Research Article |
Corresponding author: Ventsislava Petrova ( vpetrova@biofac.uni-sofia.bg ) Academic editor: Roumiana Metcheva
© 2022 Polya Marinovska, Teodora Todorova, Anna Tomova, Emiliya Pisareva, Krassimir Boyadzhiev, Martin Dimitrov, Petya Parvanova, Maria Dimitrova, Stephka Chankova, Ventsislava Petrova.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Marinovska P, Todorova T, Tomova A, Pisareva E, Boyadzhiev K, Dimitrov M, Parvanova P, Dimitrova M, Chankova S, Petrova V (2022) Saccharomyces cerevisiae yeast cells as a test system for assessing Zeocin toxicity. In: Chankova S, Peneva V, Metcheva R, Beltcheva M, Vassilev K, Radeva G, Danova K (Eds) Current trends of ecology. BioRisk 17: 105-116. https://doi.org/10.3897/biorisk.17.77227
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Having unique genetic machinery and a high degree of conservation with higher eukaryotes, the yeast Saccharomyces cerevisiae is recognised as a smart experimental system for studying the modes of chemical toxicity. The present study was undertaken to elucidate the changes in the intracellular redox homeostasis and key macromolecule structure following exposure to Zeocin. Cell populations of logarithmic, quiescent (Q) and non-quiescent (NQ) cells of Saccharomyces cerevisiae BY4741 were used as a model to examine the cytotoxic effect of this radiomimetic. The levels of endogenous ROS, oxidized lipids, carbonylated proteins, and glutathione were analysed after treatment with Zeocin (IC50). An increase in ROS production and respectively increased oxidative stress was detected in all three types of cell populations, with the highest degree being observed in proliferating S. cerevisiae BY4741 cells. The stress response of both proliferating and stationary phase (Q and NQ) cells to Zeocin included an overexpression of glutathione. The quiescent cells also showed very low DNA susceptibility to high Zeocin concentration (100–300 µg/ml), presented as no induced double-strand breaks (DSBs) in the macromolecule. Based on our research it could be concluded that the cellular physiological state is a critical factor determining the resistance to environmental stress with Q cells being the most robust.
Quiescence, stress response, yeast, Zeocin
The worldwide use of chemicals, drugs and other pharmaceuticals is significant. However, they represent toxic pollutants and their presence in the environment seriously endangers human health. Pharmaceutical residues can interact with biological targets and thus exert their different toxic effects at very low concentrations. These often-irreversible interactions triggered serious damages in lipids, proteins, and DNA molecules (
Yeast strain Saccharomyces cerevisiae BY4741 (MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0) was used, obtained from the EUROSCARF Frankfurt collection (Germany). Yeast cells were grown on a liquid YPD medium at 30 °C on a reciprocal shaker (205 rpm) for 168 hours. Samples were withdrawn at exponential (24 hours) and late stationary phase (168 hours). The biomass was harvested by centrifugation at 5000 rpm for 10 min at 4 °C. The pellet was washed twice with distilled water and used for further analyses.
Isolation of Q and NQ S. cerevisiae BY4741 stationary phase yeast cells (168 h) was performed in Percoll density gradient according to the protocol described by
IC50 dose of Zeocin (Cayman Chemical Company, USA) was previously determined using a spot analysis (
Washed biomass of unexposed and exposed to Zeocin log, Q and NQ cells was mechanically disrupted according to the previously described procedure (
Protein content was measured by the method of
CFGE was performed as described in
Used data represent the mean values with Standard error of the mean (±SEM) of three independent experiments. The statistical analysis was performed using MICROSOFT OFFICE 365 EXCEL 2020 software. Differences in means were analysed using Student’s t test with independent measures. Differences were considered statistically significant at the p < 0.05 level.
A comparative study on the cytotoxic effect of Zeocin in logarithmic, quiescent, and non-quiescent S. cerevisiae BY4741 cells has been conducted. After their exposure to 50 µg/ml Zeocin, the induced intracellular changes were determined based on the level of generated reactive oxygen species and their harmful oxidative effect on key cellular macromolecules - proteins and lipids. Results presented in Fig.
Next, the presence of carbonyl groups after exposure to the toxic agent has been studied (Fig.
Comparative analysis of ROS levels in untreated (control) and Zeocin-treated proliferating, Q and NQ cells of S. cerevisiae BY4741. Each value represents the mean ± SEM (n = 3). Significant differences (* p < 0.05; ** p < 0.001) are presented.
The data obtained after the measurement of malonaldehyde equivalents in the three cell types showed that Zeocin could also impair the prooxidant balance and activate the process of lipid peroxidation (Fig.
We also sought to investigate the effect of the radiomimetic Zeocin on the non-enzymatic defence mechanisms, in particular, glutathione. Obtained results revealed that the total levels of this tripeptide in all the treated yeast cell populations were higher compared to the control ones. The most significant increase was observed in proliferating cells - about 2.5 times (Fig.
Comparative analysis of oxidized lipids in untreated (control) and Zeocin-treated Log, Q and NQ cells of S. cerevisiae BY4741. Each value represents the mean ± SEM (n = 3). Significant differences (* p < 0.05; ** p < 0.001) are presented.
The spontaneous DSB levels were found to depend on the growth phase. Around 1.5-fold higher DSBs levels were measured in non-quiescent cells compared to the logarithmic and quiescent ones (Fig.
Furthermore, the growth phase was estimated as a very important factor for the DNA susceptibility of S. cerevisiae to Zeocin (Fig.
Spontaneous levels of DBSs depending on the growth phase. Error bars represent standard error of the mean from at least three independent experiments. Where no error bars are evident, they are equal of less than the symbols. Statistical significance of differences is indicated with an asterisk (** p < 0.01; ns p > 0.05).
DSBs induced in S. cerevisiae BY4741, depending on growth phases after Zeocin treatment in a concentration range 100–300 μg/ml A cells in logarithmic phase B quiescent cells C non-quiescent cells. 1, 2 - control; 3, 4 - treatment with 100 μg/ml Zeocin; 5, 6 - treatment with 200 μg/ml Zeocin; 7, 8 - treatment with 300 μg/ml Zeocin D induction of DSBs after treatment with different concentrations of Zeocin calculated as FDR. The significance in the differences where ns p > 0.05, **p < 0.01. Where no error bars are evident, they are equal or less than the symbols.
The best expressed repair capacity was calculated for the logarithmic cells when 60 min recovery time was given. On the other side, NQ cells were found unable to repair Zeocin induced DSBs despite the recovery time (Table
Repair capacity of logarithmic and non-quiescent cells calculated after the treatment with Zeocin at concentrations 100, 200 and 300 µg/ml with 30- and 60-min recovery time.
Logarithmic | Non-quiescent | |||
---|---|---|---|---|
Recovery time (min) | Recovery time (min) | |||
Zeocin concentration (µg/ml) | 30 | 60 | 30 | 60 |
100 | 1.25 | 1.41 | 0.84 | 0.87 |
200 | 2.41 | 1.86 | 1.20 | 1.02 |
300 | 2.29 | 3.21 | 0.99 | 0.88 |
Currently, the yeast Saccharomyces cerevisiae remains one of the highly important experimental models in the field of toxicogenomics. Studying the biology of yeast cells, especially those in quiescence, could reveal the potential of this microorganism for investigating the cellular response to a particular environment. The evaluation of toxicological response and stress mechanisms in this microorganism could further be helpful in the clarification of equivalent mechanisms in higher eukaryotes. The environmental conditions can change dramatically, which includes progressive depletion of nutrients, rising ambient temperatures, or sudden contamination with xenobiotics. Regardless of their nature, such changes in the environment invariably cause stress to the organisms, to which they must effectively adapt in order to survive. This stress is often associated with ROS accumulation (
A direct indicator of the onset of the redox balance disturbance is the appearance of carbonyl groups in the proteins. Oxidative damage to proteins affects the processes maintaining the cellular homeostasis, which compromises their viability (
The measured higher intracellular concentration of malonaldehyde in Zeocin exposed cells further confirmed that one of the cytotoxic effects of this antibiotic was related to the induction of oxidative stress in the cell. It resulted in impairment of membrane functionality and permeability, probably causing the release of the intracellular content (
To prevent the unbalanced accumulation of ROS and consecutive cellular damages, yeast cells react with specific induction of both non-enzymatic and enzymatic antioxidant defence mechanisms. Key molecule acting as first line of defence against oxidative injuries is the glutathione. That is why the total intracellular amount of this tripeptide is an important parameter for measuring the oxidative stress levels. In this study we confirmed that after exposure to IC50 Zeocin the three types of yeast populations showed increased intracellular levels of glutathione ranging from 1.5-fold (for Q cells) to 2.5-fold (for logarithmic cells) (Fig.
Furthermore, this study aimed to assess the potential of Zeocin to induce DNA double-strand breaks (DSB) in dependence to the growth phase. The highest levels of spontaneous DSBs were observed in non-quiescent cell. Such results are not surprising, considering the fact that they represent the fraction of stationary cells having very low ability to reproduce and tendency to enter apoptosis. Such differences in the spontaneous DSB levels being dependent on the growth phase were also observed previously in other strains of S. cerevisiae (
The comparative analysis of different yeast cell populations – logarithmic, quiescent, and non-quiescent – revealed that the cellular physiological state is a critical factor determining the resistance to xenobiotics. More robust to Zeocin exposure were the quiescent cells showing lower levels of ROS and DSBs. By contrast, the logarithmically grown yeasts are more susceptible to the action of this compound with observed higher damages in DNA, proteins and lipids.
The data underpinning the analysis reported in this paper are deposited at Figshare at https://figshare.com/s/e89e88bfc6f1247a4755.
This work was supported by a grant from the National Science Fund, Ministry of Education and Science, Project No. DH11/10 – 15.12.2017.