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Originally published In Press as doi:10.1074/jbc.M610078200 on January 29, 2007

J. Biol. Chem., Vol. 282, Issue 12, 8759-8767, March 23, 2007
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Extracellular Production of Hydrogen Selenide Accounts for Thiol-assisted Toxicity of Selenite against Saccharomyces cerevisiae*

Agathe Tarze{ddagger}12, Marc Dauplais{ddagger}2, Ioana Grigoras{ddagger}3, Myriam Lazard{ddagger}, Nguyet-Thanh Ha-Duong{ddagger}4, Frédérique Barbier§, Sylvain Blanquet{ddagger}, and Pierre Plateau{ddagger}5

From the {ddagger}Laboratoire de Biochimie, UMR CNRS 7654, Département de Biologie, Ecole Polytechnique, 91128 Palaiseau Cedex and the §Service Central d'Analyse, USR CNRS 59, Chemin du Canal, BP 22, 69390 Vernaison, France

Received for publication, October 27, 2006 , and in revised form, January 24, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Administration of selenium in humans has anticarcinogenic effects. However, the boundary between cancer-protecting and toxic levels of selenium is extremely narrow. The mechanisms of selenium toxicity need to be fully understood. In Saccharomyces cerevisiae, selenite in the millimolar range is well tolerated by cells. Here we show that the lethal dose of selenite is reduced to the micromolar range by the presence of thiols in the growth medium. Glutathione and selenite spontaneously react to produce several selenium-containing compounds (selenodiglutathione, glutathioselenol, hydrogen selenide, and elemental selenium) as well as reactive oxygen species. We studied which compounds in the reaction pathway between glutathione and sodium selenite are responsible for this toxicity. Involvement of selenodiglutathione, elemental selenium, or reactive oxygen species could be ruled out. In contrast, extracellular formation of hydrogen selenide can fully explain the exacerbation of selenite toxicity by thiols. Indeed, direct production of hydrogen selenide with D-cysteine desulfhydrase induces high mortality. Selenium uptake by S. cerevisiae is considerably enhanced in the presence of external thiols, most likely through internalization of hydrogen selenide. Finally, we discuss the possibility that selenium exerts its toxicity through consumption of intracellular reduced glutathione, thus leading to severe oxidative stress.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In mammals, selenium is translationally incorporated in a small number of proteins under the form of selenocysteine. Several of these selenoproteins display antioxidant activities. Others are involved in immune function, sperm motility, or production of thyroid hormones (1). As a clue of the miscellaneous role of selenoenzymes, selenium deficiency is implicated in various pathologies (1).

Selenium deserved additional interest when an inverse correlation between cancer mortality rates and geographic distribution of selenium in forage crops was observed (2). Later, numerous epidemiological data, as well as animal studies and supplementation trials, have reinforced the idea of anticarcinogenic properties of selenium (3–5).

Although anticarcinogenic properties of selenium have become a field of intensive investigations, their causes remain elusive. Different non-exclusive explanations have been proposed, including (i) inhibition of DNA synthesis by selenocompounds (6, 7), (ii) induction of DNA repair (8), (iii) induction of apoptosis in cancer cells (9), and (iv) action of selenoproteins (10). However, because the cancer-protective effects keep on increasing at selenium intake levels exceeding that required to maximize the activity of selenoproteins (11), it is unlikely that selenoproteins alone mediate the protection against cancer.

The boundary between cancer-protecting and toxic levels of selenium is extremely narrow. Indeed, protection was observed with a daily allowance of 200 µg of total selenium (4), whereas the tolerable upper intake level is estimated to be 400 µg for adults (12). The use of selenium-enriched supplements may therefore be beneficial but becomes dangerous if doses are too high. The mechanisms of selenium toxicity remain largely unknown and need to be further investigated. To this end, we chose the yeast Saccharomyces cerevisiae to study the effect of selenite on viability. This model organism has the advantage to be devoid of selenoproteins (13). Therefore, its growth can be compared in the presence or absence of selenium without any interfering effect of selenoproteins. The impact of selenium on yeast has already been studied (14–20). Dual effects of selenium are observed. Low concentrations protect the cell against mutagenesis, whereas millimolar concentrations are toxic.

In many animal cell types, thiols have been recognized to be involved in the cytotoxicity of selenite (21–25). Therefore, to elucidate the underlying mechanisms, we have investigated the capacity of S. cerevisiae cells to withstand exposure to selenite upon addition to the growth medium of glutathione, L-cysteine, or dithiothreitol. We found that all assayed thiols drastically enhanced selenite toxicity. Several selenium-containing compounds as well as reactive oxygen species derive from the spontaneous reduction of selenite by thiols. Eventually, we show that extracellular production of hydrogen selenide, a short-lived product of the reduction of selenite, must be at the origin of the thiol-assisted selenite toxicity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Glutathione, sodium selenite, L-cysteine, dithiothreitol, DL-selenocystine, xanthine, 2-morpholinoethanesulfonic acid (MES),6 milk xanthine oxidase, Escherichia coli thioredoxin reductase were from Sigma. HNO3 (Suprapur) was from Merck. NADH, NADPH, lactate dehydrogenase from rabbit muscle, catalase from beef liver and superoxide dismutase (SOD) from bovine erythrocytes were purchased at Roche Applied Science. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) was from Promega (Madison, WI).

Strain and Media—The S. cerevisiae strain DTY7 (MAT{alpha} ura3–52 leu2–3,112 his6 CUP1R-3) was kindly provided by Dr. D. J. Thiele (University of Michigan Medical School). Rich YT medium contained 1% yeast extract (Difco), 1% Bacto-Tryptone (Difco), and 2% glucose. Synthetic dextrose (SD) minimal medium contains 0.67% yeast nitrogen base (Difco), 2% glucose and 50 µg/liter of histidine, leucine, and uracil. This medium was buffered at pH 6.0 by the addition of 50 mM MES-NaOH.

Preparations of Selenodiglutathione and of Red Elemental Selenium—Selenodiglutathione was obtained from selenite and glutathione as described previously (26), with the following modifications. The reaction mixture was made by successive additions of 30 µl of 0.2 M HCl, 10 µl of 50 mM sodium selenite, and 20 µl of 100 mM glutathione. After 10-min incubation at room temperature, reaction products were applied on an Alltima C18 high-performance liquid chromatography column (0.32 x 15 cm, 5 µm, from Alltech) equilibrated in 0.05% (v/v) acetic acid/water. Elution was performed at a flow rate of 0.4 ml/min using a linear gradient from 0.05% acetic acid to 100% methanol in 20 min. Concentration of selenodiglutathione was calculated using a light absorption coefficient of 1.87 A263 units.mM–1 (27).

To obtain red elemental selenium, 1 ml of 20 mM potassium phosphate (pH 6.5) containing 1 mM sodium selenite and 10 mM glutathione was incubated for 5 min at room temperature and then centrifuged for 10 min at 18,000 x g. The pellet was washed twice with 20 mM potassium phosphate (pH 6.0) and resuspended in 0.5 ml of this buffer. Concentration of elemental selenium was determined after oxidation into selenite by concentrated nitric acid and fluorometric quantitation of resulting selenite with the help of diaminonaphtalene (28).

Purification of Proteins—E. coli thioredoxin was purified from E. coli JM101TR cells (29) harboring plasmid pFP1TRX, as described previously (30). Concentration of thioredoxin was calculated using a light-absorption coefficient of 12.65 A280 units.mM–1 (30). E. coli D-cysteine desulfhydrase was purified from E. coli XL1-Blue cells (Stratagene) harboring plasmid pKKyedO (31). Specific activity of purified D-cysteine desulfhydrase was 34 s–1, when assayed in the presence of 250 µMD-cysteine under previously described conditions (31). Concentration of D-cysteine desulfhydrase was calculated using a light-absorption coefficient of 0.599 A280 units.mg–1.ml and a Mr of 70,044 (31). According to SDS-PAGE analysis, thioredoxin, and D-cysteine desulfhydrase were at least 95% homogeneous.

Toxicity Assays—S. cerevisiae strain DTY7 was pregrown overnight at 30 °C in SD minimal medium. Cells were then inoculated in the same medium to obtain an OD650 of 0.02 and left to grow at 30 °C. When the OD650 reached 0.1, the compounds under study were added to the culture. After 1 h at 30 °C under agitation, samples were diluted 1000-fold in water. An aliquot of 200 µl of this dilution was plated onto rich YT agar plates to monitor cell viability. Plates were left to grow for 2 days at 30 °C prior to scoring. When toxicity assays included extracellular presence of enzymes, bovine serum albumin (25 µg/ml) was added to the medium to prevent adsorption on the culture tube walls.

Total Selenium Determination—Selenium incorporated in cells was determined by inductively coupled plasma mass spectrometry. To ensure dissolution of elemental selenium particles that may be produced extracellularly during the reduction of selenite by thiols, harvested cells (1.5 ± 0.7 mg of dry weight) were washed three times with 0.1 M potassium sulfite, a solvent of elemental selenium (32), then rinsed with water and lyophilized.

Samples were digested in a closed Ethos Touch Control microwave device from Milestone. Before use, vessel was cleaned by addition of 10 ml of 65% HNO3 and heating at 200 °C during 20 min. Blanks were performed to verify the absence of reagent pollution. Each sample was placed into a 50-ml Teflon digester. Then, 1 ml of 69% HNO3, 2 ml of 30% H2O2 (Trace Select, from Fluka), and 5 ml of water were added. The digester was closed, progressively heated until 180 °C in the microwave (30 min), and then maintained at 180 °C for 15 min. After cooling, mineralized samples were placed in volumetric flasks and analyzed by inductively coupled plasma mass spectrometry on a PQ Excell spectrometer from VG Elemental. The mass spectrometer was fixed on the ratio m/z = 82. Standard used for determinations was the inductively coupled plasma mass spectrometry certified multielement solution 2A, provided by Spex. Intracellular selenium concentrations were calculated by assuming that 1.0 OD650 (Shimadzu spectrophotometer UV-2101PC) corresponds to 0.4 µl of intracellular volume (33).

Enzymatic Assays—Activity of D-cysteine desulfhydrase was monitored through measurement of pyruvate produced from D-cysteine or DL-selenocystine by using a spectrophotometric method involving lactate dehydrogenase and NADH (31). The overall reaction was performed at 30 °C in a reaction mixture containing 130 µM NADH, 10 units/ml lactate dehydrogenase, 250 µMD-cysteine or 40 µMDL-selenocystine, and various concentrations of desulfhydrase. Consumption of NADH was deduced from the decrease in absorbance at 340 nm.

SOD activity was deduced from the inhibitory effect of this enzyme on Pyrogallol auto-oxidation (34). Reaction mixtures (490 µl) contained 50 mM Tris-HCl (pH 8.2), 1 mM EDTA, and catalytic amounts of SOD. The reaction was started by the addition of Pyrogallol (10 µl) at a final concentration of 200 µM. The initial rate of Pyrogallol oxidation was calculated by following light absorbance at 420 nm during 10 min.

Initial rates of catalase activity were obtained by measuring the consumption of hydrogen peroxide at 240 nm (35). Initial concentration of hydrogen peroxide in the reaction mixture was 10 mM.

Assay of Superoxide Radical Formation—Production of superoxide ions was monitored spectrophotometrically at 490 nm using the tetrazolium dye MTS (36). Assays were performed in SD medium containing MTS at 190 µM, in a double beam spectrophotometer. A reference cuvette with 190 µM MTS alone in SD medium was systematically used.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Glutathione Exacerbates the Toxicity of Selenite toward S. cerevisiae Cells—To evidence an effect of glutathione on the sensitivity of S. cerevisiae to selenite, cell survival was estimated after 1-h incubation in SD minimal medium containing varying concentrations of both glutathione (0–400 µM) and sodium selenite (0–50 µM). Cells were plated on rich medium and left to grow for 2 days. Their ability to form colonies was used as an indicator of viability. In the absence of glutathione, selenite concentrations as high as 5 mM reduced survival by <60% (Fig. 1A). Glutathione addition strongly enhanced the toxicity of selenite (Fig. 1B). For instance, in the presence of 400 µM glutathione and 20 µM selenite, only 12% of the cells survived. Besides, under such conditions of high mortality, we noted that a large proportion of survivors (over 25%) displayed the phenotype of the petite mutant (37).

To know whether the effect of glutathione was mediated by its thiol function, cell survival was monitored after incubation in SD medium containing selenite and supplemented by either L-cysteine or dithiothreitol. Like glutathione, these thiol-containing compounds dramatically increased the toxicity of selenite (Table 1). On the other hand, oxidized glutathione or L-cystathionine, which do not carry reduced thiol groups, did not enhance the toxicity of selenite (Table 1).


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  		TABLE 1
Effect of various sulfur-containing compounds on the toxicity of selenite against S. cerevisiae

Strain DTY7 was grown in minimal SD medium. When the optical density reached 0.1 at 650 nm, the culture was complemented with 20 µM selenite plus one of the assayed compounds at the indicated concentration. After 1-h incubation at 30 °C, samples were plated onto YT agar to monitor cell viability. Results are expressed as percentage of survival compared to control samples incubated in SD medium alone. Ranges were calculated from two independent experiments.

 
Selenite (SeO2–3) is known to react spontaneously with glutathione (GSH) (27, 38–40). The first product is selenodiglutathione (GS-Se-SG, Reaction 1 below). In the presence of excess glutathione, selenodiglutathione is further reduced by glutathione into glutathioselenol (GS-SeH/GS-Se–, Reaction 2). Glutathioselenol either spontaneously dismutates into elemental selenium (Se(0)) and glutathione (Reaction 3) or is further reduced by glutathione to yield volatile hydrogen selenide (H2Se/HSe–/Se2–, Reaction 4). Finally, H2Se is readily oxidized by oxygen into elemental selenium (Reaction 5). Excess glutathione can shield hydrogen selenide from oxidation (Reaction 6):

Formula 1REACTION 1

Formula 2REACTION 2

Formula 3REACTION 3


Formula 4REACTION 4

Formula 5REACTION 5

Formula 6REACTION 6


Figure 1
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  		FIGURE 1.
Toxicity of selenite against S. cerevisiae. Strain DTY7 was grown in SD minimal medium. When the optical density reached 0.1 at 650 nm, selenite (A) or selenite plus glutathione (B) were added to the culture. After 1-h incubation at 30 °C, samples were plated onto YT agar to monitor cell viability. In B, glutathione concentrations were as follows: {circ}, 0 µM; {square}, 50 µM; {triangleup}, 100 µM; {diamond}, 150 µM; •, 200 µM; {blacksquare}, 300 µM; and {blacktriangleup}, 400 µM. Results are expressed as percentage of survival compared with control samples incubated in SD medium alone. Bars represent mean and range for two independent experiments.

 
Along this series of reactions, reactive oxygen species (ROS) such as superoxide ions (Formula 6), hydrogen peroxide (H2O2), and hydroxyl radicals (OH·), are likely to be produced (24, 41–43). Further experiments described below were designed to evidence which product(s) may account for the high toxicity of selenite:glutathione mixtures.

Extracellular Formation of Reactive Oxygen Species Is Not at the Origin of the High Toxicity of Selenite in the Presence of Glutathione—First, we asked whether ROS were indeed produced upon mixing selenite and glutathione in the synthetic minimal medium. For this purpose, putative superoxide ion production was searched for by using MTS as spectrophotometric probe. This probe is highly sensitive to superoxide at pH 6.0, the pH of the growth medium (36). Incubation of MTS in SD medium containing 20 µM selenite and 400 µM glutathione resulted in a time-dependent increase in the absorbance at 490 nm (Table 2). This signal was quenched by the addition of SOD (Table 2), thus evidencing the generation of superoxide ions by the selenite:glutathione mixture. A part of the MTS signal resisted to SOD addition (Table 2). This remaining signal possibly reflects the reaction of the MTS probe with other reducing agents such as glutathione, glutathioselenol, or hydrogen selenide. In agreement with this idea, we found that incubation of MTS with glutathione alone in the SD medium promoted a slight but continuous increase in the absorbance at 490 nm (Table 2).


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  		TABLE 2
Generation of superoxide ions by selenite:glutathione and xanthine: xanthine oxidase mixtures

Generation of superoxide ions was measured at 490 nm after 1-h incubation of 190 µM MTS in SD medium containing various combination of compounds. Experiments were performed in the presence or absence of SOD at a concentration of 125 units/ml. The SOD-quenchable signal corresponds to the difference between the signals registered in the absence or in the presence of SOD. When indicated, xanthine oxidase was added at a final concentration of 7.5 x 10-3 units/ml. All experiments contained 25 µg/ml bovine serum albumin. Standard deviations were calculated from four independent experiments.

 
Superoxide ions can be produced by mixing xanthine and xanthine oxidase. Using the MTS assay, we established that a mixture of 15 µM xanthine and 7.5 x 10–3 units/ml xanthine oxidase sustained, within 1 h, a signal at 490 nm equivalent to the SOD-quenchable signal produced by the mixture of 400 µM glutathione with 20 µM selenite (Table 2). Cells in SD medium at 30 °C were exposed for 1 h to these concentrations of xanthine and xanthine oxidase, and their survival was surveyed. Yeast cells fully resisted this treatment (Table 3). An additional experiment was performed where xanthine concentration was increased 5-fold. Despite the subsequent increase in ROS production, cell survival remained equal to nearly 100%.


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  		TABLE 3
Toxicity of various selenite:glutathione and xanthine:xanthine oxidase mixtures

S. cerevisiae cells were incubated for 1 h in SD medium containing the indicated compounds. Results are expressed as percentage of survival compared to control samples incubated in SD medium alone. When indicated, SOD, xanthine oxidase, and catalase were added at final concentrations of 125 units/ml, 7.5 x 10-3 units/ml, and 500 units/ml, respectively. Ranges were calculated from two independent experiments.

 
In another set of experiments, free radical scavengers were added to the growth medium at the same time as selenite, glutathione, and S. cerevisiae cells. Neither the Formula 6 scavenger SOD, the H2O2 scavenger catalase, nor the OH• scavenger mannitol, alone or in combination, lowered the toxicity of the selenite: glutathione mixture (Table 3). We verified that added catalase and SOD had remained fully active after the incubation in the presence of the cells. Altogether, these experiments enabled us to conclude that enhancement of selenite toxicity by glutathione cannot be ascribed to the presence of ROS in the growth medium.

Toxicity of Selenodiglutathione for S. cerevisiae Is Low in the Absence of Glutathione—The first step of the reduction of selenite by glutathione is the formation of selenodiglutathione. Because this compound can be easily isolated (27), we monitored S. cerevisiae survival after 1-h incubation in SD medium containing varying concentrations (0–50 µM) of selenodiglutathione. Whatever the added concentration of selenodiglutathione, survival remained larger than 90% (Fig. 2), thus establishing the low toxicity of selenodiglutathione. Nevertheless, in the presence of additional 10–400 µM glutathione, selenodiglutathione induced high mortality (Fig. 2). These results indicate that selenite toxicity in the presence of glutathione must arise from a step beyond the formation of selenodiglutathione.

Actually, such a low toxicity of selenodiglutathione could be expected from the results in Fig. 1. In the presence of 200 µM glutathione, paradoxically, 50 µM selenite is significantly less toxic than 10 µM selenite. Four molecules of glutathione are required to produce one molecule of selenodiglutathione from one molecule of selenite. Consequently, at a 200:50 selenite: glutathione stoichiometry, the main reaction product is selenodiglutathione (39). At a 200:10 stoichiometry, selenodiglutathione is also produced but is allowed to combine downstream with excess glutathione. Products of the further reactions can be expected to contain the agent(s) responsible for the mortality of the cells.

Toxicity of Elemental Selenium for S. cerevisiae Is Negligible—Elemental selenium is one of the products of the reaction between selenite and glutathione (Reaction 5). Incubation of yeast cells for 1 h in SD medium containing elemental selenium (0–200 µM) did not cause any significant lethality (Fig. 3). Addition of glutathione (400 µM) jointly with elemental selenium did not change this behavior (Fig. 3). On the other hand, elemental selenium protected the cells against the toxicity of a selenite: glutathione mixture. For instance, addition of 200 µM elemental selenium to SD medium containing 20 µM selenite and 400 µM glutathione significantly increased the survival of the cells (Fig. 3). This protective role of elemental selenium will be debated in the discussion part.


Figure 2
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  		FIGURE 2.
Toxicity of selenodiglutathione in the presence of varying concentrations of glutathione. Strain DTY7 was grown in SD minimal medium. When the optical density reached 0.1 at 650 nm, various combinations of selenodiglutathione and glutathione were added to the culture. After 1 h at 30 °C, samples were plated onto YT agar to monitor cell viability. Results are expressed as percentage of survival compared with control samples incubated in SD medium alone. Glutathione concentrations were as follows: {circ}, 0 µM; •, 10 µM; {blacksquare}, 20 µM; and {blacktriangleup}, 400 µM. Bars represent mean ± S.D. of three independent experiments.

 


Figure 3
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  		FIGURE 3.
Effect of elemental selenium on the viability of S. cerevisiae. Strain DTY7 was grown in SD minimal medium. When the optical density reached 0.1 at 650 nm, varying concentrations of elemental selenium were added to the culture, either alone ({circ}) or in the presence of 400 µM glutathione (•) or of 400 µM glutathione plus 20 µM selenite ({blacksquare}). After 1 h at 30 °C, samples were plated onto YT agar to monitor cell viability. Results are expressed as percentage of survival compared with control samples incubated in SD medium alone. Bars represent mean and range for two independent experiments.

 
Hydrogen Selenide Is Toxic for S. cerevisiae—In the cascade of reactions between selenite and glutathione, the products responsible for toxicity are thus located after selenodiglutathione and before elemental selenium. Beyond the formation of selenodiglutathione, the reaction between glutathione and selenite produces intermediary products such as glutathioselenol or hydrogen selenide (Reactions 2 and 4). These compounds are thought to be short-lived and so should be the toxicity of a selenite:glutathione mixture if they were at its origin. To assess a possible involvement of short-lived products in toxicity, cells were added in tubes in which the reaction between 20 µM selenite and 400 µM glutathione had been triggered earlier. Fig. 4 shows that a selenite:glutathione mixture prepared less than 30 min before addition of the cells was fully toxic. Indeed, after incubation of cells for 1 h in such tubes, survival was <10%. When the mixtures were older than 30 min, toxicity progressively decreased with their age. For instance, upon exposure to a 45-min-old mixture, cell survival exceeded 70%. Hence, toxicity must be associated with transitory products of the reduction pathway.


Figure 4
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  		FIGURE 4.
Toxicity of a selenite:glutathione mixture decreases with the age of the mixture. 1-ml aliquots of SD medium containing 20 µM selenite and 400 µM glutathione were incubated for various times at 30 °C before being inoculated with exponentially growing S. cerevisiae DTY7 cells. After a further 1-h incubation at 30 °C, samples were plated to determine cell survival. Results are expressed as percentage of survival compared with control samples incubated in SD medium alone. Bars represent mean ± S.D. of three independent experiments.

 
Thioredoxin reductase catalyzes reduction of selenite into hydrogen selenide according to the following global reaction (Reaction 7 (26)).

Formula 7REACTION 7
We observed that incubation of S. cerevisiae cells in SD medium containing 20 µM selenite, 500 µM NADPH, 1.4 µg/ml E. coli thioredoxin reductase, and thioredoxin concentrations higher than 1 µM resulted in a high mortality (Fig. 5). Control experiments without NADPH or without thioredoxin reductase did not indicate any significant lethality. The toxicity of the above mixture is thus likely to result from hydrogen selenide. Because reduction of selenite by an excess of thiols also produces H2Se, we suspected this compound to be at the origin of the exacerbation of selenite toxicity by thiols.

To further assess the toxicity of H2Se, we produced this compound directly in the growth medium through the catalytic action of E. coli D-cysteine desulfhydrase on D-selenocystine (44) in Reaction 8.

Formula 8REACTION 8
At first, the specific activity of D-cysteine desulfhydrase in SD medium containing 40 µMDL-selenocystine was measured by following formation of pyruvate from DL-selenocystine. This activity was found equal to 0.03 s–1 at 30 °C. Then, we evaluated survival of S. cerevisiae cells after 1-h incubation in SD medium containing 40 µM DL-selenocystine and concentrations of D-cysteine desulfhydrase ranging from 0 to 150 nM. A high mortality was observed at D-cysteine desulfhydrase concentrations larger than 50 nM (Fig. 6). We estimated that such an enzyme concentration catalyzes the overall production of nearly 5 µM H2Se within 1 h. Finally, we verified that cells survived if DL-selenocystine or D-cysteine desulfhydrase were omitted. Altogether, the above results evidence that H2Se is highly toxic toward S. cerevisiae. Noticeably, H2Se doses causing toxicity, as evaluated here, lie in the same range of concentrations as the lethal doses of selenite in toxic selenite:glutathione mixtures.


Figure 5
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  		FIGURE 5.
Enhancement of selenite toxicity by the thioredoxin/thioredoxin reductase system. Strain DTY7 was grown in SD minimal medium. When the optical density reached 0.1 at 650 nm, 20 µM selenite, 500 µM NADPH, 1.4 µg/ml thioredoxin reductase, and various concentrations of thioredoxin were added. Results are expressed as percentage of survival compared with control samples incubated in SD medium alone. Bars represent mean and range for two independent experiments. Control experiments performed at 20 µM thioredoxin but in the absence of NADPH or thioredoxin reductase yielded no toxicity (not shown).

 


Figure 6
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  		FIGURE 6.
Effect of DL-selenocystine plus D-cysteine desulfhydrase on the viability of S. cerevisiae. Strain DTY7 was grown in SD minimal medium. When the optical density reached 0.1 at 650 nm, DL-selenocystine (40 µM) and various concentrations of D-cysteine desulfhydrase were added to the culture. After 1 h at 30 °C, samples were plated onto YT agar to monitor cell viability. Results are expressed as percentage of survival compared with control samples incubated in SD medium alone. Bars represent mean ± S.D. of four independent experiments.

 


Figure 7
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  		FIGURE 7.
Selenium uptake by S. cerevisiae. Strain DTY7 was grown in SD minimal medium. When the optical density reached 0.1 at 650 nm, 10 mM sodium selenite ({blacksquare}), 20 µM sodium selenite ({circ}), or 20 µM sodium selenite plus 400 µM glutathione (•) were added to the culture. At various times, cells were harvested and assayed for internalized selenium. Confidence limit for each experimental value is estimated to be 25%.

 
Production of H2Se Causes a Large Uptake of Selenium—To further investigate the mechanism of the toxicity, uptake of selenium by yeast cells was followed during a 4-h incubation in growth medium containing either selenite alone or selenite plus glutathione. In the presence of 20 µM or 10 mM selenite alone, selenium in the cells reached 75 µM and 5 mM, respectively (Fig. 7). If selenite (20 µM) was in the presence of glutathione (400 µM), then cellular selenium went up to 5 mM within 4 h (Fig. 7). Other assayed thiols stimulated selenium uptake in a similar manner (Table 4).


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  		TABLE 4
Uptake of selenium in the presence of various selenium- and thiol-containing compounds

Strain DTY7 was grown in minimal SD medium. When the optical density reached 0.1 at 650 nm, the culture was complemented with various selenium- and thiol-containing compounds. When indicated, thioredoxin was added at final concentration of 1 µM, with 1.4 µg/ml thioredoxin reductase and 500 µM NADPH. After 1-h incubation at 30 °C, cells were harvested and assayed for internalized selenium, as described under "Experimental Procedures." Selenium concentration in yeast cells was calculated assuming an intracellular volume of 0.4 µl/OD650 (33). Ranges were calculated from two independent experiments.

 
Thioredoxin, in the presence of thioredoxin reductase and NADPH, was shown above to promote cellular mortality in the presence of selenite. Thus, we investigated whether selenium uptake by the cells was associated with this mortality. Upon 1-h incubation in the presence of selenite (20 µM), thioredoxin (1 µM), thioredoxin reductase and NADPH, internalized selenium amounted to more than 3 mM (Table 4).

Finally, to evidence whether hydrogen selenide was the precursor of the selenium uptakes observed above, cells were left to incubate for 1 h in the presence of both DL-selenocystine (40 µM) and D-cysteine desulfhydrase (50 nM). A marked uptake of selenium (11 mM) was observed. Incubation in the absence of the desulfhydrase resulted in a much smaller increase (0.3 mM) (Table 4).

These results show that hydrogen selenide enters in the cell much more efficiently than selenite. A massive accumulation of selenium may explain the thiol-assisted toxicity of selenite.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Role of H2Se in the Thiol-assisted Toxicity of Selenite—Spontaneous reaction between thiols and selenite generates ROS (24, 41–43). However, extracellular production of ROS is unlikely to be at the origin of the enhancement of selenite toxicity. First, under our experimental conditions, H2O2 concentrations are expected to remain in the micromolar range (28). Because millimolar concentrations of H2O2 are required to kill S. cerevisiae (45), an involvement of H2O2 in the toxicity of a selenite:glutathione mixture can be ruled out. Second, we show here that superoxide ions produced from xanthine and xanthine oxidase are not toxic. Finally, addition of ROS scavengers such as mannitol, catalase or SOD, alone or in combination, does not protect S. cerevisiae against the effect of the selenite: glutathione mixtures.

Reduction of selenite by glutathione produces compounds such as selenodiglutathione, glutathioselenol, elemental selenium, and hydrogen selenide (27, 38–40). The toxicity of either selenodiglutathione or elemental selenium is shown here to be negligible. On the other hand, hydrogen selenide is strongly toxic, as evidenced by the high mortality of yeast cells exposed to hydrogen selenide enzymatically generated from DL-selenocystine. Furthermore, we observed that elemental selenium relieves the toxicity of the selenium:glutathione mixtures. Because it catalyzes the oxidation of hydrogen selenide by oxygen (46, 47), elemental selenium is likely to protect the cells by quenching the accumulation of hydrogen selenide originating from the reaction of selenite with glutathione.

A strong argument in favor of an involvement of hydrogen selenide in toxicity comes from our measurements of selenium intake. Incubation of yeasts in the presence of either (i) selenite and glutathione or (ii) selenite and thioredoxin plus thioredoxin reductase or (iii) DL-selenocystine and D-cysteine desulfhydrase always resulted in a marked uptake of selenium by the cells. Because these various mixtures have in common the capacity to generate H2Se, it may be proposed that enhancement of selenite toxicity by thiols results from the entry of selenium inside the cell under the form of hydrogen selenide.

Another short-lived product of the reaction of selenite with glutathione is glutathioselenol. This unstable compound reacts with glutathione to form H2Se. Consequently, it is difficult to assess whether glutathioselenol contributes by itself to the massive entry of selenium in the presence of glutathione. In this study, we observed that addition to selenite of thioredoxin plus thioredoxin reductase caused high toxicity and promoted a marked increase in cellular selenium concentration. Because of the large size of thioredoxin, it is difficult to imagine that any selenol derivative of thioredoxin corresponding to glutathioselenol can be massively taken up by the cell. Being insoluble, elemental selenium, also, is not likely to cross the cell membrane. Therefore, at least in this case, the only compound likely to enter the cell is H2Se.

Exposure of yeast cells to micromolar concentrations of selenite in the absence of added glutathione results in a weak selenium uptake (Table 4). A transport of selenite in S. cerevisiae has already been reported (19), but no transporter has been identified in this organism so far. The question thus arises to know whether internalization of selenium from selenite is still mediated by H2Se, although no thiol is added in the growth medium. Possibly, the yeast cells display or excrete some thiol-containing compounds able to react with selenite extracellularly and to reduce it into H2Se. Another possibility would be that membrane-bound sulfhydryl groups participate to the transport. Interestingly, in agreement with this idea, an increase in the amount of sulfhydryl groups of such proteins in brush-border membrane vesicles from chick duodenum enhances selenite uptake (48). Otherwise, an H2Se-independent pathway for selenite internalization has to be discovered. Anyway, in the latter case, once selenite is internalized, it should be allowed to react with intracellular thiols, thus producing H2Se inside the cell. Finally, H2Se might be involved in toxicity also when selenite is added without glutathione.

Fates of H2Se inside the Cell—As soon it comes through the membrane of the cell, hydrogen selenide is expected to undergo various chemical or enzymatic transformations. Below, we consider major stable products, which are formed upon such transformations, and discuss their potential to exert cellular toxicity. A first type of modification comes out from the resemblance between selenium and sulfur. Thus, H2Se may enter the sulfur assimilation pathway and be at the origin of selenoamino acids. Actually, selenomethionine has been shown to accumulate in yeast cells grown in the presence of selenite (49). Once there, selenomethionine can take the place of methionine in polypeptides (50). However, yeast cells appear rather insensitive to the incorporation of this amino acid into their proteins (50). On the other hand, selenocysteine does not accumulate in selenized yeast cells, and its incorporation into proteins is negligible (50). Oxidation of H2Se by oxygen yields elemental selenium. A reddening of yeast cells upon incubation with selenite alone has been reported (19). This color most probably comes from the formation of red elemental selenium inside the cell. Because elemental selenium is not soluble, it is not likely to exert any toxicity unless it has been previously reduced.

Cellular Uptake of Selenium in Yeast Might Cause Oxidative Stress—Hydrogen selenide not only enters the cell with an efficiency far larger than that of selenite, but also is susceptible to be the species responsible for toxicity. Possibly H2Se promotes a change in the balance between reduced and oxidized glutathione. Indeed, according to the Reaction 6 above, the presence of hydrogen selenide in the cell is likely to cause oxidation of glutathione. The cycle involving oxidation of hydrogen selenide by oxygen (Reaction 5), and formation of glutathioselenol from elemental selenium and glutathione (reversal of Reaction 3) (27), followed by reduction of glutathioselenol by glutathione into hydrogen selenide (Reaction 4), may drive continuous consumption of glutathione. Moreover, ROS generated from the reaction of hydrogen selenide with molecular oxygen inside the cell (43) may also contribute to the oxidation of glutathione. Eventually, a decrease in the pool of intracellular reduced glutathione could yield an oxidative stress, thus possibly accounting for the genotoxic effects observed in yeast upon exposure to selenite or selenite:glutathione mixtures (14, 15).

Other mechanisms sustaining selenium toxicity may involve reactions of hydrogen selenide with metal-containing proteins. Indeed, the iron of lipoxygenase of human monoclonal B-lymphocytes has been shown in vitro to be sensitive to H2Se (51). Apart from that, by analogy with the action of H2S in rat hepatocyte mitochondria (52), H2Se could promote inhibition of heme-containing enzymes belonging to the respiratory chain. Reaction of H2Se would therefore lead to leakage of electrons and their capture by molecular oxygen and, eventually, to formation of superoxide ions. Superoxide ions, whether due to redox cycling or to inhibition of enzymes of the respiratory chain, might then be amplified by mitochondria, by analogy with what occurs in human hepatoma cells (53). In favor of an involvement of mitochondria in the toxicity of selenite:glutathione mixtures, we observed a high proportion of petite mutants in the survivors after exposure of yeast cells to selenite plus glutathione.

The Case of Mammals—Selenium as a diet supplement can be administrated under inorganic (selenite) or organic (selenomethionine, selenized yeast cells) forms. At one step or another of their metabolism, all these forms can produce hydrogen selenide, as we will discuss below.

With selenite as the selenium source, reduction occurs early in the absorption process. When administrated by perfusion inside the intestine, selenite is mainly recovered intact in the vasculature (54). However, small metabolites such as selenodiglutathione or selenodicysteine, as well as selenium associated to proteins, become also detectable in the intestine vasculature (54). All the above mentioned selenocompounds, including selenite, are likely to be further reduced in the vessels. Indeed, after intravenous injection, selenite is taken up within minutes by the red blood cells where it is reduced to selenide (55). Selenide is then released by the cells in the plasma. Eventually, through transport by blood, selenium can be widely distributed.

When administrated orally to pigs, selenomethionine is transported across the brush-border membranes of intestine (56) and addressed to nearly all organs in its intact form (57). Inside the cells, selenomethionine can be transformed in selenocysteine by the sulfur pathway (50) and then into alanine and elementary selenium by the selenocysteine beta-lyase (58). Elementary selenium can be reduced in selenide by intracellular thiols.

Finally, selenized yeast cells contain various forms of selenium. Selenomethionine in proteins is by far the major form (59). Digestion of proteins and metabolization of selenomethionine will supply cells with hydrogen selenide.

Concluding Remarks—In liver cells, detoxification of selenium is ensured by the enzymatic conversion of hydrogen selenide into several methylated species (60–64). Such metabolic pathways do not occur in S. cerevisiae (65). However, according to the available literature, the toxicity of selenium toward animal cells and yeast cells shares many features. For instance, with animal cells: (i) thiols have been observed to enhance the accumulation of selenium (66–68) and the toxicity of selenite (21, 66, 69); (ii) hydrogen selenide toxicity has been recognized very early (70); and (iii) redox phenomena (24, 25, 71) as well as mitochondria (72) are involved in the toxicity mechanisms of selenocompounds. Interestingly, in bacteria also, redox phenomena are involved in the toxicity of selenite (73). In particular, in E. coli, the gene of SOD is essential to the defense against selenite (74).

We may therefore propose that the toxicity of selenite follows general rules involving perturbation by hydrogen selenide of the redox balance of the cell. In animal cells, the choice between apoptosis and proliferation is linked to the redox state. By decreasing the reducing power of the cell through hydrogen selenide action, selenite might exert its anticarcinogenic properties.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Recipient of a fellowship from the French national Programme inter-organismes "Toxicologie Nucléaire." Back

2 Both authors contributed equally to this work. Back

3 Current address: Institut des Sciences du Végétal, CNRS, Avenue de la Terrasse, Bât. 23, 91198 Gif-sur-Yvette Cedex, France. Back

4 Current address: Laboratoire Interfaces, Traitements, Organisation et Dynamique des Systèmes, Université Paris VII, 1, rue Guy de la Brosse, 75005 Paris, France. Back

5 To whom correspondence should be addressed. Tel.: 33-1-69-33-41-81; Fax: 33-1-69-33-30-13; E-mail: plateau{at}bioc.polytechnique.fr.

6 The abbreviations used are: MES, 2-morpholinoethanesulfonic acid; ROS, reactive oxygen species; SD, synthetic dextrose minimal medium; SOD, superoxide dismutase; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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