (AmB) induced concentration-dependent effects on Saccharomyces cerevisiae which were separable into two distinct stages. At low concentrations the drug inhibited the growth of the yeast and reversible changed cell permeability to Na+ and K+. At high levels it was lethal. The intracellular K+ concentration of cells with reversible damage (stage I) could be increased by addition of K+ to the medium, but cells irreversibly damaged (stage II) were not able to retain K+. The addition of K+ to the medium did not influence the growth-inhibitory or killing action of AmB. Addition of Mg2+ to cultures increased S. cerevisiae resistance to the killing effects of AmB. At low concentrations of AmB, growth inhibition was also decreased by extracellular Mg2+, but at higher concentration of AmB, growth inhibition was increased, probably because the prevention by Mg2+ of the lethal effect allowed expression of the inhibitory effect in a greater range. Simultaneous addition of K+ and Mg2+ markedly decreased both the inhibitory and lethal action of AmB at all concentrations. Filipin, a pentaene macrolide, had only lethal effects, which were unaffected when K+ was added to the medium but were diminished when medium was supplemented with Mg2+.

Inhibition of Amphotericin B (Fungizone) Toxicity to Cells by Egg Lecithin-Glycocholic Acid Mixed Micelles

Mixed micelles prepared from egg lecithin and the sodium salt of glycocholic acid markedly inhibited amphotericin B toxicity to mammalian cells without significantly affecting the antifungal effects of the drug.

Treatment of murine candidiasis and cryptococcosis with amphotericin B incorporated into egg lecithin-bile salt mixed micelles.

Amphotericin B (AmB) with deoxycholate (Fungizone) and AmB incorporated into mixed micelles (AmB-mixMs) composed of egg lecithin with glycocholate, deoxycholate, or taurocholate were compared as treatments for murine infections. For mice infected with Candida albicans, treatment consisted of a single intravenous injection; for mice infected with Cryptococcus neoformans, treatment consisted of two intravenous injections. The maximal tolerated doses of AmB as Fungizone were 1.25 mg/kg of body weight in mice with candidiasis and 2.5 mg/kg of body weight in mice with cryptococcosis. The AmB-mixMs were nontoxic to mice at doses of 80 and 100 mg/kg of body weight and were therapeutically more active than the maximal tolerated dose of Fungizone in both models of infection. However, when Fungizone or AmB-mixMs were administered at equivalent doses of AmB, AmB-mixMs were more active in treating murine candidiasis, whereas Fungizone was more active in treating murine cryptococcosis.

Interference with effects of amphotericin B on Candida albicans cells by 2-chloroethyl-1-nitrosoureas.

Two nitrosoureas, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) and 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), with strong carbamoylating and weak alkylating activities, interfered with the induction of potassium leakage and lethal action of amphotericin B (AmB) on Candida albicans. 2-Cyclohexyl isocyanate, the product of decomposition of CCNU, and 2-chloroethyl isocyanate, the product of decomposition of BCNU, also interfered with the anticandidal actions of AmB. In contrast, two nitrosoureas with weak carbamoylating and strong alkylating activities, 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperydyl)-1-nitrosourea and 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-glucopyranose, did not affect AmB action against C. albicans. These results indicate that the inhibitory action of CCNU and BCNU on the anticandidal effects of AmB is associated with the carbamoylating activity of these nitrosoureas.