Physicochemical cell damage in relation to lethal amphotericin B action

Abstract

A direct relationship between the concentration-dependent rate of amphotericin B-induced K+ release from Candida albicans and the concentration-dependent rate of killing by the drug was established. This relationship together with the observed rapidity of both release and killing action supports the conclusion that the lethal action of amphotericin B is primarily physicochemical in nature.There is abundant evidence to suggest that if amphotericin B is to kill susceptible fungal organisms, it must bind, via hydrophobic interaction, to ergosterol in the cell membrane (2,5,7, 10). This interaction presumably causes distortion and structural rearrangement to an extent that the membrane is destabilized and cannot function normally. Abnormal function is evidenced, for example, by efflux of cellular potassium ions (K+). The action is basically physicochemical in nature, and although it is generally considered to be the primary mode of amphotericin B action, it might not be the only mechanism involved or even the primary mechanism for that matter. Brajtburg and coworkers (2) have raised the possibility that the lethal action of amphotericin B involves something more than simply disturbance in membrane permeability. They found (2), as have others (1, 3), that K+ release per se is not a lethal event. In essence, their hypothesis is that subsequent to hydrophobic binding to ergosterol, amphotericin B undergoes autoxidation, with the production of reactive intermediates (e.g., free radicals and peroxides) that cause lethal oxidative damage to fungal cells. Since autoxidation of amphotericin B requires some significant period of time (8), inherent in this proposal is a series of time-dependent chemical changes preceding the lethal cell-damaging interaction. In this study the direct physicochemical cell membrane damage hypothesis has been examined further.Candida albicans 11651 was purchased from the American Type Culture Collection, Rockville, Md., and maintained on slants of Sabouraud dextrose agar (Difco Laboratories, Detroit, Mich.). Prior to each experiment, yeast cells were grown at 35 to 37°C with rotary shaking (150 rpm) in 20-ml volumes of a filter-sterilized (pH 7) synthetic broth containing 6.7 g of yeast nitrogen base (Difco), 1.5 g of L-asparagine, and 10 g of glucose in 1 liter of deionized water. Amphotericin B was purchased from Sigma Chemical Co., St. Louis, Mo., and working solutions were prepared in Me2SO at 100 times the final concentrations required for particular determinations.To prepare cells for an experiment, 4-ml volumes of a culture grown to early stationary phase (18 h) were diluted into flasks containing 16 ml of fresh medium and were incubated for 2 h with shaking at 35 to 37°C. Cells were collected by filtration, washed three times with several milliliters of saline, and suspended in 0.1 M NaCl (as recommended by the K+ electrode manufacturer, Orion Research) at -3 x 107 CFU/ ml. Forty milliliters of the suspension was then placed in either a 125-ml Erlenmeyer flask or a 100-ml beaker.In each o...

Antifungal activity of ibuprofen alone and in combination with fluconazole against Candida species

“…In support of this assumption, a high concentration of the metabolic inhibitor sodium azide induced K leakage at a much slower rate than ibuprofen. The results of the present study regarding the K ef¯ux induced by ibuprofen do not necessarily mean that the loss of intracellular K per se is responsible for the cidal action of the drug, as membrane disorganisation may result in multiple perturbations that would eventually lead to a lethal outcome [16].…”

“…At intervals, treated and untreated (control) suspensions were ®ltered through 0.45-ìm Millipore ®lters and the ®ltrates were assayed for K with a K -sensitive glass electrode connected to a Spotlyte analyser (Menarini Diagnostics). The results are represented as percentage of K leaked by cells boiled for 30 min [16,17]. The K ef¯ux was also analysed for Candida cells treated with 10 mM sodium azide for 5±30 min.…”

Salivary Histatin 5 and Human Neutrophil Defensin 1 Kill Candida albicans via Shared Pathways

“…The modes of action of currently used drugs in treatment of candidiasis, including polyene antimycotics and azole derivatives, have been attributed to their ability to alter yeast membrane permeability. Polyene antimycotics complex with ergosterol of the plasma membrane, resulting in a release of cellular potassium (3,4). The azole-based drugs inhibit the biosynthesis of ergosterol (42) and induce release of K ϩ and 260-nm-wavelength absorbing materials from C. albicans cells (41).…”

No evidence of oxidant events in amphotericin B cytotoxicity versusL. infantumpromastigotes

“…The pore forming polyene antibiotic amphotericin B, generally used to cure serious systemic fungal infections, has been shown efficient against various Leishmania species and successfully applied to the treatment of both cutaneous and visceral leishmaniasis (Croft et al, 1991;Davidson et al, 1994 et al, 1994;Ramos et al, 1996). However, experiments performed on unicellular fungi and mammalian cells have suggested that possible oxidative stress in relation to amphotericin B autooxidation could also complete amphotericin B toxicity, since protection against amphotericin B could be achieved by antioxidant compounds such as catalase and enhanced toxicity could be produce by prooxidants such as ascorbic acid (Sokol-Anderson et al, 1986, 1988Bratjburg et al, 1990).…”