Photonics/BMG LABTECH/Fluorescence Analysis of Reactive Oxygen Species (ROS) Generated by Six Isolates of Aspergillus fumigatus

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Fluorescence Analysis of Reactive Oxygen Species (ROS) Generated by Six Isolates of Aspergillus fumigatus

Fluorescence Analysis of Reactive Oxygen Species (ROS) Generated by Six Isolates of Aspergillus fumigatus


Reactive oxygen species (ROS) are essential intermediates in oxidative metabolism. Nonetheless, when generated in excess, ROS can damage cells by peroxidizing lipids and disrupting structural proteins, enzymes and nucleic acids. Excess ROS are generated during a variety of cell stresses, including ischemia/reperfusion, exposure to ionizing and ultraviolet radiation and/or infl ammation. Reactive oxygen species (ROS) may contribute to infl ammation and tissue damage. The generation processes of reactive oxygen species can be monitored using luminescence analysis or fl uorescence methods. The intracellular ROS generation of cells can be investigated using 2’,7’-dichlorfl uorescein-diacetate (DCFH-DA), which is an established compound to detect and quantify intracellularly produced H2O2. 1 The conversion of nonfl uorescent 2’,7’-dichlorfl uoresceindiacetate (DCFH-DA) to the highly fl uoresecent compound, 2’,7’- dichlorfl uorescein (DCF), happens in several steps. First, DCFH-DA is transported across the cell membrane and deacetylated by esterases to form the nonfl uorescent 2’,7’-dichlorfl uorescein (DCFH). This compound is trapped inside of the cells. Next, DCFH is converted to DCF through the action of peroxide, which is generated by the presence of peroxidase.2 Aspergillus species are of interest in the pathogenesis of several dermatological diseases. It is uncertain whether Aspergillus itself may generate ROS and therefore actively induce tissue damage. The present study investigates whether Aspergillus species are capable of producing ROS by themselves and if there are differences between the several strains.

Materials and Methods

Six isolates of Aspergillus fumigatus (AF 65, AF 71, AF 72, AF 91, AF 210, AF 294 ) cultured 5 weeks on Sabouraud-Glucose-Agar (BAG, Lich, Germany) were investigated. After addition of isotonic NaCl solution and centrifugation with 100 rpm for 10 min, the blastospore concentration could be estimated by counting in CASY 1 (Schärfesystem GmbH, Germany). These cell suspensions with concentrations of 105 to 107 cells/mL were measured in the FLUOstar (BMG LABTECH, Germany) using 100 µL of fungal cell suspension after incubation with 100 µL DCFHDA (0.4 nM) for each single test. To eliminate LBS induced effects, polymyxin B (3 mg/mL) was added to all experimental suspensions.

Each measurement was done at least sixteen times in duplicate for calculation of the mean and the standard error of the mean.

Results and dicussion

The ability of various Aspergillus species to generate reactive oxygen species (ROS) was investigated. For all fungal cells, a linear increasing fl uorescence activity could be observed depending on the incubation time with DCFA-DA. By using a calibration curve, the measured fl uorescence signals were converted to H2O2 concentrations and one example is given for AF 71 with different incubation periods. Because of the small sized Aspergillus fumigatus (2-3 mm), detectable fl uorescence was observed only at concentrations >105 cells /mL. The ROS generation showed a linear and direct proportional dependence on cell numbers and the results were reproducible on 3 different days with a fi xed incubation period of 2.5 hours at 37°C. The highest value could be found at concentrations of 107 cells /mL.

The isolates of A. fumigatus AF 91 and AF 72 are resistent against itraconazole (antifungal agent) and AF 65 is resistant to amphotericin B (antifungal agent).3,4 We investigated whether or not there are connections between the resistance and the ROS generation. Interisolate differences could be found.


The morphological event of fungi, usually acknowledged as a major factor of virulence, is associated with increased intracellular ROS formation, which are most likely secreted. Together with phospholipases, ROS are capable of destroying the host cell membranes. This process may be contributed to the invasiveness of A. fumigatus and to the inflammatory response of the host. Clearly, the pathogenicity of Aspergillus species is a function of a multitude of parameters working together in a sequential and cooperative manner to establish infection. It has been shown by several authors, that water as well as superoxide, hydrogen peroxide and OH-radicals can be generated in the course of the mitochondrial electron transport process.5 Fungi also possess the normal and the alternative pathway of electron transport. The interesting connection between the monovalent oxygen reduction and the energy conservation in isolated chloroplastes was described.5 The hydrogen peroxide formation at the phosphorylation points I and II is disposed by a cytochrome c oxidation process. In this study, the method of ROS fluorescence measurement was utilized on different unstimulated Aspergillus species for the first time. There were linear correlations found between ROS levels and blastospore concentrations. A pathophysiological meaning of the released oxygen metabolites in the complicated system of inflammatory reactions, as an additional factor of virulence is not to be excluded, which was also estimated in Saccharomyces cerevisiae. 6


[1] Cathcart R, Schwiers E, Ames BN.; (1983); Detection of picomole levels of hydrogenperoxide usinga fluorescent dichlorofluorescein assay. Anal. Biochem.; 134: 111-116. [2] LeBel CP, Ischiropoulos H, Bondy SC.; (1992); Evaluation of the probe 2’,7’- dichlorofluorescein as an indicator of reactive oxygen species formation and oxidative stress. Chem. Res. Toxicol.; 5: 227-231. [3] Denning DW, Radford SA, Oakley KL, Hall L, Johnson EM, Warnock DW.; (1997); Correlation between in-vitrosusceptibility testing to itraconazole and in-vivo outcome of Aspergillus fumigatus infection. J. Antimicrob Chemother.; 40: 401-414. [4] Oakley KL, Moore CB, Denning DW.; (1998); In vitro activity of the echinocandin antifungal agent LY 303,366 in comparison with itraconazole and amphotericin B against Aspergillus sp. Antimicrob. Agents Chemother.; 42: 2726-2730. [5] Ruotsalainen M, Hyvärinen A, Nevalainen A, Savolainen KM.; (1995); Production of reactive oxygen metabolites by opsonized fungi and bacteria isolated from indoor air and their interactions with soluble stimuli, fMLP or PMA. Environ. Res.; 69: 122-31. [6] Jakubowsky W, Bartosz G.; (1997); Estimation of oxidative stress in Saccharomyces cerevisiae with fluorescent probes. Int. J. Biochem. Cell Biol.; 29: 1297-1301.

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