Scanning Electron Microscopy of Candida albicans After In Vitro Treatment with Miconazole

Miconazole, a potent antifungal agent, labilizes rat liver lysosomes. Its labilizing effect is followed by measuring the release of lysosomal hydrolases, namely, acid phosphatase, (-glucuronidase, and arylsulfatase A. The effect of miconazole is concentration dependent in the range of 10-5 to 1.2 x 10-4 M. However, at higher concentrations, miconazole inhibits enzyme release but does not inhibit enzyme activities per se. The effect of miconazole depends on the drug/ lysosome ratio and is influenced by the pH of the incubation media, being minimal at alkaline pH. Membrane-active drugs such as nystatin, 2-phenethylalcohol, hexachlorophene, and digitonin have been compared with miconazole for their lysosome-labilizing action. The effect of miconazole on the lysosomal membrane is confirmed by a decrease in turbidity of the lysosomal suspension.Miconazole {1-[2,4-dichloro-,8-(2,4-dichlorobenzyloxy)-phenethyl]imidazole nitrate} has a broad spectrum of antimicrobial activity against pathogenic and nonpathogenic yeasts, dermatophytes, numerous saprophytic fungi, and gram-positive bacteria (13,25,31). Chemotherapeutic activity of miconazole as a topical applicant is well documented in the treatment of skin and nail infections and vaginal candidiasis (2,5,14,29,32). Studies on the mechanism of biological action of miconazole have been recently reported (9, 10, 28, 33; K. H. Sreedhara Swamy, M. Sirsi, and G. Ramananda Rao, Biochem. Pharmacol., in press). The drug induces leakage of intracellular materials from cells of Candida albicans (28), and at low concentrations it selectively inhibits the uptake of purines and glutamine into these cells (33). Electron microscopic examination of cells of C. albicans exposed to miconazole revealed that the earliest drug-induced alterations are seen at the plasma membrane (9, 10). Further, miconazole has been shown to induce hemolysis of mammalian erythrocytes and binds strongly to erythrocyte membrane lipoproteins (Sreedhara Swamy et al., in press). These investigations clearly indicate that the paramount feature of the biological action of miconazole is its interaction with cell membrane of sensitive organisms, resulting in the impairment of membrane function and eventually cell death.In an attempt to delineate the mode of action of miconazole on cellular and organellar membranes and to obtain a more comprehensive view of biological action, we have carried out studies on the interaction of miconazole with rat liver lysosomes. The present paper describes the effect of miconazole on the integrity of lysosomal membrane, providing further evidence that miconazole interacts with biological systems by impairing membrane function. MATERIALS AND METHODSChemicals. Miconazole nitrate was a gift sample from Ethnor Ltd., Bombay, India. Hexachlorophene, digitonin, 2-phenethylalcohol, p-nitrophenylphosphate, p-nitrocatechol sulfate, phenolphthalein-,-nglucuronide, Triton X-100, and tris(hydroxymethyl)-aminomethane (Trizma base) were purchased from Sigma Chemical Co., St. Louis, Mo. Nystatin was kin...

Section: Discussionsupporting

confidence: 59%

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confidence: 99%

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Mechanism of Action of Miconazole: Labilization of Rat Liver Lysosomes In Vitro by Miconazole

Swamy

Joshi

Rao

1976

“…At the ultrastructural level, C. albicans cells show changes in a dosedependent manner when grown in the presence of 10`8 to 10-M concentrations of miconazole. These changes range from slight alterations in the cell periphery and increase in the number of peroxisomes and lipid globules, to progressive cytoplasmic deterioration, prominent shape changes, and complete cellular necrosis (11,12; S. De Nollin, Ph.D thesis, University of Brussels, Brussels, Belgium, 1976). Recent investigations with scanning and transmission electron microscopy (12) on C. albicans cultures treated with a fungicidal dose of miconazole (10-4 M) have shown that the sequence of alterations differs totally from that obtained with lower-dose treatments.…”

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confidence: 99%

Cytochemical and Biochemical Studies of Yeasts After In Vitro Exposure to Miconazole

Nollin

Belle

Goossens

et al. 1977

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Yeast cells exposed to different doses of the antimycotic agent miconazole revealed important cytochemical changes in the topographic distribution of the phosphatases. A strong effect was observed on the behavior of oxidative and peroxidative enzymes. Decreased cytochrome c oxidase and peroxidase activity and increased catalase activity were seen after treatment with a fungistatic dose ofmiconazole, whereas a complete disappearance ofthese enzymes was observed after treatment with a minimal fungicidal dose of miconazole. This was in complete agreement with the quantitative biochemical data. A hypothesis is advanced concerning the possible involvement of peroxidase and catalase in the mechanism of action of this drug., is an imidazole nitrate that has demonstrated broad-spectrum activity against most pathogenic fungi and gram-positive bacteria (7,10,18,19,36). The studies on the uptake and utilization of substances in Candida albicans by Van den Bossche (37) indicated that miconazole induced changes in the permeability of the plasmalemma and the cell wall. At the ultrastructural level, C. albicans cells show changes in a dosedependent manner when grown in the presence of 10`8 to 10-M concentrations of miconazole. These changes range from slight alterations in the cell periphery and increase in the number of peroxisomes and lipid globules, to progressive cytoplasmic deterioration, prominent shape changes, and complete cellular necrosis (11, 12; S. De Nollin, Ph.D thesis, University of Brussels, Brussels, Belgium, 1976). Recent investigations with scanning and transmission electron microscopy (12) on C. albicans cultures treated with a fungicidal dose of miconazole (10-4 M) have shown that the sequence of alterations differs totally from that obtained with lower-dose treatments.The cytochemistry of untreated yeast cells has already been described by several authors (2, 3, 13, 22, 40; S. De Nollin, Ph.D. thesis, 1976). In earlier experiments (16, 13), we tried to optimize the preparatory conditions for the cytochemical examination of yeasts cells by using a modification of the conventional procedure for preservation. This yields, besides a good micromorphology of the cells, sufficient enzyme activities and allows an adequate penetration of substrates and captation ions during the incubation. For the biochemical studies, Van Belle, Goossens, and Van Roy (in preparation) have recently developed a method for obtaining adequate homogenization of the very rigid cells of yeast, thereby yielding reliable enzyme activities that are directly proportional to the number of cells (more than 99.9% of the cells are thoroughly homogenized). Using these modifications of the cytochemical and biochemical preparative techniques, we have tried in this study to evaluate the effects of miconazole on the activities and the distribution pattem of phosphohydrolases and of oxidative and peroxidative enzymes in C. albicans and Saccharomyces cerevisiae. Our particular aim was to see whether there was any correlation between the observed morph...

“…Miconazole and AmB exert their antifungal effect by alteration of fungal cell membrane permeability (4,8,20 Superoxide anion release assay. Superoxide anion (02-) was measured spectrophotometrically at a wavelength of 550 nm by the superoxide dismutase-inhibitable reduction of ferricytochrome c (24).…”

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Miconazole and amphotericin B alter polymorphonuclear leukocyte functions and membrane fluidity in similar fashions

Yasui

Masuda

Matsuoka

et al. 1988

decreased the membrane fluidity in a similar manner. These results demonstrate that miconazole and AmB alter selected in vitro membrane properties of human PMN.Amphotericin B (AmB) remains the drug of choice for most systemic fungal infections. The interaction of AmB with ergosterol in fungal cell membranes has been thoroughly studied (10, 14, 161. There are fewer studies of the interaction of AmB with human cell membranes.Most reported studies of the effect of AmB on human polymorphonuclear leukocytes (PMN) have been in vitro investigations (1,2,11,12). The clinical significance of the reported in vitro AmB-induced changes in PMN function is unclear. However, clinical reports of lethal pulmonary reactions after simultaneous administration of AmB and leukocyte transfusions in patients with profound neutropenia (22,23) are consistent with drug-induced alteration of PMN functions. A possible explanation for these pulmonary reactions is drug-induced alteration of the PMN membrane with subsequent PMN aggregation in the lungs leading to tissue damage (2, 11).Miconazole, an imidazole derivative, is an intravenous antifungal drug that is often used as an alternative to AmB. Miconazole and AmB exert their antifungal effect by alteration of fungal cell membrane permeability (4,8,20 Superoxide anion release assay. Superoxide anion (02-) was measured spectrophotometrically at a wavelength of 550 nm by the superoxide dismutase-inhibitable reduction of ferricytochrome c (24). The total volume of all reaction mixtures was 1 ml; they contained 100 pAl of PMN suspension (107/ml), 50 ,ul of 2 mM ferricytochrome c, and 25 ,ul of buffer (sample tube) or superoxide dismutase (reference tube). After the reaction mixture was temperature equilibrated