1. Eglė Lastauskienė, Auksė Zinkevičienė*, Donaldas Čitavičius
Apoptosis in Candida spp. and Malassezia furfur induced by acetic acid and carbonates
Eglė Lastauskienė, Auksė Zinkevičienė*, Donaldas Čitavičius
Department of Microbiology and Biotechnology, Faculty of Natural Sciences, Vilnius University
*Centre of Diagnosis and Treatment of Allergic Diseases
Introduction
Apoptosis is highly regulated programmed cell death. During yeast cell apoptosis amino acids and peptides are released and can stimulate regeneration of human epithelium cells. Detection of chemical compounds inducing apoptosis in yeast and nontoxic for humans is of great medical relevance. Acetic acid is known as an inducer of apoptosis in yeast. It is known, that sodium bicarbonate and potassium carbonate have antifungal activity as well as week organic acids. However, the type of induced cell death of these substances in Candida and Malassezia yeast is unknown.
Results
Minimal inhibitory concentrations of tested chemical compounds are presented in table 1. Potassium carbonate, acetic acid and formic acid exhibited the most intense antifungal activity (Fig.1). Treatment of C. albicans and C. guilliermondii cells with 2.5% of sodium carbonate or 2.5% potassium carbonate stimulated yeast to hyphae switching (Fig. 2). Palmitic acid, oleic acid, potassium acetate, sodium acetate and ammonium acetate had no antifungal activity even at 10% concentration and weren’t used for further research.
Table1. Minimal inhibitory concentrations
Fig. 1. Viability of C. guilliermondii during growth in SC medium supplemented with MIC of analyzed compounds.
NaHCO3
K2CO3
Acetic acid
Formic acid
Citric acid
Litium acetate
C. albicans 5%
2.5%
0.25%
0.1% 8% 1%
C.guilliermondii
0.3%
0.2%
C. lusitaniae
M. furfur
For apoptosis analysis yeast cells were incubated with MIC of tested compounds for 2, 4 and 24 hours. Apoptosis analysis revealed, that formic acid induced apoptosis in more the 85% of yeast cells. Similar percentage of apoptotic cells were detected after treatment with acetic acid. MIC of sodium bicarbonate and potassium carbonate induced apoptosis in 75.88% and 55.18% of cells, respectively. All the tested compounds caused caspase-dependent apoptosis, except potassium carbonate. 2.5% of potassium carbonate induced apoptosis in caspase-independent way. Results of TUNEL and AnnexinV-Fluos staining used for apoptosis phenotype detection showed good correlation (Fig. 4, Fig. 5).
Flow-cytometry was used to analyze the influence of tested compounds on the growth of mammalian cell lines. Formic acid had the lowest effect on the survivability on Jurkat and NIH 3T3 cells (Fig. 6). Citric acid, litium acetate, sodium bicarbonate, potassium carbonate caused massive wave of cell death in mammalian cell lines.
Objective
The detection of chemical compounds inducing apoptosis in Candida spp. and M. furfur with the lowest toxicity to the mammalian cells.
Fig. 3. Hyphae formation in C. guilliermondii after treatment with 2.5% NaHCO3.
Materials and methods
C. guilliermondii, C. lusitaniae and M. furfur were isolated from the skin of patients with clinical diagnosis of atopic dermatitis. The minimal inhibitory concentrations (MIC) of acetic acid, sodium bicarbonate, potassium carbonate, citric acid, litium acetate, potassium acetate, sodium acetate, ammonium acetate, palmitic acid, oleic acid and formic acid, were determined by a microdilution method. Viability was assessed by using clonogenic assay. After exposure to tested agents markers of apoptosis were identified: appearance of phosphatidylserine in the outer layer of the cytoplasmic membrane was revealed by FITC-annexin V staining; DNA strands breaks and chromatin condensation were demonstrated by TUNEL assay; caspase activity was determined by staining active caspase with FITC-VAD-FMK. Apoptosis results were verified by CFU analysis.
Influence of analyzed compounds on the mammalian cells growth was tested using Jurkat and NIH 3T3 cell lines. The untreated and 0.5% acetic acid or 0.2% formic acid treated Jurkat and NIH 3T3 cells were incubated in RPMI medium at +37 oC up to 3 days. The cells were analyzed after 1, 2 and 3 days. Apoptotic cells were stained with 7AAD and analyzed by flow-cytometry. For the analysis of cell proliferation CFSE was added to the Jurkat and NIH 3T3 cells at a final concentration of 1µM. The labeled cells were analyzed by flow-cytometry.
Jurkat cells
NIH 3T3 cells 10 20 30 40
Cells x104/ ml 80
% of apoptotic cells
1 day
2 days
3 days
CFSE, MFI
Cells x104/well 60
control
acetic acid
formic acid
Fig. 4. Externalization of phosphatidylserine in C. lusitaniae cells after 4 hours incubation with 0.1% of formic acid. Early apoptosis (1), late apoptosis (2).
Fig. 5. TUNEL staining. (A) Negative control. Yeast cells incubated without terminal deoxynucleotidyltransferase. (B) Positive control. Yeast cells were additionally treated with DNaseI. (C) Analyzed cells. Green stained cells indicates presence of DNA strand brakes.
Fig. 6. The influence of acetic and formic acids on the growth of mammalian cell lines.
Jurkat and NIH 3T3 cells were stained with vital fluorescence dye CFSE and cultured in the presence of 0.5% acetic or 0.2% formic acid for 3 days. The cell proliferation and apoptosis was analyzed by flow cytometry as described in materials and methods. Decreasing mean fluorescence intensity (MFI) of CFSE shows the proliferation of the cells.
Conclusions
Apoptotic markers in yeast cells were detected in a dose and time dependent manner after treatment with week acids. Cell death was accompanied by activation of caspase.
doses induced necrosis.
Formic acid had the lowest effect on the growth of Jurkat and NIH 3T3 cells.
Its was revealed in this study, that formic acid posses remarkable toxic activity against yeast and main assume pharmacological importance.
MIC of potassium carbonate and sodium bicarbonate induced cell apoptosis, while higher
Acknowledgments
We are thankful to dr. Irutė Girkontaitė for excellent scientific assistance.