Effect of Sodium Chloride and Cadmium on the Growth,Oxidative Stress and Antioxidant EnzymeActivities of Zygosaccharomyces rouxii

LI Chunsheng,XU Ying,JIANG Wei,LV Xin,and DONG Xiaoyan

College of Food Science and Engineering,Ocean University of China,Qingdao 266003,P.R.China

? Ocean University of China,Science Press and Spring-Verlag Berlin Heidelberg 2014

1 Introduction

Cadmium(Cd)is a toxic heavy metal,which is known as an important environmental pollutant and a potent human carcinogen.It interferes with many cellular processes,and one of the main consequences is the enhanced production of reactive oxygen species(ROS)related to oxidative stress(Guoet al.,2007; Latinwoet al.,2006).These ROS,i.e.,superoxide radical(O2?),hydrogen peroxide(H2O2)and hydroxyl(?OH),are highly toxic to the cells and rapidly attack all biomolecules such as DNA,lipids,proteins and amino acids(Finkel and Holbrook,2000; Woodmansee and Imlay,2002),eventually leading to cell death.To mitigate the ROS-induced oxidative damage,yeast cells have developed a complex antioxidative defense system,including non-enzyme scavengers(e.g.,glutathione,uric acid,vitamin C and vitamin E)and antioxidant enzymes(e.g.,superoxide dismutase,SOD; catalase,CAT; and peroxidases,POD)(Anet al.,2012; Kumaret al.,2011).Of these,SOD is the major O2?scavenger whose enzymatic action results in the formation of H2O2and O2,while the accumulation of H2O2in cells is prevented by CAT and POD(Yuanet al.,2011; Zhanget al.,2007).

Along with rapid industrial development,Cd pollution poses a great threat to marine environments,especially aquaculture ecosystems.Due to biomagnification and accumulationviathe food chain,Cd pollution may result in an increasing risk of seafood contamination(Stankovic and Jovic,2012; Whyteet al.,2007).The biosorption method,which takes advantage of microbial cells,is considered promising for solving Cd pollution problems because of the low cost,high removal efficiency,regeneration of the biosorbent,and the possibility of metal recovery(Cruzet al.,2004).Presently,the application of microbial cells as biosorbents in seawater and highly salty food is restricted due to the high salt environment.Research has shown that sodium chloride(NaCl)stress can lead to oxidative damage to microbial cells through an increase in ROS production(Mittovaet al.,2002; Qunet al.,2007; Srivastavaet al.,2005).Up to now,there has been a total lack of information in the literature regarding the effect of salt on microorganisms under Cd stress.

Zygosaccharomyces rouxiiis a salt-tolerant yeast species commonly used for aroma development in the processes of soy and fish sauce fermentation.It can tolerate higher salt concentrations than another most useful yeastSaccharomyces cerevisiae(Liet al.,2013; Tomitaet al.,2000).Our previous study showed thatZ.rouxiiwas capable of removing Cd from aqueous solution(Xuet al.,2009).However,the effects of Cd and NaCl on the physiology ofZ.rouxiiremain poorly understood.Therefore,the aims of this study were:1)to determine the effects of NaCl and Cd on the growth and oxidative stress ofZ.rouxiiand 2)to explore the antioxidative defense mechanism based on enzyme activity assays of antioxidant SOD,CAT,and POD inZ.rouxii.The results will provide physiological clues to the possible use ofZ.rouxiias a biosorbent for Cd removal from seawater and liquid highly salty food.

2 Materials and Methods

2.1 Yeast Strain and Pre-Culturing

Z.rouxii(CICC1379)used in this study was purchased from the China Center of Industrial Culture Collection(CICC)and maintained in YEPD agar slants(1% yeast extract,2% peptone,2% glucose,and 2% agar,pH 5.0)at 4℃ prior to use.Pre-culturing ofZ.rouxiiwas done by activating cells twice.TheZ.rouxiicells were first transferred to a fresh YEPD agar slant and incubated at 30℃in a constant temperature incubator for 24 h.Thereafter,a loopful ofZ.rouxiicells were transferred to 250 mL Erlenmeyer flasks containing 50 mL YEPD liquid medium and incubated in an incubator set at 30℃ and 180 r min?1for 24 h.

2.2 Evaluation of the Effects of NaCl and Cd on the Growth of Z.rouxii

The stock Cd solution(100 mg L?1)was prepared by dissolving CdCl2?2.5H2O(Sinopharm Group,China)in ultrapure water.The pre-culturedZ.rouxiiwas grown in YEPD liquid medium supplemented with different concentrations of Cd and NaCl for 24 h.The absorbance of 10-fold diluted culture sample was measured at 600 nm with a spectrophotometer(UV-2800,Unico)to quantify the cell biomass.The cell growth inhibition rate(reduction rate of cell biomass compared with control)was used to analyze the effects of NaCl and Cd on the growth ofZ.rouxii.

2.3 Flow Cytometry Assays

The effects of NaCl and Cd on the oxidative stress ofZ.rouxiiwere studied by flow cytometry assay of the ROS level and cell death rate after double staining with 2’,7’-dichlorofluorescein diacetate(DCFH-DA,Sigma)and propidium iodide(PI,Sigma).TheZ.rouxiicells incubated with different concentrations of Cd and NaCl for 24 h were washed with 0.85% NaCl solution twice and resuspended in 1 mL of 0.85% NaCl at about 1×107cells mL?1.After incubation with 100 μmol L?1DCFH-DA at 37℃and 180 r min?1for 50 min in the dark,the samples were rapidly placed on ice,washed twice,resuspended in 1 mL of 0.85% NaCl,and then stained with 10 μg mL?1PI.The fluorescence of cell suspensions was recorded with a flow cytometer(Beckman Coulter Cytomics FC 500 MPL)equipped with an air-cooled 488 nm argon laser.Green fluorescence of the cells stained with 2’,7’-dichlorofluorescein(DCF)was collected in the FL1 channel(525 nm ± 20 nm),and red fluorescence of the cells labeled with PI was collected in the FL3 Channel(625 nm ± 20 nm).A total of 20000 events were registered per sample and the obtained data were analyzed with the CXP Analysis 2.1 software.

2.4 Antioxidant Enzyme Activity Assays

Z.rouxiicells cultured with various concentrations of Cd and NaCl for 24 h were centrifuged and washed twice with 0.85% NaCl solution.The cells were ground with liquid nitrogen,resuspended in 0.05 mmol L?1phosphatebuffered solution(pH 7.0),and then centrifuged at 7000 r min?1for 10 min.The supernatant was collected and used for enzymatic activity assays of SOD,CAT,and POD spectrophotometrically with a commercial reagent kit(Nanjing Jiancheng Bioengineering Institute,China)(Yuanet al.,2011).According to the manufacturer’s instructions,the activity of SOD was assayed at 550 nm using the xanthine and xanthine oxidase system.One unit(U)of SOD activity was defined as the amount of SOD required for 50% inhibition of the xanthine and xanthine oxidase system reaction in 1 mL of enzyme extraction of 1 mg of protein at 37℃.The activity of CAT was determined based on the rate of H2O2decomposition by the enzyme.The samples were treated with excess H2O2at 37℃,and the absorbance of remaining H2O2was measured at 405 nm.One unit of CAT activity was defined as 1 μmol H2O2decomposed in 1 mg of protein per second.According to the reaction principle of POD catalysis of H2O2,the activity of POD was determined by detection of changes in the absorbance at 420 nm.One POD unit was defined as the amount of enzyme needed to catalyze 1 μg of substrate per minute per milligram of total protein at 37℃.

2.5 Statistical Analyses

All data presented here are the mean ± SD(n=3).Statistical analyses were performed with one-way analysis of variance(ANOVA).A multiple comparison Tukey test was applied to evaluate the differences among treatments.In all cases the level of statistical significance was set atP< 0.01.

3 Results

3.1 Growth-Inhibiting Effect of NaCl and Cd on the Growth of Z.rouxii

When grown in the YEPD medium,the growth ofZ.rouxiiwas inhibited in the presence of NaCl or Cd alone,but the growth-inhibiting effect of high-level Cd(e.g.,> 6 mg L?1)was reduced in the presence of 2%–8% NaCl(Fig.1).For example,the growth inhibition rate ofZ.rouxiiincubated with 20 mg L?1Cd for 24 h decreased from 78.4% to 45.0% when the YEPD medium was supplemented with 6% NaCl(Fig.1).

Fig.1 Effect of NaCl and Cd at different concentrations on the growth of Z.rouxii after 24 h incubation.

3.2 Effect of NaCl and Cd on ROS Production and Cell Death of Z.rouxii

Although 2%–6% NaCl did not significantly affect the ROS production ofZ.rouxii,the ROS-positive cell rate significantly increased in the presence of 8% NaCl compared with the control(Fig.2a).There was no significant difference in the death rate ofZ.rouxiiin the presence of 2%–8% NaCl(Fig.2b).As compared with NaCl,Cd had a greater effect on the ROS production and cell death ofZ.rouxii(Fig.2).The intracellular ROS level and death rate ofZ.rouxiisignificantly increased with the Cd concentration from 0 mg L?1to 8 mg L?1(Figs.2a,2c).With continuous increases in the Cd concentration,a significant shift of cells to quadrant K2 and K1 was observed,indicating the increase in cell death rate(Figs.2b,2c).In quadrant K3,there remained 38.4% living cells in the presence of 50 mg L?1Cd despite that high-level Cd could cause serious oxidative stress and cell death(Fig.2c).

The Cd-induced ROS production ofZ.rouxiicells was significantly attenuated after incubation with NaCl and Cd for 24 h.The ROS-positive cell rates ofZ.rouxiigrown with 8 mg L?1Cd in the presence of 2%,4%,6% and 8% NaCl were 3.9%,4.0%,5.1% and 5.9%,respectively,obviously lower than that in the absence of NaCl(40.0%)(Fig.2a).In the presence of NaCl,the death rate ofZ.rouxiiunder Cd stress significantly decreased(Fig.2).The death rate ofZ.rouxiitreated with 50 mg L?1Cd respectively decreased from 62.7% to 26.5%,25.4%,20.7% and 26.5% in the presence of 2%,4%,6% and 8% NaCl.

3.3 Effect of NaCl and Cd on the Activities of SOD,CAT and POD of Z.rouxii

The effects of NaCl and Cd on the SOD activity ofZ.rouxiiare summarized in Fig.3.The SOD activity first increased and then decreased with an increasing concentration of NaCl or Cd.However,the combined use of NaCl and Cd produced different experimental results.In the presence of 20 mg L?1Cd,the SOD activity ofZ.rouxiifirst decreased and then increased with an increasing concentration of NaCl(Fig.3a).In the presence of 6% NaCl,the SOD activity ofZ.rouxiisignificantly increased compared with the control but showed no significant differences among Cd treatments at various concentrations(Fig.3b).

The effects of NaCl and Cd on the CAT activity ofZ.rouxiiare shown in Fig.4.In the presence of 2%–6% NaCl,the CAT activity ofZ.rouxiisignificantly increased after 24 h incubation.As compared with that of control,the CAT activity ofZ.rouxiislightly increased in the presence of 8 mg L?1Cd but decreased in the presence of 20 mg L?1and 50 mg L?1Cd.Under Cd stress,the CAT activity ofZ.rouxiisignificantly increased in the presence of NaCl,especially at 6%.The CAT activity ofZ.rouxiiin the presence of 6% NaCl combined with 8,20 and 50 mg L?1Cd was 54.3%,154.7% and 320.7% higher than that in the absence of NaCl,respectively.However,there was no significant difference in the CAT activity ofZ.rouxiiamong the tested Cd concentrations(0–50 mg L?1)in the presence of 6% NaCl,similar to the observations of SOD activity.

The POD activity ofZ.rouxiiin the presence of different concentrations of NaCl and Cd is shown in Fig.5.In the absence of Cd,the POD activity ofZ.rouxiiincreased with the NaCl level and peaked in the presence of 6% NaCl,consistent with the changes in the CAT activity.As compared with that of control,the POD activity ofZ.rouxiirelatively decreased after Cd treatment(Fig.5a)but was significantly improved after combined use of 6% NaCl.The POD activity ofZ.rouxiishowed 163.9%,234.1% and 88.0% increases in presence of 6% NaCl combined with 8,20 and 50 mg L?1Cd,respectively.

4 Discussion

4.1 Increased Antioxidant Enzyme Activities May Contribute to the High Salt Tolerance of Z.rouxii

It has been proposed thatZ.rouxiican tolerant highlevel NaCl mainlyviatwo mechanisms:1)the adaptation to high osmotic pressure and 2)the intracellular cation homeostasis.When the surrounding medium contains high-level NaCl,large amounts of osmotically compatible solutes,such as glycerol and arabitol,can be synthesized and accumulated inZ.rouxiicells to acclimate to the salt stress(Reedet al.,1987; Yoshikawaet al.,1995).Z.rouxiican efficiently excrete Na+into the extracellular medium via Na+-ATPase and Na+/H+-antiporter in order to counteract the potential of metabolic failure caused by the Na+influx into cells(Pribylovaet al.,2008; Watanabeet al.,2003).In the present study,results showed that the enhanced antioxidant enzyme activities possibly contributed to the salt tolerance ofZ.rouxii.When NaCl was added to the medium,the activities of major antioxidant enzymes(i.e.,SOD,CAT and POD)significantly increased even in the presence of 6% NaCl(Figs.3a,4a and 5a),while the corresponding ROS levels showed no significant increases compared with the control.The increased SOD and POD activities have been shown to protect arbuscular mycorrhizae plants from salinity injury and to improve their salt tolerance(Qunet al.,2007).However,we observed that the SOD,CAT and POD activities ofZ.rouxiidecreased in the presence of 8% NaCl(Figs.3a,4a and 5a).It suggested that there was no sufficient antioxidant enzyme activities to scavenge excess O2?and H2O2accumulated inZ.rouxii,leading to a significant increase in the ROS level at 8% NaCl(Fig.2a).

Fig.2 Effect of NaCl and Cd on the ROS production(a)and cell death(b)of Z.rouxii after 24 h incubation.Bars with different letters are significantly different at P < 0.01.(c)Representative flow cytometry dot plots of Z.rouxii cells after incubation with NaCl and Cd.Quadrant K1:DCF?/PI+,no(or low)ROS accumulation,dead cells; quadrant K2:DCF+/PI+,high ROS accumulation,dead cells; quadrant K3:DCF?/PI?,no(or low)ROS accumulation,living cells; quadrant K4:DCF+/PI?,high ROS accumulation,living cells.

Fig.3 Effect of NaCl(a)and Cd(b)on the SOD activity of Z.rouxii after 24 h incubation.Bars with different letters are significantly different at P < 0.01.

Fig.4 Effect of NaCl(a)and Cd(b)on the CAT activity of Z.rouxii after 24 h incubation.Bars with different letters are significantly different at P < 0.01.

Fig.5 Effect of NaCl(a)and Cd(b)on the POD activity of Z.rouxii after 24 h incubation.Bars with different letters are significantly different at P < 0.01.

4.2 Effect of NaCl on Alleviating Cd Toxicity to Z.rouxii

In this study,the presence of NaCl or Cd alone obviously inhibited the cell growth ofZ.rouxii(Fig.1).The results showed that Cd was more toxic toZ.rouxiidue to the increase in cellular ROS level,leading to more significant cell death(Fig.2).However,the growth-inhibiting effect of Cd onZ.rouxii(Fig.1)as well as the Cdinduced ROS production and cell death ofZ.rouxii(Fig.2)were reduced by combined use of NaCl.The overproduction of ROS is a common consequence of different stress factors,e.g.,high-level Cd or NaCl.Microbial cells have developed a complex antioxidative defense system to maintain metabolic functions under stress conditions,and the stimulation of antioxidant enzyme activities is one of the most important pathways through which this can be done.As the first enzyme of the enzymatic antioxidative pathway,SOD plays a key role in the cellular defense system against ROS.The activity of SOD modulates the relative amounts of O2?and H2O2while decreasing the risk of ?OH radical formation(Bowleret al.,1992).In the present study,the SOD activity increased with the concentrations of NaCl and Cd in combination,but was lower in the presence of low-level NaCl combined with Cd than in the absence of NaCl(Fig.3).A possible reason is that the two metal ions mutually decreased their penetration intoZ.rouxiicells and thus reduced NaCl and Cd accumulation in cells(Jianget al.,2013; Liet al.,2013; Lefèvreet al.,2009; Navarreteet al.,2009; Martynaet al.,2000),further preventing the stimulation of SOD in the presence of low-level NaCl and Cd but enhancing the stimulation of SOD in the presence of high-level NaCl and Cd.

The product of SOD decomposition,i.e.,H2O2,remains toxic to cells and must be eliminated by subsequent conversion to H2O.Both CAT and POD are important enzymes that regulate intracellular H2O2levels.In our work,the CAT activity ofZ.rouxiiunder Cd stress increased after the addition of NaCl,suggesting that NaCl has a similar protective effect onZ.rouxiicells despite the presence of Cd(0–50 mg L?1)(Fig.4b).These results indicate that NaCl has a greater effect on CAT activity and can more significantly increase the CAT activity compared with Cd.Under NaCl and Cd stress,the enhanced CAT activity ofZ.rouxiicontributed to the decrease in ROS production.It was reported that CAT activity in coordination with SOD activity played a central role in accelerating the scavenging of O2?and H2O2(Badawiet al.,2004; Lianget al.,2003; Mittovaet al.,2002).

As one of the principal enzymes involved in the elimination of ROS,POD is more efficient than CAT in H2O2scavenging because of its high substrate affinity(Zhanget al.,2007).In the present study,the presence of NaCl significantly alleviated Cd-induced inhibition of POD activity(Fig.5),thus increasing the efficiency in detoxifying H2O2.This might be another important mechanism for the decrease in Cd-induced ROS production and cell death ofZ.rouxiiafter incubation with NaCl and Cd.

Acknowledgements

The authors would like to thank for the financial support of the National Natural Science Foundation of China(Grant Nos.31101330 and 30972289),the Natural Science Foundation of Shandong Province in China(Grant No.ZR2010CM043),the International Joint Research Program(Grant No.2010DFA31330),and the Program for Changjiang Scholars and Innovative Research Team in University(Grant No.IRT1188).

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