Dimethyl sulfoxide induces oxidative stress in the yeast

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

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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

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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

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(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


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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

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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


Relative fluorescence [a.u./number of cells × 108]

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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.

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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

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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.

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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


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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


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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