Evaluation of Cytotoxic Effect of Nystatin on Yeast Cells By Electrochemical Monitoring of Intracellular NADH with Double Mediator System
Authors: Alim Mohammad Abdul , Shinohara Hiroaki , Suga Minoru 
Published: December 2020

Background and purpose

Environmental safety concern needs in medical diagnosis and food test to rapidly identify pathogenic organisms and their susceptibilities to medical agents and toxic chemicals have encouraged to develop more easy and precise new diagnostic methods. Bio-electrochemical techniques are very convenient and useful for monitoring cell viability, the identification of intracellular function and understanding the biological process [1-5]. NAD+ /NADH is a crucial cofactor for enzymatic reaction and ATP production in cytosol and mitochondria. This biomaterial is one of the most important markers for sensing these mentioned processes.
Recently, we have proposed a rapid and sensitive bio-electrochemical method for yeast cell counting in broader range as compared with conventional optical method through the monitoring of intracellular NADH by using a double mediator system with a screen-printed carbon electrode (SPCE). Now by applying this method, we have succeeded to evaluate the fast-acting toxicity of nystatin (NYT) at very lower concentration on yeast cell. Near future this method could be also applicable to the bacterial cell viability.

Experimental method

Fission yeast Schizosaccharomyces pombe cell suspension with HBSS was taken in each well of 96 well plates and final conc. of 500 μM potassium ferricyanide and 10 μM 1-mPMS solution were added in a drop wise to the cell suspension. After incubation for 10 minutes, SPCE was vertically immersed into the cell suspension and chronoamperometric measurement was immediately performed by potential application at +0.5V. Prior to the electrochemical measurement, yeast cell was grown in YE medium (0.5% Difco yeast extract and 3% glucose) supplemented with 150 µg/mL leucine to a density of cells/ml at 30 [6]. After culturing the cell, the optical density of cell suspension was measured at 592 nm to estimate the number of yeast cells. The cells were washed with HBSS and adjusted to the desired concentration.

Result and discussion

The oxidation current was measured in each yeast cell suspension by chronoamperometry of ferrocyanide generated by intracellular NADH with redox mediation of 1-mPMS. Fig.1 and Fig.2 shows that the decrease of oxidation current was clearly dependent on the concentration of NYT (final conc. was 1, 2, 4, 8, 16, 32 mg/mL). It was indicated that intracellular NADH level corresponding to the cell viability was rapidly decreased by NYT effect. On the other hand, we have also investigated the toxic effect of NYT by double staining (SYT09 + PI) fluorescence method. Generally, NYT forms a pore-like structure on the cell membrane and thereby the integrity of the cell membrane is lost [7]. Though NYT caused disruption of fungal cell membrane at higher concentration but at very lower concentration PI could not enter into the cell and therefore PI staining was not occurred. That means, in this method no effect was observed (data will be shown in the conference). In electrochemical method, the cell viability decreased at very lower concentration maybe due to apoptotic pathway or the inhibition of unspecific enzyme by NYT inside of yeast cell but not for the disruption of cell membrane. It was now considered that NYT to the cells with pore formation in cell membranes is the secondary inhibitory mechanism and the primarily action may be due to intracellular oxidative damage and interaction with organelles [7].
Though evaluation of nystatin effect at very low concentration was not possible by the double staining method but it was possible to detect it by our electrochemical method.

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Figure 1