RESEARCH ARTICLE
Dimethyl sulfoxide induces oxidative stress in the yeast
Saccharomyces cerevisiae
1 & Grzegorz Bartosz1,2
Izabela Sadowska-Bartosz1, Aleksandra Pa˛czka1, Mateusz Mołon
w, Rzeszo
w, Poland; and 2Department of Molecular Biophysics, University of
Department of Biochemistry and Cell Biology, University of Rzeszo
dz, Ło
dz, Poland
Ło
1
Correspondence: Izabela Sadowska-Bartosz,
Department of Biochemistry and Cell Biology,
w, Zelwerowicza
University of Rzeszo
w, Poland.
4, 35-601 Rzeszo
Tel.: +48 17 8755408;
fax: +48 17 8721425;
e-mail: [email protected]
Received 15 May 2013; revised 30 July 2013;
accepted 3 September 2013.
Final version published online 11 October
2013.
DOI: 10.1111/1567-1364.12091
Editor: Ian Dawes
Keywords
DMSO; glutathione; reactive oxygen species;
succinate dehydrogenase.
Abstract
Dimethyl sulfoxide (DMSO) is used as a cryoprotectant for the preservation of
cells, including yeast, and as a solvent for chemical compounds. We report that
DMSO induces oxidative stress in the yeast. Saccharomyces cerevisiae wt strain
EG-103 and its mutants Dsod1, Dsod2, and Dsod1 Dsod2 were used. Yeast were
subjected to the action of 1–14% DMSO for 1 h at 28 °C. DMSO induced a
concentration-dependent inhibition of yeast growth, the effect being more pronounced for mutants devoid of SOD (especially Dsod1 Dsod2). Cell viability
was compromised. DMSO-concentration-dependent activity loss of succinate
dehydrogenase, a FeS enzyme sensitive to oxidative stress, was observed. DMSO
enhanced formation of reactive oxygen species, estimated with dihydroethidine
in a concentration-dependent manner, the effect being again more pronounced
in mutants devoid of superoxide dismutases. The content of cellular glutathione was increased with increasing DMSO concentrations, which may represent
a compensatory response. Membrane fluidity, estimated by fluorescence polarization of DPH, was decreased by DMSO. These results demonstrate that
DMSO, although generally considered to be antioxidant, induces oxidative
stress in yeast cells.
YEAST RESEARCH
Introduction
Dimethyl sulfoxide (methyl sulfoxide, methylsulfinylmethane; DMSO) has been extensively studied since 1860’s
(Capriotti & Capriotti, 2012). DMSO is present in the
environment as a waste product of the paper industry
and from the production of dimethyl sulfide (DMS) and
is also formed by the degradation of sulfur-containing
pesticides. Moreover, DMSO occurs naturally from photooxidation of DMS in the atmosphere and from degradation of DMS by phytoplankton in the marine
environment. Because DMSO has low volatility and is
highly hygroscopic, it is rapidly scavenged from the atmosphere by rain and returned to earth and thereby plays a
role in the global sulfur cycle (Murata et al., 2003).
DMSO is a small amphiphilic molecule with a hydrophilic sulfoxide group and two hydrophobic methyl
groups. Its amphiphilic nature appears to be an important characteristic defining its action on membranes. It is
an effective penetration enhancer and is routinely used as
ª 2013 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
a cryoprotectant (Kashino et al., 2010; Kwak et al., 2010).
A vast array of literature describes the membranepermeabilizing activities of DMSO, and studies have
shown its effectiveness in promoting membrane permeation by both hydrophilic and lipophilic compounds
(Hao et al., 2010; Nocca et al., 2012). DMSO can induce
formation of water-permeable pores in dipalmitoyl phosphatidylcholine bilayers, and this is a possible pathway
for the enhancement of penetration of other molecules
through lipid membranes (Nocca et al., 2012).
DMSO is widely used as a cryoprotectant. Cryopreservation is commonly accepted as a suitable method for longterm storage of various types of cell. The cryopreservation
media contain DMSO (5–10%) as the active substance
(e.g. CryoMaxx I medium with 5% DMSO or CryoMaxx
II with 10% DMSO, from PAA Laboratories, Austria). As
a cryoprotectant, DMSO exerts its effect on the hydrophilic region of membrane lipids and prevents decrease in
membrane fluidity at temperatures at which cells otherwise
sustain freezing injury (Thirumala et al., 2010).
FEMS Yeast Res 13 (2013) 820–830
821
Effect of DMSO on yeast
There are numerous reports on the radical-scavenging
activity of DMSO. This compound is regarded as a highly
effective scavenger of the hydroxyl radical with a secondorder rate constant k2 of 8.16 9 109 M1 s1. This can
result in radioprotection and cryoprotection, as it is
thought that reactive oxygen species (ROS) generated by
freezing or thawing processes cause additional damage to
the biological material. DMSO is employed in cell biology
to induce cell fusion and cell differentiation. Its radicalscavenging properties can underlie also these effects, as
there is evidence that signaling by ROS is involved
(Homer et al., 2005).
DMSO has found medical applications, generally fallen
into three functional categories encompassing tissue/organ
preservation, penetration-enhancing solvent excipients,
and active pharmaceutical agents, primarily anti-inflammatory, which is again ascribed to its radical-scavenging
properties (Capriotti & Capriotti, 2012). Nevertheless, the
physiological and pharmacological properties and effects
of DMSO are not completely understood.
Although the cryoprotective action of DMSO has been
partly ascribed to its antioxidant action, there are divergent
data on its pro-oxidant/antioxidant action in cellular
systems. It has been reported that DMSO affects the oxidative stress-induced cytotoxicity in two completely different
ways on yeast and mammalian cells (Kwak et al., 2010).
The reasons for these cellular differences in DMSO effects
are unclear (Qi et al., 2008). In sharp contrast to its cytotoxic effect in yeast under oxidative stress, DMSO showed a
protective effect on oxidative stress-induced cytotoxicity in
human SK-Hep1 cells (Kwak et al., 2010). DMSO reduced
also arsenite- or hydrogen peroxide (H2O2)-induced intracellular ROS production in human hamster hybrid and
mouse embryo cells and was capable of trapping nitric
oxide free radicals in human umbilical vein endothelial
cells. In the yeast, DMSO was found to inhibit in vivo
methionine-S-sulfoxide reduction by competitively inhibiting methionine sulfoxide reductase A activity (Kwak et al.,
2010). On the other hand, DMSO was observed to reduce
the protein-carbonyl content in yeast cells in the absence of
H2O2 treatment, suggesting its ROS-scavenging activity
even under normal culture conditions.
Saccharomyces cerevisiae is an appropriate model eukaryotic organism to study physiological parameters that affect
cell ability to survive freeze–thaw injury and oxidative
stress. A wide range of mutants is available that exhibit
altered cellular responses to various types of stress that may
be incurred during freeze–thaw injury, and these may be
exploited to study the nature of freeze–thaw injury and
how to avoid it (Pereira et al., 2003; Momose et al., 2010).
The aim of this study is to ascertain the effect of DMSO
on redox equilibrium in yeast cells. We analyzed biochemical markers of oxidative stress (such as ROS generation,
FEMS Yeast Res 13 (2013) 820–830
activity of succinate dehydrogenase and glutathione
content), membrane fluidity as well as cell viability and
survival of S. cerevisiae, treated with DMSO (1–14%). To
better elucidate the effect of DMSO, the following yeast
strains carrying deletions in superoxide dismutase (SOD)
genes were used: S. cerevisiae wild-type (wt) strain EG-103
and its mutants Dsod1, Dsod2 and Dsod1 Dsod2.
The cytoplasmic Cu/Zn-superoxide dismutase (Cu/ZnSOD), which is encoded by the SOD1 gene, appears to be
a key enzyme involved in the regulation of intracellular
levels of ROS and in protecting cells from the toxicity of
exogenous oxidant agents. Cellular antioxidant defenses
include also several other important elements, such as the
mitochondrial Mn-SOD, encoded by the SOD2 gene. It
protects mitochondria from ROS generated during respiration and exposure to ethanol. Glutathione (GSH) act as
a radical and metal scavenger and is critical for removal
of peroxides by glutathione peroxidases, thus protecting
cells against oxidation (Pereira et al., 2003). Succinate
dehydrogenase (EC 1.3.99.1) in the yeast S. cerevisiae is a
mitochondrial respiratory chain enzyme that utilizes the
cofactor, FAD, to catalyze the oxidation of succinate and
the reduction of ubiquinone. The succinate dehydrogenase enzyme is a heterotetramer composed of a flavoprotein, an iron–sulfur protein, and two hydrophobic
subunits and, as an iron–sulfur protein, is especially
sensitive to oxidative stress (Robinson & Lemire, 1996).
Materials and methods
Chemicals
Dimethyl sulfoxide (DMSO; DMS666, Purity: ≥ 99.9%
Sterile Filtered) produced by BioShop Canada Inc.
(Burlington, Ontario, Canada) was purchased from Lab
Empire (Rzesz
ow, Poland). Dihydroethidine (DHE) was
from Molecular Probes (Leiden, Netherlands). All other
reagents, if not stated otherwise, were purchased from
Sigma (Poznan, Poland) and were of analytical grade.
Components of culture media were from BD Difco
(Becton Dickinson and Company, Spark) except for glucose (POCh, Gliwice, Poland).
Yeast strains, media and growth conditions
The following yeast strains were used: wild-type strain
EG103 (DBY746) (MATa, leu2–3, his3D1, trp1-289, ura352), EG118 (EG103 with sod1D::URA3) (Gralla & Valentine, 1991), EG110 (EG103 with sod2D::TRP1) and EG133
(EG103 with sod1D::URA3, sod2D::TRP1) (Liu et al., 1992).
Yeast was grown in a standard liquid YPD medium (1%
yeast extract, 1% yeast Bacto-peptone, 2% glucose) on a
rotary shaker at 150 r.p.m. at a temperature of 28 °C.
ª 2013 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
822
Effect of DMSO on cell proliferation
Cells from 15-h cultures of yeast (OD600 1) in YPD
medium were washed twice with PBS and suspended in
YPD medium, at a density of 5 9 106 cells mL1. Such
cell suspensions were aliquoted into 96-well transparent
polystyrene plates (Grainier) and were treated with various concentrations of DMSO (0%, 1%, 2%, 4%, 6%, 8%,
10%, 12% or 14%; total volume of the working solution
of 200 lL per well). Cells growth was monitored turbidimetrically at 600 nm in an Anthos 2010 type 17550
microplate reader directly after transfer of the suspensions
of cells to a plate and every 2 h during 18 h as well as
after 24 h.
Effect of DMSO on cell survival
Yeast cultures (OD600 1) in YPD medium were treated
with various concentrations of DMSO at 28 °C. After
incubation (28 °C with shaking) for 1 h, 100 lL of each
of yeast cultures were suspended in Milli-Q Ultrapure
water at a density of 107 cells mL. Then, the samples were
vortexed and centrifuged (12 100 g, 5 min, 28 °C). Eight
hundred microlitre of supernatant of each sample were
discarded and 200 lL of methylene blue solution
(100 lg mL1; methylene blue dissolved in 2% sodium
citrate dihydrate in a volume ratio of 1 : 1) was added to
200 lL of yeast cell suspension for 10 min. A total of
1000 cells per each sample triplicate were analyzed in an
optical microscope, and the percentage of stained cells
was determined.
In parallel, the effect of DMSO exposure on cell survival
was assessed by the colony formation assay. Briefly, after
incubation with DMSO, the cells were centrifuged (8100 g,
2 min) and washed with the YPD medium to remove
DMSO, resuspended in the medium, counted, and diluted
to a concentration 1000 cells mL1. One hundred microlitre of so-prepared suspension was evenly distributed on
the surface of a Petri dish and incubated at 30 °C. After
48 h, the number of colonies was counted.
Determination of ROS level
Dihydroethidine (DHE) enters the cell and is oxidized by
ROS, particularly superoxide, to yield the fluorescent
ethidium. Ethidium binds to DNA (Eth-DNA), which
further amplifies its fluorescence. Thus, increases in DHE
oxidation to Eth-DNA (i.e. increases in Eth-DNA fluorescence) are indicative of superoxide generation (Carter
et al., 1994). After incubation with DMSO, 1 mL of each
of yeast cultures was washed twice with phosphatebuffered saline (PBS), pelleted, and suspended in
100 mM phosphate buffer, pH 7, containing 0.1% glucose
ª 2013 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
I. Sadowska-Bartosz et al.
and 1 mM EDTA, at a density of 108 cells mL1. Two
hundred microlitre per well of yeast cells resuspended in
buffer was transferred to 96-well black polystyrene plates
(Greiner) and DHE was added to a final concentration of
18 lM from a stock solution (1 mg mL1 DMSO) to
each well. The kinetics of fluorescence increase, due to
the oxidation of the fluorogenic probe, was measured
using a TECAN Infinite 200 microplate reader. Measurement conditions were the following: kex = 518 nm and
kem = 605 nm; the temperature was 28 °C. Readings were
registered at 2-min intervals for 10 min. Results were
read from a linear portion of curve. ROS production was
expressed as a relative rate of fluorescence increase (arbitrary units)/number of cells 9 108. No cross-reactivity
between the fluorogenic probe and DMSO was observed
in blank samples in the absence of cells.
Succinate dehydrogenase (SDH) activity assay
SDH activity was measured using the whole cells in situ
assay (Kregiel et al., 2008) with some modifications.
Briefly, 0.5 mL of each of yeast cultures was collected
from cultivation medium by centrifugation (720 g,
10 min, 28 °C) and washed twice with PBS in the same
manner. Supernatant was discarded and the biomass was
added: 0.3 mL of 0.5 M substrate dissolved in water,
3 mL of 0.68 mM nitro blue tetrazolium dissolved in
water and one small crystal of PMS. The mixture was
then incubated at constant temperature of 28 °C with
shaking, and the reaction was stopped after 1 h by the
addition of 0.4 mL of 37% formaldehyde. The samples
were centrifuged (2000 g, 8 min, 28 °C), supernatants
were discarded, and the pellets were resuspended in 2 mL
of undiluted DMSO for extraction of formazan crystals
formed in yeast cells during the assay. Three hundred
microlitre of samples per well was transferred to 96-well
transparent polystyrene plates (Greiner). The final absorbance of DMSO extracts was measured at 540 nm using a
TECAN Infinite 200 microplate reader and calculated as
nmoles formazan/108 cells, using an absorption coefficient
of 0.72 mM1 mm1 (Wang et al., 1998).
Each experiment was performed in triplicate, and each
data were the mean of six measurements.
Estimation of GSH and GSSG content
GSH content was estimated using o-phthalaldehyde (Senft
et al., 2000; Robaszkiewicz et al., 2008). After incubation
with DMSO, yeast cultures were washed twice with 100
mM phosphate buffer pH 6.9, pelleted, and suspended in
100 lL of cooled RQB-TCA buffer (20 mM HCl, 5 mM
diethylenetriaminepentaacetic acid). Then, the mixture
was vortexed, cooled on ice for 15 min, and centrifuged
FEMS Yeast Res 13 (2013) 820–830
823
Effect of DMSO on yeast
(9900 g, 4 °C, 10 min). Pellets were dissolved in 1 mL
5% SDS and 0.1 M NaOH in the ratio of 1 : 4 and were
used to determine protein content (Lowry et al., 1951).
The supernatant was taken for the GSH assay. Two microlitre of deproteinized supernatant diluted to 25 lL
with RQB-TCA was put on two wells (denoted ‘+’ and
‘’) of a 96-well black plate. The sample ‘’ was added
with 4 lL of 7.5 mM N-ethylmaleimide in RQB-TCA;
both samples were added with 40 lL of 1 M potassium
phosphate 100 mM phosphate buffer, pH 7, mixed for
1 min, and incubated at room temperature for 5 min.
Then, 160 lL of 0.1 M potassium phosphate buffer was
added, followed by 25 lL of 0.5% o-phtalaldehyde in
methanol. After 30-min incubation in total darkness
(room temperature, with shaking), the fluorescence was
read at 355 nm/460 nm using a TECAN Infinite 200
microplate reader.
The value obtained for the ‘’ sample was subtracted
from that obtained for the ‘+’ value, and GSH concentration was read from a calibration curve obtained with
glutathione as a standard.
For determining GSSG concentration, two paired
samples, ‘+’ and ‘’, each containing 25 lL of deproteinized supernatant, were added with 4 lL of 7.5 mM
N-ethylmaleimide in RQB-TCA and 40 lL of 1 M potassium phosphate buffer. Then, 5 lL of 100 mM sodium
dithionite in RQB-TCA was introduced into the sample
‘+’. The mixture was incubated at room temperature for
60 min. The remaining part of the procedure was the
same as for GSH estimation. The calibration curve was
prepared with GSSG.
Membrane fluidity measurement
The fluidity of the yeast membranes was measured in the
whole cells using 1,6-diphenyl-1,3,5-hexatriene (DPH)
dissolved in DMSO (stock concentration 2 mM)
(Obrenovitch et al., 1978; Chen et al., 2000). Optical
characteristics of DPH strongly depend on the environment; the dye is almost nonfluorescent in aqueous solutions, while binding to the hydrophobic region of
membranes results in a sharp increase in the fluorescence
signal, with an excitation maximum in the UV range.
After incubation with DMSO, yeast cultures were washed
twice with PBS, pelleted, and suspended in 100 mM
phosphate buffer, pH 7, containing 0.1% glucose and
1 mM EDTA, at a density of 108 cells mL1. Suspensions
of yeast cells were labeled with DPH in the dark at 28 °C
for 20 min (20 lL of 2 mM DPH/mL yeast culture at a
density of 108 cells mL1). The same volume of the solvent (DMSO) was added to the cells as a control. Fluorescence polarization was measured on a Hitachi F2500
fluorescence spectrophotometer equipped with a polarFEMS Yeast Res 13 (2013) 820–830
izer. The excitation and emission wavelengths were 360
and 431 nm, respectively. The measured fluorescence
intensities were corrected for background fluorescence
and light scattering from the unlabelled sample, (a sample
without DPH). The fluorescence anisotropy (r) was calculated using the following equation:
r¼
Ivv Ivh G
Ivv þ 2Ivh G
where Ivv and Ivh are the intensities of fluorescence emitted, respectively, parallel and perpendicular to the direction of the vertically polarized excitation light, and G is
the correction factor (G = Ihv/Ihh) for the optical system
given by the ratio of vertically to the horizontally polarized emission components when the excitation light is
polarized in horizontal direction. An increase in fluorescence anisotropy (r) means a decrease in membrane
fluidity.
Statistical analysis
Data were given in the form of arithmetical mean values
and standard deviations. Differences between means were
analyzed using Kruskal–Wallis test with Tukey’s post hoc
analysis. The statistical analysis of the data was performed using StatSoft, Inc. (2011), STATISTICA, version 10,
www.statsoft.com. P-values of < 0.05 were considered
significant.
Results
Initially, we checked how exposure to DMSO affected the
survival and growth of various yeast strains. DMSO
inhibited the growth of yeast in liquid culture in a concentration-dependent manner; superoxide dismutasedeficient strains were more sensitive to growth inhibition
by DMSO. While 12% DMSO inhibited the growth of
the yeast completely in the wt strain (EG-103), the
growth of sod1D and sod2D strains (EG-118 and EG-110,
respectively) was completely inhibited by 10% DMSO,
and the growth of the double mutant sod1D sod2D
(EG-133) was blocked by 8% DMSO (Fig. 1). The growth
inhibition was due to prolongation of the doubling time.
One-hour exposure of yeast cells did not evoke significant mortality when estimated by methylene blue staining, only a slight loss of survival being noted for most
strains at the highest DMSO concentration used (14%)
(Fig. 2a). However, colony forming assay demonstrated a
significant loss of viability, more pronounced in
SOD-deficient strains and most severe in the sod1D sod2D
(EG-133) strain (Fig. 2b). These results evidence that the
chosen conditions of exposure (lack of significant
ª 2013 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
I. Sadowska-Bartosz et al.
824
(a)
OD 600 nm
1.2
1.0
0.8
0.6
1.8
1.4
1.2
1.0
0.8
0.6
0.4
0.4
0.2
0.2
0.0
0
2
4
6
8
10
12
14
16
18
20
22
0.0
24
EG-118 (sod1Δ::URA3)
Control
1% DMSO
2% DMSO
4% DMSO
6% DMSO
8% DMSO
10% DMSO
12% DMSO
14% DMSO
1.6
OD 600 nm
1.4
(b)
EG-103 (wt)
Control
1% DMSO
2% DMSO
4% DMSO
6% DMSO
8% DMSO
10% DMSO
12% DMSO
14% DMSO
1.6
0
2
4
6
8
10
Time (h)
EG-110 (sod2Δ::TRP1)
(c)
1.6
1.6
1.0
0.8
0.6
1.2
1.0
0.8
0.6
0.4
0.4
0.2
0.2
0.0
0
2
4
6
8
10
12
16
18
20
22
24
20
22
24
Control
1% DMSO
2% DMSO
4% DMSO
6% DMSO
8% DMSO
10% DMSO
12% DMSO
14% DMSO
1.4
OD 600 nm
OD 600 nm
1.2
14
EG-133 (sod1Δ::URA3, sod2Δ::TRP1)
(d)
Control
1% DMSO
2% DMSO
4% DMSO
6% DMSO
8% DMSO
10% DMSO
12% DMSO
14% DMSO
1.4
12
Time (h)
14
16
18
20
22
24
0.0
0
2
4
Time (h)
6
8
10
12
14
16
18
Time (h)
Fig. 1. Effect of DMSO on the growth of various yeast strains. Yeast growth in YPD medium, in suspensions of initial density of 5 9 106 cells
mL1, with shaking, was monitored turbidimetrically at 28 °C.
immediate mortality) were appropriate to study the biochemical/biophysical effects of DMSO on the yeast.
To examine the effect of DMSO on the redox equilibrium of yeast cells, its effect on the generation of reactive
active species, activity of succinate dehydrogenase, a FeS
enzyme sensitive to oxidative stress, and the content of
glutathione, the main redox buffer of the cells was
assessed.
Exposure to DMSO-induced oxidative stress as
evidenced by enhanced production of reactive oxygen
species in yeast cells. Oxidation of dihydroethidine, specific
for the superoxide radical, was enhanced with increasing
DMSO concentrations, the increase being higher for the
sod1D, sod2D, and sod1Dsod2D strains (Fig. 3).
SDH activity decreased with increasing DMSO concentration. In the control and at the highest DMSO concentrations, the decrease was significantly lower in the
sod1Dsod2D strain than in the control EG-103 strain
(Fig. 4).
The concentration of reduced glutathione increased after
DMSO treatment, the increase being higher in the wt
EG-103 strain than in its SOD-deficient mutants (Fig. 5).
The concentration of oxidized glutathione was also augmented with increasing DMSO concentration (Fig. 6). No
significant changes in the GSSG/GSH ratio were observed.
In view of the known membrane action of DMSO, the
effect of this compound on membrane fluidity was studied
ª 2013 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
by measurements of DPH anisotropy. Anisotropy of the
probe increased after DMSO treatment in all strains studied, indicating a loss of fluidity (Fig. 7).
Discussion
DMSO has been used as a cryoprotective and radioprotective agent. Both these actions have been ascribed to the
radical-scavenging effects of this compound (Homer
et al., 2005; Kashino et al., 2007).
Although early studies found that organic solvents
mainly destroy the integrity of cell membranes by accumulating in the lipid bilayer of plasma membranes, the
cellular metabolic responses to the presence of an organic
solvent remain unclear. Srivastava and Smith studied the
effect of cryoprotective agents on the growth and ultrastructure of S. cerevisiae. As compared to cells grown in
the absence of antifreeze compounds, electron microscopy
of the cells which grew at 30 °C in the presence of antifreeze compounds showed thicker cell walls, highly convoluted plasmalemma, vacuoles filled with electron-dense
fibrous material, spherosomes, poorly developed mitochondria, and many vesicles (Srivastava & Smith, 1980).
Yee et al. demonstrated that while exposure of yeast to
increasing concentrations of DMSO resulted in decreasing
cell viability, it did not cause cell lysis or protein leakage
from the cells. Additionally, the inclusion of DMSO in
FEMS Yeast Res 13 (2013) 820–830
825
Effect of DMSO on yeast
(a)
110
EG-103 (wt)
EG-118 (sod1Δ::URA3)
EG-110 (sod2Δ::TRP1)
EG-133 (sod1Δ::URA3, sod2Δ::TRP1)
100
90
a
c
b
Survival [%]
80
70
60
50
40
30
20
10
0
0
1
2
4
6
8
10
12
14
DMSO concentration [%]
(b)
120
EG-103 (wt)
EG-118 (sod1Δ::URA3)
EG-110 (sod2Δ::TRP1)
EG-133 (sod1Δ::URA3, sod2Δ::TRP1)
110
100
Survival [%]
90
80
Δ
a
70
60
b
*
a b
a
Δ
c
50
40
30
20
10
0
0
1
2
4
6
8
10
12
14
DMSO concentration [%]
Fig. 2. Effect of exposure to DMSO on the survival of various yeast strains, estimated with methylene blue staining (a) and colony forming assay
(b). Statistically significant differences: a, EG-103, vs. control (0% DMSO), b, EG-118, vs. control and 1% DMSO; c, EG-133 vs. control and 1%
DMSO (a); EG-110 vs. control (0% DMSO) and 1% DMSO; b, EG-133 vs. control; c, EG-133 vs. control (0% DMSO) and 1% DMSO. Interstrain
differences: Δ, with respect to EG-103 and EG-118; *, vs. EG-103 (b).
the growth medium resulted in the conversion of yeast
cultures to respiratory deficient petites. This mutagenic
effect requires cell growth for its expression (Yee et al.,
1972). Panek et al. reported that DMSO is able to permeate glucose and cAMP. The effects of glucose and cAMP
were enhanced by pre-incubating the cells in the presence
of DMSO. No effects were observed during the heat shock,
suggesting that the solvent acts on the cell membrane
(Panek et al., 1990). Murata et al. proposed that DMSO
treatment induces membrane proliferation in yeast cells to
alleviate the adverse affects of this chemical on membrane
integrity. Yeast exposed to DMSO increased phospholipid
biosynthesis through up-regulated gene expression. It was
confirmed by northern blotting that the level of INO1
and OPI3 gene transcripts, encoding key enzymes in
FEMS Yeast Res 13 (2013) 820–830
phospholipid biosynthesis, was significantly elevated
following treatment with DMSO. Furthermore, the phospholipid content of the cells increased during exposure to
DMSO as shown by conspicuous incorporation of a lipophilic fluorescent dye (3,3’-dihexyloxacarbocyanine iodide)
into cell membranes (Murata et al., 2003). Zhang et al. used
microarray analysis of c. 6200 yeast open reading frames
(ORFs) to monitor the global gene expression profiles of
Saccharomyces cerevisiae BY4743 grown in media with a
high concentration of DMSO. Genomic analyses showed
that 1338 genes were significantly regulated by the presence
of DMSO in yeast. Among them, only 400 genes were previously found to be responsive to general environmental stresses, such as temperature shock, amino acid starvation,
nitrogen source depletion, and progression into stationary
ª 2013 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
I. Sadowska-Bartosz et al.
Relative fluorescence [a.u./number of cells × 108]
826
800
EG-103 (wt)
700
EG-118 (sod1Δ::URA3)
600
EG-110 (sod2Δ::TRP1)
Δ
*e
*c *f
EG-133 (sod1Δ::URA3, sod2Δ::TRP1)
b
a
a
500
b
*
*
*d e
400
300
200
100
0
0
1
2
4
6
8
DMSO concentration [%]
10
12
14
Fig. 3. Effect of DMSO exposure on the generation of reactive oxygen species in yeast cells, estimated with dihydroethidine. Statistically significant
differences: Within-strain differences: a, EG-103, vs. control and 1% DMSO; b, EG-118, vs. 1%, 2% and 4% DMSO; c, EG-118, vs. 1% and 2%
DMSO; d, EG-110, vs. control and 1% DMSO; e, EG-133, vs. control and 1%, and 2% DMSO; f, EG-133, vs. control and 1% DMSO. Interstrain
differences: Δ, significant differences with respect to EG-103, EG-110 and EG-118, *, significant differences with respect to EG-103.
EG-103 (wt)
14
Succinate dehydrogenase activity
[nM/number of cells × 108]
EG-118 (sod1Δ::URA3)
12
EG-110 (sod2Δ::TRP1)
EG-133 (sod1Δ::URA3, sod2Δ::TRP1)
10
8
**
**
**
**
6
b
c
**
*
*
**
f
a d
*
h
e
g
4
2
0
0
1
2
4
6
8
DMSO concentration [%]
10
12
14
Fig. 4. Effect of DMSO exposure on the succinate dehydrogenase activity of yeast cells. Within-strain differences: a, EG-103, vs. control, 1% and
2% DMSO, b, EG-103, vs. control, and 1% DMSO; c, EG-103, vs. control; d, EG-118, vs. control and 1% DMSO; e, EG-110, vs. control, 1%,
2%, and 4% DMSO; f, EG-110, vs. control and 1% DMSO; g, EG-133, vs. control, 1%, 2% and 4% DMSO; h, EG-133, vs. control and 1%
DMSO. Interstrain differences: *, vs. EG-103 and EG-118.
phase. The DMSO-responsive genes were involved in a variety of cellular functions, including carbohydrate, amino acid
and lipid metabolism, cellular stress responses, and energy
metabolism. Most of the genes in the lipid biosynthetic
pathways were down-regulated by DMSO (Zhang et al.,
2013). The study of Momose et al. supported that view that
exposure to cryoprotectants prior to freezing not only
reduces the freeze–thaw damage but also affects various processes to the recovery from freeze–thaw damage. These
ª 2013 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
authors found that DMSO increased the expression of genes
involved in protein refolding and trehalose increased the
expression of genes involved in spore formation (Momose
et al., 2010). Nishida et al. suggested recently that overexpression of genes encoding proteins effective for tolerance
of specific organic solvents would enable enhanced tolerances for practical use (Nishida et al., 2013).
Due to its anti-inflammatory therapeutic effects ascribed
to the ROS-scavenging action, DMSO has been proposed
FEMS Yeast Res 13 (2013) 820–830
827
Effect of DMSO on yeast
Reduced glutathione content [nM/mg protein]
9000
8000
7000
6000
EG-103 (wt)
EG-118 (sod1Δ::URA3)
EG-110 (sod2Δ::TRP1)
EG-133 (sod1Δ::URA3, sod2Δ::TRP1)
*
*
*
*
*
b
*
*
b
a
a
5000
4000
3000
2000
1000
0
0
1
2
4
6
8
DMSO concentration [%]
10
12
14
Oxidized glutathione content [nM/mg protein]
Fig. 5. Effect of DMSO exposure on the reduced glutathione content of yeast cells. Statistically significant differences: a, EG-103, vs. control; b,
EG-118, vs. control; *, significant difference with respect to all other strains.
2000
1800
1600
EG-103 (wt)
EG-118: EG-103 with sod1Δ
EG-103 with sod2Δ
EG-133: EG-103 with sod1Δ::URA3 sod2Δ::TRP1
c
c
a b
a
c
a
d
d
b
b
1400
1200
1000
800
600
400
200
0
0
1
2
4
6
8
DMSO concentration [%]
10
12
14
Fig. 6. Effect of DMSO exposure on the oxidized glutathione content of yeast cells. Statistically significant differences: a, EG-103, vs. control
(0% vs. DMSO); b, EG-118, vs. control; c, EG-110 vs. control; d, EG-133 vs. control.
as a remedy for several gastrointestinal disorders (Salim,
1991). DMSO has been suggested for the treatment of
dermatological disorders, intractable urinary frequency
(Sehtman, 1975; Okamura et al., 1985; Goto et al., 1996),
and manifestations of amyloidosis (Morassi et al., 1989).
Furthermore, DMSO can cross the blood–brain barrier and
has a beneficial effect in the treatment of traumatic brain
edema (Ikeda & Long, 1990) and Alzheimer’s disease
(Regelson & Harkins, 1997). This solvent has the ability to
antagonize thrombocyte adhesion and aggregation,
suppress the tissue factor expression, reduce thrombus formation, and inhibit vascular smooth muscle cell proliferation and migration (Markvartova et al., 2013).
FEMS Yeast Res 13 (2013) 820–830
However, more recent studies casted doubt on the antioxidant action of DMSO in vivo. This compound has been
found to have no effect on the oxidative stress induced by
ovalbumin sensitization in a guinea pig model of allergic
asthma (Mikolka et al., 2012). DMSO alleviated oxidative
stress in the serum and kidney but not in the liver of irradiated rats (Cosar et al., 2012). The radioprotective action of
DMSO has been reinterpreted and ascribed to facilitation
of DNA double-strand break repair rather than free-radical
scavenging (Regelson & Harkins, 1997).
DMSO was found to protect membranes of mammalian
cells against lipid peroxidation, preventing peroxidationdependent effects (Till et al., 1985; Rauen et al., 1997).
ª 2013 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
I. Sadowska-Bartosz et al.
828
0.6
0.5
r
0.4
EG-103 (wt)
EG-118 (sod1Δ::URA3)
*
*
*
*
*
**
*
*g
g
d
j
b
*f
b
i
a
c e
h
0.3
0.2
0.1
0.0
0
1
2
4
6
8
DMSO concentration [%]
10
12
14
Fig. 7. Effect of DMSO exposure on yeast membrane lipid anisotropy. Statistically significant differences: Within-strain differences: a, EG-103, vs.
control, 1% DMSO and 2% DMSO; b, EG-103, vs. control; c, EG-118, vs. control, 1% and 2% DMSO; d, EG-118, vs. control and 1% DMSO; e,
EG-110, vs. control, 1% and 2% DMSO; f, EG-110, vs. 1% DMSO; g, EG-110, vs. control; h, EG-133, vs. control, 1% and 2% DMSO; i,
EG-133, vs. control and 1% DMSO; j, EG-133, vs. control. Interstrain differences: *, significant vs. EG-103 and EG-133.
However, this protective action was not observed in all
systems studied and it has been suggested that secondary
radicals of DMSO generated in reactions with reactive oxygen species may be of sufficient reactivity to initiate lipid
peroxidation (Bartosz & Leyko, 1981; Miller & Raleigh,
1983). While the protection against lipid peroxidation
might contribute to the antioxidant activity of mammalian
cells, this phenomenon would be of minor importance in
yeast cells that lack the ability to synthesize polyunsaturated
fatty acids and their membranes contain mainly monounsaturated fatty acid residues which are poor substrates for
peroxidation (Uemura, 2012). On the other hand, DMSO
is known to induce differentiation of HL-60 cells, expression of NADPH oxidase and respiratory burst, which is a
pro-oxidant action (Jiang et al., 2006). Also, this effect is
absent from yeast cells but may be of concern when studying animal cells in culture.
The present results demonstrate that DMSO, when
present in the growth medium, inhibits the growth of
yeast cells in a dose-dependent manner and SOD-deficient
strains in the EG-103 background are more sensitive to
this action. As the viability loss is moderate after 1-h
exposure to DMSO, the inhibition of cell growth seems to
be due to cell cycle arrest. The higher sensitivity of
SOD-deficient strains suggests involvement of oxidative
stress in this effect of DMSO (Fig. 1). Indeed, 1-h exposure to DMSO caused a concentration-dependent increase
in the generation of reactive oxygen species. Dihydroethidine is specific for the superoxide radical anion, so the
results demonstrate increased generation of superoxide,
higher in SOD-deficient mutants of EG-103.
ª 2013 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
Another evidence of DMSO-induced oxidative stress
was the inactivation of SDH (Fig. 4), a FeS enzyme, being
a sensitive target for free-radical oxidation (Ishii et al.,
2011). The level of glutathione was enhanced in yeast
exposed to DMSO, which seems to represent a compensatory reaction to oxidative stress (Figs 5 and 6).
Which can be the mechanism of induction of oxidative
stress by DMSO? It has been postulated that the action of
various antibiotics, of different mechanisms of action,
results in oxidative stress due to derangement of the
tricarboxylic acid cycle, a transient depletion of NADH,
destabilization of iron–sulfur clusters, and stimulation of
the Fenton reaction (Kohanski et al., 2007). Seemingly,
this mechanism can be generalized to propose that any
serious derangement of cellular structure and function is
likely to induce oxidative stress, as proper cell functioning
is aimed at minimizing undesired oxidative stress. Treatment of cells with a rather high concentration of DMSO
may lead to derangement of cellular fine structure,
including organization of mitochondria, which may easily
lead to oxidative stress.
Estimation of membrane fluidity showed a progressive
increase in membrane anisotropy (an inverse measure of
membrane fluidity) with increasing DMSO concentration.
This, again, seems to represent a compensatory response
to increase in membrane permeability induced by this
compound (Hao et al., 2010; Nocca et al., 2012; Hazen,
2013).
In summary, the present data demonstrate that DMSO
induces oxidative stress in yeast cells. DMSO is a useful
cryoprotectant employed also for freezing of yeast cells.
FEMS Yeast Res 13 (2013) 820–830
Effect of DMSO on yeast
However, its cryoprotective action does not have to
involve antioxidant effects but be based, for example, on
membrane-permeabilizing properties preventing transient
formation of destructive transmembrane concentration
gradients during freezing and thawing process and prevention of crystal formation (Pegg, 2007).
Acknowledgements
The authors declare no conflict of interest.
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