Research Article |
Corresponding author: Teodora Ivanova Todorova ( tedi_todorova@yahoo.com ) Academic editor: Roumiana Metcheva
© 2022 Polya Galinova Marinovska, Teodora Ivanova Todorova, Krassimir Plamenov Boyadzhiev, Emiliya Ivanova Pisareva, Anna Atanasova Tomova, Petya Nikolaeva Parvanova, Maria Dimitrova, Stephka Georgieva Chankova, Ventsislava Yankova 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 PG, Todorova TI, Boyadzhiev KP, Pisareva EI, Tomova AA, Parvanova PN, Dimitrova M, Chankova SG, Petrova VY (2022) Cellular susceptibility and oxidative stress response to menadione of logarithmic, quiescent, and nonquiescent Saccharomyces cerevisiae cell populations. In: Chankova S, Peneva V, Metcheva R, Beltcheva M, Vassilev K, Radeva G, Danova K (Eds) Current trends of ecology. BioRisk 17: 127-138. https://doi.org/10.3897/biorisk.17.77320
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The aim of the present study was to compare cellular susceptibility and oxidative stress response of S. cerevisiae logarithmic (log), quiescent (Q), and non-quiescent (NQ) cell populations to menadione – a well-known inducer of oxidative stress. Three main approaches were used: microbiological – cell survival, molecular – constant field gel electrophoresis for detection of DNA double-strand breaks (DSB), and biochemical – measurement of reactive oxygen species (ROS) levels, oxidized proteins, lipid peroxidation, glutathione, superoxide dismutase (SOD) and catalase on S. cerevisiae haploid strain BY4741. The doses causing 20% (LD20) and 50% (LD50) lethality were calculated. The effect of menadione as a well-known oxidative stress inducer is compared in the log, Q, and NQ cells. Survival data reveal that Q cells are the most susceptible to menadione with LD50 corresponding to 9 µM menadione. On the other hand, dose-dependent DSB induction is found only in Q cells confirming the results shown above. No effect on DSBs levels is observed in log and NQ cells. Further, the oxidative stress response of the cell populations is clarified. Results show significantly higher levels of SOD and ROS in Q cells than in log cells after the treatment with 100 µM menadione. On the other side, higher induction of oxidized proteins, malondialdehyde, and glutathione is observed after menadione treatment of log cells. Our study provides evidence that Saccharomyces cerevisiae quiescent cells are the most susceptible to the menadione action. It might be suggested that the DNA damaging and genotoxic action of menadione in Saccharomyces cerevisiae quiescent cells could be related to ROS production.
Menadione, quiescence, Saccharomyces cerevisiae, stress response
Organisms have developed strategies to trigger a stress response when exposed to environmental challenges in order to restore cellular homeostasis (
Based on this understanding cellular quiescence is of great importance, especially since studies performed on quiescent cells are still scarce. Such studies in multicellular organisms are difficult because of the complexity of the signals that control them. One of the possible solutions is the application of quiescent yeast cells as it is believed that they function similarly to the mammalian and human cells and share similar mechanisms and the same set of genes involved in the quiescence (
Saccharomyces cerevisiae is a widely used test system for studying oxidative stress and its related consequences. Results obtained on S. cerevisiae could be easily extrapolated at mammalian, including human level because of homology in genes and conservative functions of proteins (
The aim of the present study is to compare cellular susceptibility and oxidative stress response to menadione of S. cerevisiae logarithmic (log), quiescent (Q), and non-quiescent (NQ) cell populations.
Saccharomyces cerevisiae BY4741 (MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0) obtained from the EUROSCARF collection was used in the present work. The growth curve of Saccharomyces cerevisiae BY4741 on YEPD medium is provided as a Suppl. material
Samples were withdrawn at exponential (24 h) and late stationary phase (168 h). Quiescent (G0) and non-quiescent cells were isolated from stationary phase yeast population (168 h) according to the protocol described by
Cell suspensions with concentration 1×107 cells/ml were treated with various concentrations of menadione (2-methyl-l,4-naphthoquinone, synthetic form of vitamin K) in the range 1–200 µM for 60 min at 30 °C, 200 rpm. Cells were then centrifuged (825 g), the supernatant was removed and the pellet was resuspended in a liquid YEPD medium. Cells were plated on a solid YEPD medium and incubated at 30 °C for 3 days to evaluate the survival. Doses of lethality (LD20, and LD50) were calculated (
lgLD50=lgA+(lgB-lgA)/((50-A)/(B-A))
lgLD20=lgA+(lgB-lgA)/((20-A)/(B-A)),
where A – the closest smaller than 50 or 20%, respectively, lethality percentage; lgA- lg of the concentration corresponding to A; B – the closest higher than 50 or 20%, respectively, lethality percentage; lgB- lg of the concentration corresponding to B.
Isolation of cell-free extracts from log, Q, and NQ cells was carried out according to the procedure described by
CFGE for detection of DNA double-strand breaks (DSBs) was applied as described in
The redox state of logarithmic, quiescent, and non-quiescent yeast cells was assessed through measurement of intracellular levels of accumulated ROS (
The measurement of intracellular glutathione was carried out according to the procedure of
Superoxide dismutase (SOD) and catalase (CAT) enzyme activities were determined spectrophotometrically according to
Total intracellular protein was determined according to
The experiments were repeated at least three times from independently grown cultures. Data points in all the figures are mean values. Error bars represent standard errors of mean values. Where no error bars are evident, errors were equal to or less than the symbols. All the calculations were done with GraphPad Prism program, version 6.04 (San Diego, USA). The statistical analysis included the application of Student’s t-test and One-way ANOVA followed by Bonferroni’s post hoc test. P<0.05 was accepted as the lowest level of statistical significance.
Our first step was to determine the cell survival of the three cell populations after treatment with 100 µM menadione. Data revealed that the log cells are the most resistant to menadione action (Fig.
Cell survival after menadione treatment A effect of 100 µM menadione on log, Q, and NQ cell populations B effect of menadione in a concentrations’ range of 1–200 µM on log and Q cells. Each value represents the mean ± SEM (Standard error of the mean) (n = 3).
Two levels of lethality were calculated: LD20 and LD50 (Table
Further, the levels of DSB induced were compared. Our results confirmed the ones obtained for cell survival. Dose-dependent DSB induction is measured only in quiescent cells (Fig.
Cell populations | LD20 (µM) | LD50 (µM) |
---|---|---|
Log | 35 | 199 |
Quiescent | 0.65 | 9 |
DSBs induced by various concentrations (50–150 µM) of menadione A induction of DSB presented as normalized FDR B Q cells C log cells D NQ cells.
Further experiments were focused on studying the potential differences in the susceptibility based on various markers for oxidative stress – reactive oxygen species, oxidized proteins, malondialdehyde, intracellular glutathione, superoxide dismutase, and catalase.
The ROS measured in the three cell populations are presented in Fig.
Comparative analysis of the levels of reactive oxygen species A oxidized proteins B malondialdehyde C and total glutathione D in S. cerevisiae logarithmic (log), quiescent (Q), and non-quiescent (NQ) cell populations after the treatment with menadione. Each value represents the mean ± SEM (Standard error of the mean) (n = 3). Significant differences (* p < 0.05; ** p < 0.001) are presented.
The constitutive levels of ROS, oxidized proteins, and MDA in NQ cells were significantly higher than those measured in log and Q cells. Treatment with 100 µM menadione resulted in significant induction of oxidized proteins and glutathione (Fig.
Data presented in fig. 3B provides information concerning the concentration of protein carbonyl groups. Comparing the constitutive levels, around 7-fold higher levels were measured in Q cells in comparison with the log ones. This could be explained as a result of the cells’ starvation. Although, the highest quantity – 14 µM/mg was determined in Q cells the induction was only around 2-fold. Higher induction – around 6-fold was measured in the log cells.
Concerning the MDA, comparatively equal constitutive levels were observed between Q and log cells (Fig.
The GSH concentration in untreated Q cells was 3-fold higher than that in log cells. Interestingly, menadione treatment did not result in a significant induction of GSH compared to the untreated control. The GSH concentration was only 2-fold higher (Fig.
Concerning the constitutive levels of the antioxidant enzymes superoxide dismutase and catalase, differences were obtained. The catalase levels were comparable in the three cell populations, while SOD was lower in Q cells than in the log and NQ cells (Fig.
Comparative analysis of the response to menadione based on the enzymatic antioxidant system A superoxide dismutase and B catalase presented as units/mg. Each value represents the mean ± SEM (Standard error of the mean) (n=3). Significant differences (* p < 0.05; ** p < 0.001) are presented.
Significant induction of SOD was observed in Q cells after the application of menadione (Fig.
Data presented here provide a comparative analysis of the cellular susceptibility and oxidative stress response to menadione of logarithmic, quiescent, and nonquiescent Saccharomyces cerevisiae cell populations. Differences in the cellular susceptibility are obtained depending on the endpoint used. Based on cell survival, DSBs induction, ROS, and SOD Q cells are more susceptible to menadione. On the other side, higher induction of oxidized proteins, MDA, and glutathione is observed following menadione treatment of log cells.
The measured increased ROS levels in Q cells correspond well with the decrease in cell survival and the well-expressed DSB induction. The cytotoxic mechanism of action of Menadione in G0 cells is stronger, probably due to lower metabolic activity and higher oxygen levels in the cells. This is in accordance with the report by
It is already reported that the toxicity of quinones including menadione in S. cerevisiae depends on the oxygen presence (
In the present work, log cells showed increased levels of oxidized proteins, MDA, and glutathione. This could be explained by their increased metabolic activity and a higher rate of protein synthesis (
Lipid oxidation occurs through the interaction of ROS with fatty acids in the membrane lipid layer. This changes the functionality and permeability of biological membranes and also leads to other disorders. Cell death can be caused by the release of cell contents as a result of these changes. Malonaldehyde is the end product of lipid oxidation. It accumulates in cells and is a highly reactive and toxic electrophilic compound that can form covalently bound products with different proteins. Its concentration in the cell is used as a biomarker to account for the influence of stress agents. In our work, the MDA levels remained similar in control and treated Q cells. One of the explanations could be the thicker cell wall (
Glutathione plays an important role in protecting the cell against oxidative stress by protecting it from the toxic effects of ROS through its involvement in mechanisms for detoxification and regeneration of important cellular antioxidants (
All enzymes in glutathione metabolism work in an integrated way, allowing the cell to adapt to different stress conditions (
Our study provides evidence that Saccharomyces cerevisiae quiescent cells are the most susceptible to the menadione action. It might be suggested that the DNA damaging and genotoxic action of menadione in Saccharomyces cerevisiae quiescent cells could be related to ROS production.
This work was supported by a grant from the National Science Fund, Ministry of Education and Science, Project No. DH11/10.
Figure S1
Data type: jpg file
Explanation note: Fig. S1. Growth curve of Saccharomyces cerevisiae BY4741 and glucose assimilation in batch cultivation on YEPD media at 30 °C, 204 rpm for 168 h.