BioRisk | of | 05-I | 6 (2022) Apeer-reviewed open-access journal

doi: 10.3897/biorisk.17.77227 RESEARCH ARTICLE & B te) R IS k

https://biorisk.pensoft.net

Saccharomyces cerevisiae yeast cells as a test system
for assessing Zeocin toxicity

Polya Marinovska', Teodora Todorova”, Anna Tomova', Emiliya Pisareva',
Krassimir Boyadzhiev’, Martin Dimitrov’, Petya Parvanova’,
Maria Todorova’, Stephka Chankova’, Ventsislava Petrova

I Sofia University “St. Kliment Ohridski’, Faculty of Biology, Department of General and Industrial Microbi-
ology, 8 Dragan Tsankov Blvd., 1164 Sofia, Bulgaria 2. Institute of Biodiversity and Ecosystems Research, BAS,
Department of Ecosystem Research, Environmental Risk Assessment and Conservation Biology, 2 Gagarin Str.,
1113 Sofia, Bulgaria

Corresponding author: Ventsislava Petrova (vpetrova@biofac. uni-sofia. bg)

Academic editor: Roumiana Metcheva | Received 28 October 2021 | Accepted 6 December 2021 | Published 21 April 2022

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

Abstract

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, quies-
cent (Q) and non-quiescent (NQ) cells of Saccharomyces cerevisiae BY4741 were used as a model to exam-
ine the cytotoxic effect of this radiomimetic. The levels of endogenous ROS, oxidized lipids, carbonylated
proteins, and glutathione were analysed after treatment with Zeocin (IC,,). An increase in ROS produc-
tion 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 pg/
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.

Keywords

Quiescence, stress response, yeast, Zeocin

Copyright Polya Marinovska et al. 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.

106 Polya Marinovska et al. / BioRisk 17: 105-116 (2022)

Introduction

The worldwide use of chemicals, drugs and other pharmaceuticals is significant. How-
ever, 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 mol-
ecules (Farrugia and Balzan 2012). Our ability to predict toxicological outcomes of
exposure to drugs as well as understanding the mechanisms underlying toxicity in
biological systems are of significant importance for human health and safety. Saccha-
romyces cerevisiae is one of the best-explored models of eukaryotic cells for studying
cellular mechanisms that occur under stress conditions. The main advantage of using
yeasts in drug testing is the possibility to identify sensitive biomarkers and mecha-
nisms of drug-mediated cell toxicity in the cases when the toxic compounds are still
unknown. In addition, S. cerevisiae is currently the only system that allows evaluation
of all targets in the cell, simultaneously and in vivo (Smith et al. 2010). However, most
of the yeast-based toxicological studies involve the use of proliferating Saccharomyces
cerevisiae cells (Gasch et al. 2000; Ericson et al. 2008; Dos Santos et al. 2012; Braconi
et al. 2016). Since quiescence is the most common cellular state found on Earth,
using Saccharomyces cerevisiae in quiescent state will be the more valuable tool for
prediction of cellular response to exposure of environmental toxic compounds. Cor-
respondingly, this study compares the toxic response of proliferating and stationary
phase (Q and NQ) S. cerevisiae BY4741 cells to Zeocin. With its radiomimetic prop-
erties, this compound is known to induce single (Miné-Hattab and Rothstein 2013)
and double-strand breaks (Chankova et al. 2007; Kopaskova et al. 2012; Todorova et
al. 2015a) as well as basic substitutions in the DNA molecule (Guénolé et al. 2013).
Moreover, it has been shown that Zeocin has pro-oxidative, mutagenic, and carcino-
genic effects in S. cerevisiae (Todorova et al. 2015b). Taking this into consideration,
we investigated the effect of Zeocin (IC,,) on the level of intracellular reactive oxygen
species (ROS), total intracellular glutathione, oxidized lipids, and proteins as sen-
sor molecules for measuring induced oxidative stress during drug toxicity. The DNA
damaging potential of high concentrations Zeocin on proliferating, Q and NQ cells
was also estimated.

Materials and methods

Microorganism and growth conditions

Yeast strain Saccharomyces cerevisiae BY4741 (MATa; his3A1; leu2A0; met15A0;
ura3A0) was used, obtained from the EDROSCAREF 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 sta-

Quiescent yeast cells for assessing Zeocin toxicity 107

tionary 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 quiescent (G,, Q) and non-quiescent (NQ) cells

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 Allen et
al. (2006).

Zeocin treatment

IC,, dose of Zeocin (Cayman Chemical Company, USA) was previously determined
using a spot analysis (Daskalova et al. 2021). The toxic effect of IC,, concentration
on the level of intracellular damages was studied after exposure to 50 pg/ml Zeocin.
The yeast cells were incubated with the stress-inducing agent for 60 min at room tem-
perature, and then washed twice with distilled water and subjected to disintegration.
Different concentrations of Zeocin (100, 200 and 300 ug/ml) were used in the experi-
ments for double-strand breaks (DSBs) detection.

Disintegration mode

Washed biomass of unexposed and exposed to Zeocin log, Q and NQ cells was mechan-
ically disrupted according to the previously described procedure (Daskalova et al. 2021).

Biochemical analyses

Protein content was measured by the method of Lowry et al. (1951). The concentration
of reactive oxygen species (ROS) was determined by the nitroblue tetrazolium test (NBT)
method described by Kostova et al. (2008). The levels of oxidized proteins were assessed
following the methodology of Mesquita et al. (2014). Quantitative evaluation of oxidized
lipids was done using the method described by Hodges et al. (1999). The concentration
of intracellular glutathione was determined using the method of Zhang (2000).

Constant field gel electrophoresis (CFGE) for detection of double-strand
breaks

CFGE was performed as described in Todorova et al. (2015b) and Todorova et al.
(2019). Logarithmic, Q, and NQ cell suspensions were treated with different concen-
trations of Zeocin for 1 min on ice. Cells were centrifuged and included into agarose
plugs at concentration 1x10° cells/ml. The agarose plugs were then placed in 1 ml of
lysis solution (pH = 8) containing proteinase K at final concentration 1 mg/ml. After

cell lysis, plugs were washed in Tris- EDTA (pH = 7.5) and inserted into a series of wells

108 Polya Marinovska et al. / BioRisk 17: 105-116 (2022)

in an agarose gel. Electrophoresis conditions were: 40 h at a constant field of 0.6 V/cm
(20 V). The levels of induced double-strand breaks (DSBs) represented as a fraction of
DNA released (FDR) from the wells were quantified by detecting the ethidium bromide
fluorescence using the Gene Tool Analyser G:Box Syngene. To evaluate the repair capac-
ity of cell populations 30 and 60 min recovery time was given after Zeocin treatment.

Data analysis

Used data represent the mean values with Standard error of the mean (LSEM) of three
independent experiments. ‘The statistical analysis was performed using MICROSOFT
OFFICE 365 EXCEL 2020 software. Differences in means were analysed using Stu-
dent’s t test with independent measures. Differences were considered statistically sig-
nificant at the p < 0.05 level.

Results

ROS levels

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 pg/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. 1 show the level of ac-
cumulated ROS in untreated and Zeocin-treated logarithmic, Q and NQ cells. They
clearly indicate that the action of Zeocin leads to an increase in the concentration of
toxic radicals in the three types of cells. ROS values measured in treated logarithmic
(400 uM/ml) and NQ (125 uM/ml) cells were almost four times higher than those in
untreated cells. The lowest increase in the level of ROS (1.5 times) after Zeocin treat-
ment was observed for the quiescence cells.

Levels of oxidized proteins

Next, the presence of carbonyl groups after exposure to the toxic agent has been studied
(Fig.2). Qand NQ cells of S. cerevisiae BY4741 exhibited differential responses to the action
of Zeocin. The highest increase in the level of carbonyl groups has been observed in G, cells
— about 3-fold. Logarithmically growing Zeocin-treated cells as well as non-quiescent cells
showed a slight increase in the amount of carbonyl groups compared to the control ones.

Levels of oxidized lipids

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

Quiescent yeast cells for assessing Zeocin toxicity 109

ROS level

— * *
=
S 800
=
S 600
* *

jad —__.

400 *#

200

0

log Q NQ
=Control © Treated

Figure |. 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 differ-
ences (* p < 0.05; ** p < 0.001) are presented.

Carbonylated proteins
20 * *

ray
uw

Carbonyl groups [uM/mg]
ray
=)

ui

Q NQ

=Control & Treated
Figure 2. Comparative analysis of carbonylated proteins in untreated (contol) and Zeocin-treated loga-
rithmic, Q and NQ cells of S. cerevisiae BY4741. Each value represents the mean + SEM (n = 3). Signifi-
cant differences (* p < 0.05; ** p < 0.001) are presented.

the process of lipid peroxidation (Fig. 3). The higher levels of oxidized lipids were
observed in both Zeocin-treated proliferating and quiescent cells (1.88 and 1.4 nmol/
mol, respectively). On the contrary, non-quiescent cells showed a very low level of lipid
peroxidation whether or not they are treated with Zeocin.

110 Polya Marinovska et al. / BioRisk 17: 105-116 (2022)

Oxidized lipids

= 2,5

(=)

= *

= 2

i?)

—

2 1,5

so

NS

= 1

S
0,5 * OK
‘ —

NQ

=Control @Treated
Figure 3. 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.

Intracellular glutathione levels

We also sought to investigate the effect of the radiomimetic Zeocin on the non-enzy-
matic 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 proliferat-
ing cells - about 2.5 times (Fig. 4).

Spontaneous levels of double-strand breaks

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. 5). No statistically significant difference was cal-
culated between the DSB levels in logarithmic and quiescent cells.

Zeocin induced double-strand breaks

Furthermore, the growth phase was estimated as a very important factor for the DNA
susceptibility of S. cerevisiae to Zeocin (Fig. 6). Approximately similar statistically signifi-
cant response measured as DSB induction was measured in both logarithmic (Fig. 6 A, D)
and non-quiescent cells (Fig. 6 C, D). The DSBs in logarithmic cells did not differ sta-
tistically from those in the non-quiescent cells. However, no significant effect of this ra-
diomimetic has been observed in quiescent cells, regardless of the applied concentration

(Fig. 6 B, D). The DSB levels were comparable with those in untreated cells.

Quiescent yeast cells for assessing Zeocin toxicity 111

GSH level

"OD
E 600 =e
z 400
ie)
200
0

NQ

log

=Control @Treated

Figure 4. Comparative analysis of the amount of total glutathione in untreated (control) and Zeocin-
treated Log, Q and NQ cell of S. cerevisiae BY4741. Each value represents the mean + SEM (n = 3).
Significant differences (* p < 0.05; ** p < 0.001) are presented.

log Q NQ
growth phase

Figure 5. 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).

Repair capacity depending on the growth phase

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 1). It was not
possible to calculate repair capacity of Q cells, having in mind that no statistically
significant increase of DSBs was measured after the treatment with different Ze-

ocin concentrations.

112 Polya Marinovska et al. / BioRisk 17: 105-116 (2022)

2

8
S|

1

Zeocin concentration (g/ml)

Figure 6. DSBs induced in S. cerevisiae BY4741, depending on growth phases after Zeocin treatment
in a concentration range 100-300 pg/ml A cells in logarithmic phase B quiescent cells C non-quiescent
cells. 1, 2 - control; 3, 4 - treatment with 100 pg/ml Zeocin; 5, 6 - treatment with 200 pg/ml Zeocin; 7,
8 - treatment with 300 pg/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.

Table |. Repair capacity of logarithmic and non-quiescent cells calculated after the treatment with Ze-
ocin at concentrations 100, 200 and 300 ug/ml with 30- and 60-min recovery time.

Logarithmic Non-quiescent
Recovery time (min) Recovery time (min)
Zeocin concentration (ug/ml) 30 60 30 60
100 1.25 1.41 0.84 0.87
200 2.41 1.86 1.20 1.02
300 2.29 321 0.99 0.88

Discussion

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 fur-
ther 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

Quiescent yeast cells for assessing Zeocin toxicity 113

xenobiotics. Regardless of their nature, such changes in the environment invari-
ably cause stress to the organisms, to which they must effectively adapt in order
to survive. This stress is often associated with ROS accumulation (Avery 2011).
Results obtained through this study confirmed that the exposure to Zeocin led to
disturbance of the cellular redox balance and induced an increase in the levels of
toxic oxygen radicals independently of the growth phase. The fraction of NQ cells
probably rapidly lost the ability to divide and accumulated almost 4 times higher
levels of ROS (Fig.1). Meanwhile, the stationary cells which have entered in G, cell
cycle, showed significantly higher resilience to the effect of Zeocin. The treatment
with this toxic chemical led to only a 2-fold increase in the levels of ROS, which
was probably due to their specific morphological and physiological properties. It
was well-known that S. cerevisiae quiescent cells are characterised by thicken cell
walls, condensed chromosomes and increased termo- and osmotolerance (Gray et
al. 2004). On the other hand, the NQ cells differ significantly, possessing genomic
instability, being easily lysed, and providing nutrients for those cells entering G,
(Aragon et al. 2008). As regards the logarithmic cells, they are characterised by
higher metabolic activity, elevated ROS levels and respectively increased susceptibil-
ity to toxic compounds, in that case Zeocin (Cabral et al. 2003).

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 (Farrugia and
Balzan 2012). In this respect as the Q-cells were characterised by very low metabolic
levels and lower ability to sequestered damaged molecules, the observed elevated oxida-
tive modifications in their proteins was not surprising. This might be also due to the
coordinated toxic effects of Zeocin, and stress caused by the lack of nutrients in the
environment. In NQ cells, the amount of carbonylated proteins after treatment with
Zeocin was comparable to that in control cells - 6.56 and 6.06 uM/mg, respectively
(Fig. 2). This indicated that here the appearance of oxidized proteins is rather a con-
sequence of the physiological state of the cell population and is not directly resulting
from the Zeocin-induced oxidative stress.

The measured higher intracellular concentration of malonaldehyde in Zeocin ex-
posed 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
(Hodges et al. 1999). Obviously, when the metabolic activity of the cell is higher and
the transport across the membrane - dynamic, the yeast cells are more vulnerable to the
action of xenobiotics, including the Zeocin. This eventually explains the measured excess
levels of oxidized lipids in the logarithmic cells comparing to the Q and NQ ones (Fig. 3).

To prevent the unbalanced accumulation of ROS and consecutive cellular damages,
yeast cells react with specific induction of both non-enzymatic and enzymatic antioxi-
dant 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 con-
firmed that after exposure to IC,, Zeocin the three types of yeast populations showed

114 Polya Marinovska et al. / BioRisk 17: 105-116 (2022)

increased intracellular levels of glutathione ranging from 1.5-fold (for Q cells) to 2.5-
fold (for logarithmic cells) (Fig. 4). Having in mind that in actively proliferating cells
the rate of transcription and translation are the highest, it is not surprising that in
these yeast populations the biosynthesis of glutathione was augmented with more than
100%. By contrast, in quiescent yeast cells, characterised by very low metabolic profile,
the measured increase in glutathione is only about 30%. These findings also correlated
with the observed tendency in the intracellular ROS levels of the studied yeasts. Non-
quiescent cells had the highest measured concentrations of glutathione possibly related
with the excess levels of intracellular ROS in general (Aragon at al. 2008).
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 de-
pendent on the growth phase were also observed previously in other strains of S. cerevisiae
(Todorova et al. 2015b, 2019). The statistically significant induction of DSB in logarithmic
cells after the treatment with concentrations equal or higher than 200 pg/ml Zeocin is in a
good correspondence with the one obtained on strain D7ts1 (Todorova et al. 2019). In late
stationary cells significantly higher levels of DSBs were detected in comparison with those
in exponential and early stationary cells. However, the entrance into G, cell cycle consider-
ably affects the levels of DSB, rendering quiescent cells with a low susceptibility to DNA
damages. When Zeocin is used as an inducing agent for DSB formation the tendency has
been preserved. Most robust to its action were the cells already entered the G, state. A pos-
sible explanation for their resistance to this radiomimetic chemical could be again assigned
to their specific cellular features. As mentioned above they are characterised by thickened
cell walls, highly condensed chromosomes and, as a result, restricted ability of the Zeocin to
penetrate the cell and reach its target molecule (Gray et al. 2004; Allen et al. 2006).

Conclusions

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.

Dataset deposition

The data underpinning the analysis reported in this paper are deposited at Figshare at
https://figshare.com/s/e89e8 8bfc6fl 24744755.

Quiescent yeast cells for assessing Zeocin toxicity 15

Acknowledgement

This work was supported by a grant from the National Science Fund, Ministry of Edu-
cation and Science, Project No. DH11/10 — 15.12.2017.

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