Diphenyl Diselenide Protects against Methylmercury-Induced Toxicity

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Diphenyl Diselenide Protects against Methylmercury-Induced Toxicity in Saccharomyces cerevisiae via the Yap1 Transcription Factor Fabricio Luís Lovato,† Joaõ Batista Teixeira da Rocha,*,† and Cristiane Lenz Dalla Corte*,†,‡ †

Departamento de Bioquímica e Biologia Molecular, Programa de Pós-graduaçaõ em Ciências Biológicas: Bioquímica Toxicológica, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Cep 97105-900 Santa Maria, RS, Brazil ‡ Universidade Federal do Pampa, Campus Caçapava do Sul, Av. Pedro Anunciaçaõ , 111, Vila Batista, 96570-000 Caçapava do Sul, RS, Brazil ABSTRACT: Methylmercury (MeHg) is a ubiquitous and persistent environmental pollutant that induces serious neurotoxic effects. Diphenyl diselenide [(PhSe)2], an organoseleno compound, exerts protective effects against MeHg toxicity, although the complete mechanism remains unclear. The aim of this study was to investigate the mechanisms involved in the protective effect of (PhSe)2 on the toxicity induced by MeHg using wild-type Saccharomyces cerevisiae and mutants with defects in enzymes and proteins of the antioxidant defense system (yap1Δ, ybp1Δ, ctt1Δ, cat1Δ, sod1Δ, sod2Δ, gsh1Δ, gsh2Δ, gtt1Δ, gtt2Δ, gtt3Δ, gpx1Δ, gpx2Δ, trx1Δ, trx2Δ, trx3Δ, and trr2Δ). In the wild-type strain, (PhSe)2 protected against the growth inhibition, reactive oxygen species production, and decrease in membrane integrity induced by MeHg and restored thiol levels to values indistinguishable from the control. Single deletions of yap1, sod1, sod2, gsh1, gsh2, gpx1, gpx2, trx1, trx2, and trx3 decreased the capacity of (PhSe)2 to prevent MeHg toxicity in yeast, indicating their involvement in (PhSe)2 protection. Together, these results suggest a role of (PhSe)2 in modulating the gene expression of antioxidant enzymes and ABC transporters through the action of the transcription factor YAP1, preventing the oxidative damage caused by MeHg in S. cerevisiae.

1. INTRODUCTION Mercury is an element of global importance because large quantities have been released into the environment by natural processes or anthropogenic activities.1 Consequently, humans can be exposed to Hg in different ways, including through air, water and food.2 Humans can be exposed to organic forms of Hg, particularly to the organometallic cation methylmercury ([CH3Hg]+, MeHg), by eating fish and seafood, because all forms of fish contain MeHg at different levels.3 MeHg has a strong affinity for thiol (-SH) groups, which contributes to a more uniform distribution of the compound in the body after long-term exposure.2,4,5 The neurotoxic effects of MeHg are widely known.6 Individuals intoxicated with MeHg present sensory disorders in the hands and feet, hearing and visual impairment, weakness, and, in extreme cases, paralysis and death.7 Although the exact mechanisms underlying the toxicity of MeHg are not yet completely understood, there are points of evidence indicating a central role of oxidative stress in this process, which is related to the high affinity of MeHg for endogenous -SH and selenol (-SeH) groups.8,9 Inhibitory effects of antioxidant enzymes such as glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) by MeHg have been postulated as a key mechanism for the reactive oxygen species (ROS) overproduction caused by MeHg.8,10,11 © 2017 American Chemical Society

The efficacy of different agents and nutrients for preventing or reversing MeHg toxicity has been investigated, including compounds containing selenium. The element Se is an essential nutrient for humans and animals.12 In humans, Se is present in the composition of 25 selenoproteins, which play important roles as antioxidants and in redox metabolic processes.13 Se activates antineoplastic factors, prevents heart disease, presents antiproliferative and anti-inflammatory properties, stimulates the immune system, and exerts antagonistic action against heavy metals.14 Se can also protect against some neurological consequences of Hg exposure.3 Accordingly, the affinity of Hg for Se is a million times higher than its affinity for S.15 Diphenyl diselenide [(PhSe)2], an organic compound containing Se, is a simple electrophilic intermediate used in the synthesis of various organic compounds of pharmaceutical interest.16 Of particular pharmacological significance, (PhSe)2 has antinociceptive, anti-inflammatory, antihyperglycemic, antiteratogenic, hepatoprotective, neuroprotective, gastroprotective, antidepressant, and anxiolytic properties.17−22 Previous studies in rodents have noted the protective effect of (PhSe)2 against systemic toxicity of MeHg.17,23,24 Freitas et al. (2009) suggested that the intermediate of (PhSe)2, phenyl selenol/selenolate (PhSeH/PhSe−), could interact with MeHg, Received: December 12, 2016 Published: April 18, 2017 1134

DOI: 10.1021/acs.chemrestox.6b00449 Chem. Res. Toxicol. 2017, 30, 1134−1144

Article

Chemical Research in Toxicology

Table 1. Description of S. cerevisiae Deletion Mutant Strains Used in This Study

yielding a stable complex that enhanced the excretion of this metal from the body of mice.23 The yeast Saccharomyces cerevisiae has emerged as a suitable model for toxicogenomic studies, expanding our understanding of the response mechanisms to chemical stress.25 Accordingly, the use of S. cerevisiae mutants with defects in antioxidant defenses could be an important tool to elucidate the molecular targets of various chemicals.26 Previous studies using S. cerevisiae have proposed novel mechanisms involved in MeHg toxicity. Lee et al. (2009) showed that Rip1, a component of the mitochondrial electron transport system, is potentially involved in ROS production, enhancing MeHg toxicity in yeast.27 Additional evidence obtained using S. cerevisiae points to a role of deubiquitinating enzymes in MeHg toxicity and the involvement of the Whi2/Akr1 system as a defense mechanism against the MeHg susceptibility of yeast cells.28,29 The present study aimed to investigate genes and pathways involved in the protective effect of (PhSe)2 on MeHg toxicity using S. cerevisiae wild-type and mutant strains with deletions of different enzymes and proteins related to the antioxidant defense system.

protein

gene

Yap1 (transcription factor)

YAP1

cytosol/ nucleus

adaptative response to exogenous H2O2, xenobiotics, and cadmium

Ybp1

YBP1

cytosol

oxidized Yap1p transport from the cytoplasm to the nucleus

γ-GluCys synthetase

GSH1

cytosol

GSH synthesis

glutathione synthetase

GSH2

cytosol

glutathione Stransferases

GTT1 GTT2 GTT3

cytosol cytosol −

elimination and detoxification of conjugate molecules to GSH

superoxide dismutases

SOD1

dismutation of O2− in H2O2 + O2

SOD2

cytosol, mitochondria mitochondria

catalases

CTT1 CAT1

cytosol peroxisome

degradation of H2O2 in H2O + O2

glutathione peroxidases

GPX1 GPX2

− cytosol

oxidized lipid hydroperoxides reduction

thioredoxin

TRX1 TRX2 TRX3

cytosol cytosol mitochondria

Synthesis of nucleotides, sulfate assimilation, cofactor for some peroxidases, redox control

thioredoxin redutase

TRR2

mitochondria

antioxidant defense against ROS generated in mitochondria

2. MATERIALS AND METHODS 2.1. Chemicals. Methylmercury (MeHg), diphenyl diselenide [(PhSe)2], dimethyl sulfoxide (DMSO), YPD broth, 2′,7′-dichlorofluorescein diacetate (DCFH-DA), and propidium iodide (PI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). CellTracker Green CMFDA dye was purchased from Invitrogen (San Diego, CA, USA). A stock solution of CMFDA was prepared in DMSO and diluted in phosphate-buffered saline (PBS). MeHg was dissolved in distilled water, and (PhSe)2 was dissolved in DMSO. The final volume of DMSO in the incubation medium was 0.5%, and because at this concentration DMSO did not cause toxicity in Saccharomyces cerevisiae in our experiments, these data were omitted in the results. 2.2. Strains and Storage. S. cerevisiae wild-type strain BY4741 and deletion strains (Table 1) were kindly provided by Professors Dr. Monica Lomeli and Dr. Claudio Akio Masuda, from the Leopoldo de Meis Institute of Medical Biochemistry at the Federal University of Rio de Janeiro (UFRJ). The wild-type yeast was maintained in 2 mL cryogenic tubes, and the mutants were maintained in 96-well plates, both stored at −80 °C in a solution containing Yeast Extract Peptone Dextrose (YPD, 1% yeast extract, 2% peptone, and 2% dextrose) medium and 25% glycerol. For all experiments described below, YPD medium was used. 2.3. Growth Conditions. The wild-type and deletion mutant strain cells were grown overnight (30 °C) in YPD medium with ampicillin (100 μg/mL) under continuous shaking (250 rpm). The overnight culture was used to inoculate fresh YPD to test the inhibitory effect of MeHg and/or (PhSe)2 on yeast growth. Concentrations curves of MeHg (1, 2.5, 5, 10, 20 μM) and (PhSe)2 (0.8, 1.6, 3.1, 6.25, 12.5, 25 μM) were constructed using the wild-type strain to establish the concentrations for subsequent experiments. Concentrations of 1.6 and 3.1 μM (PhSe)2 and 5 and 10 μM MeHg were chosen to study the protective effect of (PhSe)2 against MeHg toxicity in wild-type and in deletion mutant strains. Co-treatments with (PhSe)2 and MeHg were added to 1 mL of YPD medium containing diluted stationary phase cells (1 × 105), and cells were incubated at 30 °C and 250 rpm for 24 h. Samples were then diluted in water, and the cell density was measured at 600 nm in a UV−visible spectrophotometer (Shimadzu UV-1650PC) to evaluate yeast growth inhibition. Data were collected using UVProbe 2.42 software. 2.4. Quantification of Reactive Oxygen Species (ROS) Production. ROS production was examined using a method based on intracellular deacetylation and oxidation of DCFH-DA to the fluorescent compound 2′,7′-dichlorofluorescein (DCF). DCFH-DA is membrane-permeable and is trapped intracellularly following deacetylation. Concentrations curves of MeHg (1, 2.5, 5, 10, 20 μM) and (PhSe)2 (0.8, 1.6, 3.1, 6.25, 12.5, 25 μM) were constructed using the

localization

role

wild-type strain at different incubation times (0.5, 1.5, 3, 6, and 24 h) at 30 °C under constant shaking. Co-treatments with (PhSe)2 and MeHg were performed in wild-type and in deletion mutant strains. Exponentially growing cells (1 × 106) suspended in 1 mL of YPD medium were co-incubated with 1.6 and 3.1 μM (PhSe)2 and 5 and 10 μM MeHg for 24 h. After incubation, cell suspensions were centrifuged at 3000 rpm for 6 min, washed three times with PBS (pH 7.4), and resuspended in 1 mL of PBS. Cell suspensions were labeled with 40 μM DCFH-DA, incubated in the dark for 60 min, and analyzed in a flow cytometer.30 The fluorescence of the cell suspension in PBS was quantified using a BD Accuri C6 flow cytometer (BD Biosciences, California, USA). A total of 100 000 events were acquired for each sample. Data were collected using BD Accuri C6 software. 2.5. Cell Membrane Permeability Assay. Cell membrane integrity was estimated by staining with the vital dye PI. A cell membrane permeability assay was performed concomitantly with the experiments for evaluating ROS production, and the exposure protocol was the same as that described in section 2.4. After incubation, cell suspensions were centrifuged at 3000 rpm for 6 min, washed three times with PBS (pH 7.4), and resuspended in 1 mL of PBS. PI stock solution (15 μM) in PBS was added to 1 mL cell suspensions and incubated in the dark for 15 min prior to analysis.31 The PI signal was quantified using a BD Accuri C6 flow cytometer (BD Biosciences, California, USA). A total of 100 000 events were acquired for each sample. Data were collected using BD Accuri C6 software. 2.6. Labeling of Cellular Thiols with 5-Chloromethylfluorescein Diacetate (CMFDA). Thiol content in cells was determined using the fluorescent probe CellTracker Green CMFDA dye.32 As described for the previous experiments, concentration curves were constructed at different incubation times with the wild-type strain. To study the protective effect of (PhSe)2 against MeHg toxicity, 1135


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Figure 1. Effect of (A) (PhSe)2 and (B) MeHg on the growth of S. cerevisiae (wild-type) after 24 h of incubation. Data are reported as the means ± SD of duplicate determinations from three independent experiments. The data were analyzed using one-way ANOVA followed by Tukey’s test. The differences were considered to be significant when P < 0.05. * indicates comparison with control group.

Table 2. Effect of (PhSe)2 and/or MeHg on S. cerevisiae Growth (Wild-Type and Knockout Strains) after 24 h Incubationa (PhSe)2 control WT yap1Δ ybp1Δ cat1Δ ctt1Δ sod1Δ sod2A gsh1Δ gsh2Δ gtt1Δ gtt2Δ gtt3Δ gpx1Δ gpx2Δ trx1Δ trx2Δ trx3Δ trr2Δ

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

1.6 μM 97 93 94 93 97 90 91 97 93 93 89 96 100 94 97 96 96 98

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.02 0.03 0.03 0.02 0.03 0.02 0.02 0.03 0.04 0.06 0.04 0.03 0.03 0.03 0.05 0.05 0.03 0.04

5 μM MeHg

MeHg 3.1 μM

95 74 96 95 99 82 81 84 76 95 94 100 95 77 83 83 78 95

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.01 0.02* 0 03 0.02 0.04 0.01* 0.01* 0.06* 0.05* 0.04 0.03 0.03 0.03 0.03* 0.03* 0.05* 0.03* 0.04

5 μM 3 3 3 3 3 1 1 2 2 3 4 4 4 4 3 4 4 4

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.18* 0.31* 3.03* 0.18* 0.22* 0.63* 0.64* 0.21* 0.41* 0.17* 0.11* 0.15* 0.43* 0.51* 0.20* 0.14* 0.19* 0.22*

10 μM 2 1 3 3 3 0.1 1 2 2 3 3 3 2 2 3 3 4 3

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

10 μM MeHg

+ 1.6 μM (PhSe)2 + 3.1 μM (PhSe)2 + 1.6 μM (PhSe)2

0.29* 3.03* 0.25* 0.07* 0.22* 0.60* 0.43* 0.24* 0.28* 0.20* 0.07* 0.05* 0.23* 0.38* 0.19* 0.20* 0.16* 0.15*

94 2 89 95 99 2 2 2 7 99 100 97 97 46 5 4 5 96

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.01# 3.03* 0.05# 0.02# 0.04# 0.38* 0.26* 0.21* 0.20* 0.02# 0.02# 0.04# 0.02# 0.04*,# 0.21* 0.18* 0.13* 0.03*

101 103 90 90 92 82 82 92 92 86 90 91 104 102 98 93 93 93

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.01# 0.03# 0.01# 0.01# 0.01# 0.01*,# 0.01*,# 0.03# 0.04# 0.03# 0.04# 0.02# 0.03# 0.03# 0.04# 0.05# 0.04# 0.05#

2 0.5 4 3 3 0.6 1 2 2 4 4 4 2 3 3 3 5 4

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.16* 1.19* 0.54* 0.28* 3.21* 0.81* 0.30* 0.20* 0.26* 0.11* 0.11* 0.13* 0.30* 0.37* 3.12* 0.23* 0.19* 0.19*

+ 3.1 μM (PhSe)2 96 66 87 94 96 2 2 3 6 96 100 92 4 5 97 90 93 98

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.03# 0.08*,# 0.03# 0.05# 0.04# 0.36* 0.40* 0.25* 0.22* 0.03# 0.01# 0.03# 0.24* 0.27* 0.03# 0.04# 0.05# 0.03#

Results are expressed as percentage of the control group for each strain. Data are reported as the means ± SD of duplicate determinations from three independent experiments. The data were analyzed using two-way ANOVA, followed by Tukey’s test. The differences were considered to be significant when P < 0.05. *Compared with control group. #Compared with MeHg group.

a

exponentially growing cells (1 × 106) of the wild-type and deletion mutant strains yap1Δ, sod1Δ, and sod2Δ, suspended in 1 mL of YPD medium, were co-incubated with (PhSe)2 (1.6 and 3.1 μM) and MeHg (5 and 10 μM) at 30 °C under constant shaking for 24 h. After incubation, the cells were washed three times with PBS and stained with 1 μM CellTracker Green CMFDA following the manufacturer’s instructions. Cell suspensions (1 mL) were incubated with the probe for 30 min before analysis with the cytometer. A total of 100 000 events were acquired for each sample. The data were collected using BD Accuri C6 software. 2.7. Statistics. Experiments were repeated at least three times. For each individual experiment, values are expressed as the mean ± standard deviation (SD) of duplicate determinations. Data were analyzed with one-way or two-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. The results were considered statistically significant when P < 0.05. All statistical analyses were conducted using GraphPad Prism 6 (GraphPad Software, Inc., USA).

range of 0.8−3.1 μM, (PhSe)2 did not cause cell growth inhibition in the wild-type strain. On the basis of these data, concentrations of 1.6 and 3.1 μM (PhSe)2 were chosen to study the protective effect of (PhSe)2 on MeHg toxicity. All MeHg concentrations tested (1−20 μM) inhibited the cell growth of the wild-type strain when compared to the control group. Concentrations of 5−20 μM MeHg caused marked toxicity (Figure 1B). Concentrations of 5 and 10 μM MeHg were selected for further experiments to investigate potential protective effects of (PhSe)2 on MeHg-induced toxicity. Co-incubation of 3.1 μM (PhSe)2 with 5 or 10 μM MeHg abolished the MeHg-induced growth inhibition (Table 2). In the same way, co-incubation of 1.6 μM (PhSe)2 with 5 μM MeHg prevented cell growth inhibition. On the other hand, coincubation of 1.6 μM (PhSe)2 with 10 μM MeHg did not protect cells from MeHg-induced growth inhibition at a value comparable to the control group. MeHg (5 and 10 μM) treatment also inhibited the cell growth of all the deletion mutant strains tested. Co-incubation with (PhSe)2 and MeHg (1.6 μM with 5 μM; and 3.1 μM with

3. RESULTS 3.1. Effect of MeHg and/or (PhSe)2 on Yeast Growth. The results shown in Figure 1A indicate that, at a concentration 1136


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Figure 2. Effect of (A) (PhSe)2 and (B) MeHg on ROS production (DCFH oxidation) in S. cerevisiae (wild-type) at different incubation times (0.5, 1.5, 3, 6, and 24 h). Data are reported as the means ± SD of duplicate determinations from three independent experiments. The data were analyzed using one-way ANOVA followed by Tukey’s test. The differences were considered to be significant when P < 0.05. * indicates comparison with control group.

Table 3. Effect of (PhSe)2 and/or MeHg on ROS Production (DCFH Oxidation) in S. cerevisiae (Wild-Type and Knockout Strains) after 24 h Incubationa (PhSe)2 control WT yap1Δ sod1Δ sod2Δ gsh1Δ gsh2Δ gpx1Δ gpx2Δ trx1Δ trx2Δ trx3Δ

100 100 100 100 100 100 100 100 100 100 100

1.6 μM 90 90 84 93 80 91 90 91 83 86 92

± ± ± ± ± ± ± ± ± ± ±

19 17 15 20 17 34 17 11 26 17 11

3.1 μM 80 80 80.5 73 65 85 78 63 73 82 83

5 μM MeHg

MeHg ± ± ± ± ± ± ± ± ± ± ±

14 18 9 17 22 28 6 13 20 11 15

5 μM 178 286 169 184 174 175 159 177 241 200 215

± ± ± ± ± ± ± ± ± ± ±

34* 17* 13* 31* 15* 20* 13* 22* 37* 35* 16*

10 μM 288 400 274 282 311 294 267 295 367 271 266

± ± ± ± ± ± ± ± ± ± ±

22* 14* 24* 28* 22* 12* 23* 32* 15* 37* 16*

+ 1.6 μM (PhSe)2 105 103 149 103 175 161 98 129 193 144 153

± ± ± ± ± ± ± ± ± ± ±

11# 15# 12* 9# 20* 17* 16# 16# 14*,# 26*,# 25*,#

10 μM MeHg

+ 3.1 μM (PhSe)2 107 124 125 114 178 154 85 116 174 147 138

± ± ± ± ± ± ± ± ± ± ±

24# 28*,# 33# 11# 24* 33* 16# 9# 36*,# 25*,# 25*,#

+ 1.6 μM (PhSe)2 170 169 258 183 287 251 268 280 266 222 183

± ± ± ± ± ± ± ± ± ± ±

19*,# 27*,# 15* 23*,# 12* 27* 29* 21* 35*,# 28*,# 22*,#

+ 3.1 μM (PhSe)2 104 215 195 167 271 247 149 164 214 198 157

± ± ± ± ± ± ± ± ± ± ±

7# 21*,# 20*,# 22*,# 18* 26* 19*,# 9*,# 41*,# 17*,# 28*,#


a

partially protected from the growth inhibition caused by 5 μM MeHg. In the trx1Δ, trx2Δ, and trx3Δ strains, 1.6 μM (PhSe)2 could not prevent the growth inhibition induced by 5 or 10 μM MeHg, although 3.1 μM (PhSe)2 completely protected cells from the inhibitory effects caused by both concentrations of MeHg. It is important to call attention to the fact that 3.1 μM (PhSe)2 caused a slight decrease in cell growth when compared to the control for some deletion mutant strains, such as yap1Δ, sod1Δ, sod2Δ, gsh1Δ, gsh2Δ, gpx2Δ, trx1Δ, trx2Δ, and trx3Δ. 3.2. Effect of MeHg and/or (PhSe) 2 on ROS Production. (PhSe)2 caused a significant increase in ROS production when compared to the control at 1.5, 3, 6, and 24 h at concentrations of 6.25, 12.5, and 25 μM (Figure 2A). MeHg significantly increased ROS production when compared to the control group at all of the incubation times tested (0.5−24 h) (Figure 2B). At 0.5 h, only concentrations of 10 and 20 μM MeHg significantly increased ROS production. From 1.5 to 24 h, the concentrations of 5, 10, and 20 μM MeHg significantly increased ROS production. Based on these results, the incubation time of 24 h was used to study the protection of (PhSe)2 against MeHg-induced ROS production, and the concentrations of (PhSe)2 and MeHg used in the growth inhibition experiments were maintained.

5 or 10 μM) was effective to protect against MeHg-induced growth inhibition in the strains ybp1Δ, ctt1Δ, and cat1Δ; gtt1Δ, gtt2Δ, and gtt3Δ; and trr2Δ (Table 2). In the deletion mutant strain for the transcription factor YAP1, at a concentration of 3.1 μM, (PhSe)2 protected cells from growth inhibition induced by both concentrations of MeHg, 5 and 10 μM (partially). In strains sod1Δ and sod2Δ, even 3.1 μM (PhSe)2 alone caused a small inhibition of cell growth. (PhSe)2 at 1.6 μM could not prevent the toxicity of 5 μM MeHg; 3.1 μM (PhSe)2 was able to prevent the toxicity of 5 μM MeHg but was not effective against 10 μM MeHg. Both of the deletion strains gsh1Δ and gsh2Δ presented similar results as those observed for sod1Δ and sod2Δ, indicating that 1.6 μM (PhSe)2 was not able to prevent MeHg-induced cell growth inhibition. In addition, coincubation of 3.1 μM (PhSe)2 with 5 μM MeHg prevented MeHg toxicity but was not effective against 10 μM MeHg. In the gpx1Δ strain, co-incubation of 1.6 or 3.1 μM (PhSe)2 with 5 μM MeHg completely protected cells from growth inhibition. On the other hand, neither 1.6 μM nor 3.1 μM (PhSe)2 were able to protect against growth inhibition induced by 10 μM MeHg. For the gpx2Δ strain, the results were similar to those obtained with gpx1Δ, except that 1.6 μM (PhSe)2 only 1137


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Figure 3. Effect of (A) (PhSe)2 and (B) MeHg on cell membrane permeability (PI fluorescence) in S. cerevisiae (wild-type) at different incubation times (0.5, 1.5, 3, 6, and 24 h). Data are reported as the means ± SD of duplicate determinations from three independent experiments. The data were analyzed using one-way ANOVA followed by Tukey’s test. The differences were considered to be significant when P < 0.05. * indicates comparison with control group.

Table 4. Effect of (PhSe)2 and/or MeHg on Cell Membrane Permeability (PI Fluorescence) in S. cerevisiae (Wild-Type and Knockout Strains) after 24 h Incubationa (PhSe)2 control WT yap1Δ sod1Δ sod2Δ gsh1Δ gsh2Δ gpx1Δ gpx2Δ trx1Δ trx2Δ trx3Δ

100 100 100 100 100 100 100 100 100 100 100

1.6 μM 95 104 92 89 88 111 121 110 95 98 95

± ± ± ± ± ± ± ± ± ± ±

10 23 14 36 11 16 16 19 28 30 24

3.1 μM 102 112 97 97 76 115 111 107 94 l07 116

5 μM MeHg

MeHg ± ± ± ± ± ± ± ± ± ± ±

10 22 8 36 10 18 10 19 13 30 25

5 μM 214 253 146 248 142 356 431 190 333 337 330

± ± ± ± ± ± ± ± ± ± ±

52* 28* 11* 25* 15* 42* 112* 42* 64* 71* 64*

10 μM 377 455 309 670 308 678 657 552 629 640 531

± ± ± ± ± ± ± ± ± ± ±

+ 1.6 μM (PhSe)2

74* 86* 27* 53* 24* 96* 68* 64* 67* 95* 88*

102 125 111 90 105 140 116 121 118 92 103

± ± ± ± ± ± ± ± ± ± ±

12# 44# 19# 20# 6# 40# 14# 18# 230# 15# 21#

10 μM MeHg

+ 3.1 μM (PhSe)2 101 113 92 81 126 112 116 119 99 114 93

± ± ± ± ± ± ± ± ± ± ±

13# l7# 9# 23# 35# 25# 18# 22# 11# 23# 17#

+ 1.6 μM (PhSe)2 108 191 117 503 106 224 257 111 314 269 115

± ± ± ± ± ± ± ± ± ± ±

20# 64*,# 13# 34*,# 24# 52*,# 50*,# 34# 74*,# 66*,# 39#

+ 3.1 μM (PhSe)2 103 131 119 89 112 130 127 111 129 211 110

± ± ± ± ± ± ± ± ± ± ±

13# 20# 20# 23# 13# 33# 16# 18# 22# 50*,# 16#


a

In the wild-type strain, co-incubation with 1.6 μM (PhSe)2 completely prevented the increase in ROS production caused by 5 μM MeHg but only partially in relation to 10 μM MeHg. At 3.1 μM, (PhSe)2 completely prevented the increase in ROS production induced by both concentrations of MeHg (Table 3). In the yap1Δ strain, co-incubation with 1.6 μM (PhSe)2 completely prevented 5 μM MeHg-induced ROS generation, although it only partially prevented 10 μM MeHg-induced ROS production. Co-incubation of 3.1 μM (PhSe)2 with 5 and 10 μM MeHg partially protected from the ROS production increase (Table 3). In the sod1Δ strain, co-incubation with 1.6 μM (PhSe)2 could not prevent the ROS generation induced by both concentrations of MeHg (5 and 10 μM) (Table 3). At 3.1 μM, (PhSe)2 protected cells from 5 μM MeHg-induced ROS production but only partially protected cells from ROS production induced by 10 μM MeHg. For the sod2Δ strain, 1.6 μM (PhSe)2 completely protected against 5 μM MeHginduced ROS generation but only partially protected yeast from 10 μM MeHg (Table 3). Similarly, 3.1 μM (PhSe)2 protected the cells against 5 μM MeHg but only partially protected them against 10 μM MeHg. In the gsh1Δ and gsh2Δ strains, co-incubation of 1.6 and 3.1 μM (PhSe)2 with 5 and 10 μM MeHg could not protect against

MeHg-induced ROS production (Table 3). Deletion of these genes induced a higher ROS generation in both strains. In the gpx1Δ and gpx2Δ strains, co-incubation of 1.6 or 3.1 μM (PhSe)2 with 5 μM MeHg fully protected against MeHginduced ROS production (Table 3). However, co-incubation of 1.6 μM (PhSe)2 with 10 μM MeHg did not protect against ROS generation, and co-incubation of 3.1 μM (PhSe)2 with 10 μM MeHg partially prevented the increase in ROS production. In the trx1Δ, trx2Δ, and trx3Δ strains, co-incubation of 1.6 or 3.1 μM (PhSe)2 with 5 or 10 μM MeHg partially protected cells from MeHg-induced ROS production (Table 3). 3.3. Effect of MeHg and/or (PhSe)2 on Cell Membrane Permeability. To evaluate cell membrane integrity, the effect of (PhSe)2 (Figure 3A) and MeHg (Figure 3B) on PI fluorescence was examined. At 0.5 h, only 25 μM (PhSe)2 showed a significant increase in PI fluorescence when compared to the control in the wild-type strain. At 1.5 h, none of the (PhSe)2 concentrations induced alterations in PI fluorescence. At 3 and 6 h, significant increases in PI fluorescence were observed from 6.25 to 25 μM (PhSe)2. At 24 h, 12.5 and 25 μM (PhSe)2 produced a significant increase in PI fluorescence when compared to the control group in the wild-type strain. From 0.5 to 6 h of incubation, MeHg concentrations ranging from 2.5 to 20 μM induced a significant increase in PI fluorescence in the wild-type strain. At 24 h, MeHg 1138


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Figure 4. Effect of (A) (PhSe)2 and (B) MeHg on thiol content (CMFDA fluorescence) in S. cerevisiae (wild-type) at different incubation times (0.5, 1.5, 3, 6, and 24 h). Data are reported as the means ± SD of duplicate determinations from three independent experiments. The data were analyzed using one-way ANOVA followed by Tukey’s test. The differences were considered to be significant when P < 0.05. * indicates comparison with control group.

Table 5. Effect of (PhSe)2 and/or MeHg on Thiol Content (CMFDA Fluorescence) in S. cerevisiae (Wild-Type and Knockout Strains) after 24 h Incubationa (PhSe)2 control WT yap1Δ sod1Δ sod2Δ

100 100 100 100

1.6 μM 112 94 93 112

± ± ± ±

6 17 17 20

3.1 μM 131 91 93 94

5 μM MeHg

MeHg ± ± ± ±

29 13 17 17

5 μM 348 166 177 158

± ± ± ±

38* 24* 38* 31*

10 μM 512 195 164 182

± ± ± ±

46* 26* 18* 28*

+ 1.6 μM (PhSe)2 172 159 169 154

± ± ± ±

23*,# 42* 24* 20*

10 μM MeHg

+ 3.1 μM (PhSe)2 118 128 165 154

± ± ± ±

25# 21*,# 25* 36*

+ 1.6 μM (PhSe)2 190 195 154 189

± ± ± ±

12*,# 31* 31* 31*

+ 3.1 μM (PhSe)2 119 170 147 196

± ± ± ±

15# 21* 23* 43*


a

concentrations ranging from 5 to 20 μM caused a significant increase in PI fluorescence in wild-type cells. It was previously observed that membranes of yeast cells under chemical stress may develop a transient permeability to PI, which does not necessarily indicate cell death.12 This transient permeability to PI may explain the oscillations in PI fluorescence observed between MeHg concentrations from 1.5 to 6 h. Therefore, to evaluate the protective effect of (PhSe)2 on membrane damage induced by MeHg, the compound concentrations and the incubation time of 24 h were maintained the same as for DCFH-DA oxidation assay and the growth inhibition experiment. In the wild-type strain, co-incubation of 1.6 or 3.1 μM (PhSe)2 with 5 or 10 μM MeHg completely prevented the increase in membrane permeability caused by MeHg (Table 4). In the same way, in sod1Δ, gsh1Δ, gpx2Δ, and trx3Δ strains (PhSe)2 (1.6 and 3.1 μM) also protected from the increase in membrane permeability induced by MeHg (5 and 10 μM) (Table 4). Deletion of SOD1 and GSH1 genes increased PI fluorescence several-fold when compared to wild-type and other deletion mutant strains. In the yap1Δ, sod2Δ, gsh2Δ, and gpx1Δ strains, coincubation of 1.6 μM (PhSe)2 with 10 μM MeHg only partially protected cells from the MeHg-induced increase in membrane permeability, while it completely protected them from the 5 μM MeHg-induced increase in PI fluorescence (Table 4). At 3.1 μM, (PhSe)2 fully protected against the 5 or 10 μM MeHginduced increase in PI fluorescence. 3.4. Effect of MeHg and/or (PhSe)2 on Cellular Thiol Content. In the wild-type strain, (PhSe)2 caused a slight increase in thiol content only at 25 μM at 0.5 h of incubation.

At 1.5 and 6 h of incubation, (PhSe)2 from 6.25 to 20 μM caused a significant increase in the thiol levels in the wild-type strain. At 3 and 24 h of incubation, 12.5 and 25 μM (PhSe)2 significantly increased the thiol content (Figure 4A). MeHg also caused a significant increase in thiol content at all concentrations tested and in all the incubation periods in the wild-type strain (Figure 4B). Co-incubation of 1.6 μM (PhSe)2 with 5 or 10 μM MeHg only partially prevented thiol increase by MeHg (Table 5). Coexposure of 3.1 μM (PhSe)2 with 5 or 10 μM MeHg was able to maintain the thiol content at levels similar to those of the control in the wild-type strain (Table 5). For this assay, deletion mutant strains for proteins and enzymes directly involved in maintenance of the thiol redox status were not tested. The three tested deletion mutant strains, yap1Δ, sod1Δ, and sod2Δ, presented a CMFDA fluorescence several times higher than that of the wild-type strain in a 24 h incubation. In the yap1Δ strain, co-incubation of 3.1 μM (PhSe)2 with 5 μM MeHg partially prevented the MeHginduced increase in thiol levels (Table 5). In the sod1Δ and sod2Δ strains, (PhSe)2 was not able to prevent the MeHginduced increase in thiol levels (Table 5).

4. DISCUSSION The present study aimed to investigate the mechanisms involved in the protective effect of (PhSe)2 on MeHg-induced toxicity in S. cerevisiae. The results demonstrated that (PhSe)2 was able to prevent MeHg-induced growth inhibition in the S. cerevisiae wild-type strain depending on the concentrations of both compounds. Possible mechanisms involved in MeHg toxicity reduction by Se are the sequestering of Hg, an 1139


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and induce transcription of antioxidant proteins and ABC transporters that mitigate the oxidative effect of MeHg and increase MeHg detoxification, respectively.40,41 On the other hand, (PhSe)2 was able to mitigate the MeHg-induced increase in ROS and prevent alterations in membrane permeability in the YAP1 deletion mutant depending on the concentrations of the compounds (Tables 3, 4, and 6). A previous study showed that the YAP1 mutant still retained an adaptive response to stress induced by H2O2, implying the involvement of other factors in that response.42 In agreement with our data, studies in mammalian cells previously demonstrated that both MeHg and (PhSe)2 activate the transcription factor Nrf2, which is also involved in the defense mechanism against oxidative and xenobiotic stress.43,44 Ybp1 is a protein involved in the cellular response to oxidative stress. It is required for oxidation of specific cysteine residues in the transcription factor Yap1, resulting in nuclear localization of Yap1 in response to stress.45,46 However, Ybp1 does not seem to be necessary for (PhSe)2 protective action since its absence did not influence MeHg-inhibited cell growth (Tables 2 and 6). Single deletion of CTT1 and CAT1 genes that express the enzyme catalase did not interfere in the capacity of (PhSe)2 to prevent the toxic effects of MeHg (Tables 2 and 6). Catalase in yeast, may have cytosolic (catalase T, encoded by CTT1) or peroxisome localization (catalase A, encoded by CAT1).47 Hiltunen et al. (2003) demonstrated that Cat1 is not essential for yeast cells.48 In addition, CTT1 deletion mutants were not more sensitive to H2O2 than wild-type yeast, although the double mutant (CAT1 and CTT1) was hypersensitive, which points to a cooperative role of both catalases.49 In this way, our results could indicate that catalase does not participate in the (PhSe)2 protective mechanism or, as previously observed, that CAT1 can offset the absence of CTT1 and vice versa. Glutathione S-transferases (GSTs) are enzymes involved in detoxification and elimination of environmental xenobiotics, including heavy metals, via GSH conjugation.50 S. cerevisiae have two standard GSTs encoded by GTT1 and GTT2.51 GTT3 is a protein of unknown function and location, which may be involved in GSH metabolism. The deletion of any of these three genes did not alter the growth of yeast under treatment with MeHg and/or (PhSe)2 compared with the wildtype strain (Tables 2 and 6), suggesting that, at least individually, they are not essential for the (PhSe)2 effect, which does not eliminate the possibility of a cooperative role of these three enzymes. Although catalase and GST activities could protect against ROS generated in yeast cells due to intoxication with MeHg, redundancy between different detoxifying enzymes may exist. This explains the fact that the deletion of a single detoxification system does not interfere significantly in survival and cell growth. In eukaryotes, SOD presents itself in two forms: a form containing copper and zinc (Cu,Zn-SOD), encoded by SOD1 in yeast, located in the cytosol and in the mitochondrial intermembrane space and a form containing manganese (MnSOD), encoded by SOD2 in yeast, located in the mitochondrial matrix.52,53 In the present study, SOD1 and SOD2 deletion led to a decrease in the efficiency of (PhSe)2 in preventing MeHginduced cell growth inhibition (Tables 2 and 6) and ROS production (Tables 3 and 6), indicating the importance of this enzyme to the (PhSe)2 protective effects. SOD1 is involved in homeostasis of the cell wall of S. cerevisiae. Its deletion leads to

antioxidant effect, GSH synthesis, increased GPx activity, increased selenoprotein levels, and demethylation.2 In this study, we investigated whether deletion of genes that express proteins of the antioxidant system of S. cerevisiae could interfere in the capacity of (PhSe)2 to reduce MeHg toxicity. Table 6 presents a summary of these results, indicating the effect of each gene deletion on (PhSe)2 protection in the different assays. Table 6. Summary of Results Showing the Effect of Deletion of Each Gene on (PhSe)2 Protection in the Different Assaysa

yap1Δ ybp1Δ cat1Δ ctt1Δ sod1Δ sod2Δ gsh1Δ gsh2Δ gtt1Δ gtt2Δ gtt3Δ gpx1Δ gpx2Δ trx1Δ trx2Δ trx3Δ trr2Δ

yeast growth

ROS production

cell membrane permeability

X − − − X X X X − − − X X X X X −

X not used not used not used X X X X not used not used not used X X X X − not used

X not used not used not used − X − X not used not used not used X − X X X not used

thiol content X not not not X X not not not not not not not not not not not

used used used

used used used used used used used used used used used

The “X” indicates that single deletion of the gene decreased the capacity of (PhSe)2 to prevent MeHg toxicity in comparison with the wild-type strain; “−” indicates that single deletion of the gene did not affect the capacity of (PhSe)2 to prevent MeHg toxicity in comparison with the wild-type strain; and “not used” indicates that the assay was not performed with that strain. a

The absence of the YAP1 gene product decreased the effectiveness of (PhSe)2 in counteracting the MeHg-induced cell growth inhibition (Tables 2 and 6), suggesting that Yap1 is involved in the action of (PhSe)2. YAP1 deletion also resulted in a decreased ability of (PhSe)2 to restore the levels of thiols in the cells (Tables 5 and 6). The Yap1 transcription factor is one of the key proteins that regulates the response to oxidative stress induced by H2O2 and metals in S. cerevisiae.33−35 Yap1 accumulates in the nucleus when it is activated by oxidative stress due to oxidation of its cysteine residues, which modifies its conformation.36,37 The Yap1 transcription factor is involved in GSH1 transcription activation and increases the expression of SOD1 and CAT1 genes after exposure to H2O2.38,39 Yap1 was also demonstrated to regulate ATP-binding cassette (ABC) transporters in yeast. Members of the ABC superfamily catalyze the ATP-dependent transport of chemically diverse compounds across cellular membranes, including the plasma membrane or intracellular organellar membranes. Ycf1, a member of the ABC family, is a glutathione S-conjugate transporter involved in cellular detoxification, and it transports heavy metals such as cadmium, mercury, lead, and arsenite. Yap1p binds to a Yap1p response element located upstream of the YCF1 ORF and promotes increased levels of YCF1 expression.40 Both Se and Hg were able to activate Yap1 in S. cerevisiae cells, which strengthens the idea that (PhSe)2 can activate Yap1 1140


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molecule is apparently involved in this response.69 In addition, TRX2 is involved in redox regulation of Yap1 activity.70 In the present study, deletions in TRX1, TRX2, and TRX3, but not TRR2, affected the capacity of (PhSe)2 to prevent MeHginduced cell growth inhibition (Tables 2 and 6), ROS production (Tables 3 and 6), and increase in membrane permeability (Tables 4 and 6). TrxR of mammals was demonstrated to reduce (PhSe)2 to selenol which contributes to the antioxidant properties of the compound.71 However, in the present study, TRR2 does not seem to be essential to (PhSe)2 activity. Alternatively, mammalian Trx was demonstrated to increase ebselen reduction to its selenol intermediate, suggesting that a similar mechanism could be applied to (PhSe)2 in yeast.72 In the current study, both MeHg and (PhSe)2 caused an increase in thiol content in the S. cerevisiae wild-type strain. Jin et al. (2008) demonstrated that expression of genes in sulfate assimilation and GSH biosynthesis pathways is stimulated by Hg.73 Additionally, (PhSe)2 has previously been demonstrated to promote an increase in cellular GSH levels.43 An adaptive response of cells to oxidative stress, or to MeHg-induced GSH depletion, could be one explanation for the increased cellular thiol levels induced by MeHg. Thus, reduction of such levels through co-exposure of (PhSe)2 and MeHg is additional evidence of the protective role of this selenoorganic compound. The reduction in thiol levels in yeast co-treated with (PhSe)2 and MeHg could be related to the increase in GSH consumption by GSH-dependent enzymes, such as GPxs, which could have its synthesis stimulated through YAP1 activation. In addition, the thiol peroxidase activity of (PhSe)2, stimulated by the increase in ROS production induced by MeHg, could also contribute to the decreased thiol levels observed here. Because the CMFDA dye used to measure thiol levels is not specific to GSH, we are not able to infer that the increased fluorescence was solely due to an increase in GSH levels.74 It is interesting to note that, depending on the MeHg and (PhSe)2 concentrations, the protective effect of (PhSe)2 on MeHg-induced cell growth inhibition, ROS production, and membrane permeability is altered in deletion mutant strains, indicating that different mechanisms may be involved according to the MeHg:(PhSe)2 proportion. These results reinforce the hypothesis that (PhSe)2 acts via multiple mechanisms, through Hg sequestering by PhSe−, forming a complex; by modulating gene expression of antioxidant defenses and ABC transporters; or directly by neutralizing ROS.

a significant increase in chitin, a structural component of the yeast cell wall, causing an increase in sensitivity to agents that disturb the cell wall.53 It is important to note that the sod1Δ strain caused an increase in the basal PI fluorescence when compared to wild-type (Tables 4 and 6), indicating increased membrane permeability in this mutant strain. Our results showed that the absence of these enzymes led to increased levels of thiol, and (PhSe)2 was not able to prevent the MeHginduced increase in thiol levels (Tables 5 and 6). Glutathione (γ-L-glutamyl-cysteinylglycine; GSH) is the most abundant non-protein thiol tripeptide in all living organisms.54,55 GSH is involved in various cellular processes, such as control of the redox potential, protection against ROS, detoxification of electrophilic compounds, protein folding, kidnapping of toxic metals, and organic sulfur transport.56,57 GSH is synthesized in two enzymatic steps in the cytosol. The first reaction is catalyzed by γ-glutamylcysteine synthase, encoded by GSH1, and the second by glutathione synthetase, encoded by GSH2.58 The GSH1 mutants induce apoptosis, and even in a medium supplemented with GSH, they have a high tendency for loss of mitochondrial function.59,60 This explains the high emission of PI fluorescence in the gsh1Δ strain compared to the wild-type, indicating great damage to membranes (Tables 4 and 6). Moreover, these mutants showed no adaptive responses to stress induced by H2O2 during the exponential phase of growth in YPD medium.61 Here, the absence of Gsh1p and Gsh2p abolished the capacity of (PhSe)2 to prevent growth inhibition (Tables 2 and 6) and the ROS production induced by MeHg (Table 3 and 6). In addition to acting as an antioxidant and in metal sequestering, GSH is also important in the GPx-mimetic catalytic cycle of (PhSe)2 and in PhSeH/PhSe− intermediate formation, explaining the importance of GSH to the antioxidant properties of (PhSe)2.18 Studies have described the presence of three GPx proteins in yeast, which came to be called GPx1, GPx2, and GPx3.62 However, further studies demonstrated that these three proteins are PHGPx’sphospholipid hydroperoxide GPx types.63 PHGPx’s are monomeric molecules (unlike the classical GPxs, which are multimeric) and membrane associated, reducing membrane lipid hydroperoxides and acting as the major enzymes in the repair of lipid peroxidation in membranes.63 There is evidence in the literature suggesting that the GPx activity is usually increased after exposure to Hg2+.64 Additionally, (PhSe)2 increased the expression of GPX4 in aortic endothelial cells.43 In the present study, deletion of GPX1 and GPX2 abrogated 1.6 μM (PhSe)2 protection against 10 μM MeHg-induced cell growth inhibition (Tables 2 and 6) and ROS production (Tables 3 and 6), indicating a possible role of GPx in (PhSe)2 protective action. Thioredoxins (Trxs) are small proteins rich in sulfhydryl groups that are involved in antioxidant defense and in maintenance of cell redox homeostasis.65 S. cerevisiae have two cytosolic thioredoxins (Trx1 and Trx2) plus one with a mitochondrial localization (Trx3), and two TrxR, located in the cytosol (Trr1) and in the mitochondria (Trr2).66,67 TrxR is a homodimeric flavoprotein that, in the presence of NADPH, reduces oxidized Trx.68 Single deletion of TRX1 or TRX2 genes does not affect cell growth or morphology, but the deletion of both genes results in a prolongation of the S phase of the cell cycle and a marked increase in the level of oxidized glutathione.66 Deletion of the TRX2 gene results in sensitivity to H2O2, and thus, this

5. CONCLUSIONS In conclusion, the present study sheds light on the mechanisms involved in (PhSe)2 protection against MeHg toxicity and indicates a role of (PhSe)2 in activating the transcription factor Yap1, leading to the expression of important antioxidant defenses in S. cerevisiae. These data corroborate other studies in the literature, which suggest a role of (PhSe)2 in modulating gene expression of antioxidant defenses.75 Genetic and functional similarities between yeast and mammalian cells suggest that elucidation of molecular pathways through which (PhSe)2 protects against MeHg toxicity in yeast will help to direct mechanism research in mammals.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. 1141


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Chemical Research in Toxicology *E-mail: [email protected]. Tel: 55-55-32208140. Fax: 55-55-3220-8978.

(12) Zheng, W. J., and Ouyang, Z. (2001) Organic selenium compounds from plants: Their chemistry and applications in medicine, Jinan University Press, Guangzhou. (13) Kryukov, G. V., Castellano, S., Novoselov, S. V., Lobanov, A. V., Zehtab, O., Guigo, R., and Gladyhev, V. N. (2003) Characterization of mammalian selenoproteomes. Science 300, 1439−1443. (14) Alaejos, M. S., Diaz Romero, F. J., and Diaz Romero, C. (2000) Selenium and cancer: some nutritional aspects. Nutrition 16, 376−383. (15) Dyrssen, D., and Wedborg, M. (1991) The sulfur−mercury(II) system in natural waters. Water, Air, Soil Pollut. 56, 507−519. (16) Moreira Rosa, R., De Oliveira, R. B., Saffi, J., Braga, A. L., Roesler, R., Dal-Pizzol, F., Fonseca Moreira, J. C., Brendel, M., and Pêgas Henriques, J. A. (2005) Pro-oxidant action of diphenyl diselenide in the yeast Saccharomyces cerevisiae exposed to ROSgenerating conditions. Life Sci. 77 (77), 2398−2411. (17) Glaser, V., Moritz, B., Schmitz, A., Dafré, A. L., Nazari, E. M., Rauh Muller, Y. M., Feksa, L., Straliottoa, M. R., De Bem, A. F., Farina, M., da Rocha, J. B. T., and Latini, A. (2013) Protective effects of diphenyl diselenide in a mouse model of brain toxicity. Chem.-Biol. Interact. 206, 18−26. (18) Nogueira, C. W., Zeni, G., and Rocha, J. B. T. (2004) Organoselenium and organotellurium compounds: toxicology and pharmacology. Chem. Rev. 104, 6255−6286. (19) Nogueira, C. W., and Rocha, J. B. T. (2010) Diphenyl diselenide: a janus-faced molecule. J. Braz. Chem. Soc. 21, 2055−2071. (20) Nogueira, C. W., and Rocha, J. B. T. (2011) Toxicology and pharmacology of selenium: emphasis onsynthetic organoselenium compounds. Arch. Toxicol. 85, 1313−1359. (21) Hassan, W., Silva, C. E. B., Rocha, J. B. T., and LandeiraFernandez, J. (2015) Modulatory effect of diphenyl diselenide in Carioca High- and Low-conditioned Freezing rats. Eur. J. Pharmacol. 761, 341−344. (22) Rosa, R. M., Moura, D. J., Romano e Silva, A. C., Safii, J., and Pegas Henriques, J. A. (2007) Antioxidant activity of diphenyl diselenide prevents the genotoxicity of several mutagens in Chinese hamster V79 cells. Mutat. Res., Genet. Toxicol. Environ. Mutagen. 631, 44−54. (23) de Freitas, A. S., Funck, V. R., Rotta, M. S., Bohrer, D., Morschbacher, V., Puntel, R. L., Nogueira, C. W., Farina, M., Aschner, M., and Rocha, J. B. T. (2009) Diphenyl diselenide, a simple organoselenium compound, decreases methylmercury-induced cerebral, hepatic and renal oxidative stress and mercury deposition in adult mice. Brain Res. Bull. 79, 77−84. (24) Dalla-Corte, C. L., Soares, F. A. A., Aschner, M., and Rocha, J. B. T. (2012) Diphenyl diselenide prevents methilmercury-induced mitochondrial dysfunction in rat liver slices. Tetrahedron 68, 10437− 10443. (25) Dos Santos, S. C., Teixeira, M. C., Cabrito, T. R., and SáCorreia, I. (2012) Yeast toxicogenomics: genome-wide responses to chemical stresses with impact in environmental health, pharmacology, and biotechnology. Front. Genet. 3, 63. (26) Zyracka, E., Zadrag, R., Koziol, S., Krzepilko, A., Bartosz, G., and Bilinski, T. (2005) Yeast as a biosensor for antioxidants: Simple growth tests employing a Saccharomyces cerevisiae mutant defective in superoxide dismutase. Acta Biochim. Pol. 52 (3), 679−684. (27) Lee, J. Y., Hwang, G. W., and Naganuma, A. (2009) Rip1 enhances methylmercury toxicity through production of reactive oxygen species (ROS) in budding yeast. J. Toxicol. Sci. 34 (6), 715− 717. (28) Hwang, G. W., Fukumitsu, T., Ogiwara, Y., Takahashi, T., Miura, N., Kuge, S., and Naganuma, A. (2016) Whi2 enhances methylmercury toxicity in yeast via inhibition of Akr1 palmitoyltransferase activity. Biochim. Biophys. Acta, Gen. Subj. 1860 (6), 1326− 1333. (29) Hwang, G. W., Kimura, Y., Takahashi, T., Lee, J. Y., and Naganuma, A. (2012) Identification of deubiquitinating enzymes involved in methylmercury toxicity in Saccharomyces cerevisiae. J. Toxicol. Sci. 37 (6), 1287−1290.

ORCID

Cristiane Lenz Dalla Corte: 0000-0003-3478-626X Funding

Supported by FAPERGS (Fundaçaõ de Amparo a Pesquisa do Estado do Rio Grande do Sul) #2251-2551/14-7 (SIAFEM), ́ CAPES (Coordenaçaõ de Aperfeiçoamento de Pessoal de Nivel Superior), and CNPq (Conselho Nacional de Desenvolvimento ́ Cientifico e Tecnológico) #461000/2014-8 (Universal). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS CAT, peroxisomal catalase; CMFDA, 5-chloromethylfluorescein diacetate; CTT, cytosolic catalase; DCF, 2′,7′-dichlorofluorescein; DCFH-DA, 2′,7′-dichlorofluorescein diacetate; DMSO, dimethyl sulfoxide; GPx, glutathione peroxidase; GSH, reduced glutathione; GSSH, oxidized glutathione; GST, glutathione S-transferase; H2O2, hydrogen peroxide; MeHg, methylmercury; PBS, phosphate-buffered saline; PI, propidium iodide; (PhSe)2, diphenyl diselenide; ROS, reactive oxygen species; SH, sulfhydryl; SOD, superoxide dismutase; Trx, thioredoxin; TrxR, thioredoxin reductase; YPD, yeast extract peptone dextrose

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REFERENCES

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Diphenyl Diselenide Protects against Methylmercury-Induced Toxicity

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