Sodium Selenide Toxicity Is Mediated by O2-Dependent DNA Breaks

Hydrogen selenide is a recurrent metabolite of selenium compounds. However, few experiments studied the direct link between this toxic agent and cell death. To address this question, we first screened a systematic collection of Saccharomyces cerevisiae haploid knockout strains for sensitivity to sodium selenide, a donor for hydrogen selenide (H2Se/HSe−/Se2−). Among the genes whose deletion caused hypresensitivity, homologous recombination and DNA damage checkpoint genes were over-represented, suggesting that DNA double-strand breaks are a dominant cause of hydrogen selenide toxicity. Consistent with this hypothesis, treatment of S. cerevisiae cells with sodium selenide triggered G2/M checkpoint activation and induced in vivo chromosome fragmentation. In vitro, sodium selenide directly induced DNA phosphodiester-bond breaks via an O2-dependent reaction. The reaction was inhibited by mannitol, a hydroxyl radical quencher, but not by superoxide dismutase or catalase, strongly suggesting the involvement of hydroxyl radicals and ruling out participations of superoxide anions or hydrogen peroxide. The •OH signature could indeed be detected by electron spin resonance upon exposure of a solution of sodium selenide to O2. Finally we showed that, in vivo, toxicity strictly depended on the presence of O2. Therefore, by combining genome-wide and biochemical approaches, we demonstrated that, in yeast cells, hydrogen selenide induces toxic DNA breaks through an O2-dependent radical-based mechanism

Trans-sulfuration Pathway Seleno-amino Acids Are Mediators of Selenomethionine Toxicity in Saccharomyces cerevisiae.

International audienceToxicity of selenomethionine, an organic derivative of selenium widely used as supplement in human diets, was studied in the model organism Saccharomyces cerevisiae. Several DNA repair-deficient strains hypersensitive to selenide displayed wild-type growth rate properties in the presence of selenomethionine indicating that selenide and selenomethionine exert their toxicity via distinct mechanisms. Cytotoxicity of selenomethionine decreased when the extracellular concentration of methionine or S-adenosylmethionine was increased. This protection resulted from competition between the S- and Se-compounds along the downstream metabolic pathways inside the cell. By comparing the sensitivity to selenomethionine of mutants impaired in the sulfur amino acid pathway, we excluded a toxic effect of Se-adenosylmethionine, Se-adenosylhomocysteine, or of any compound in the methionine salvage pathway. Instead, we found that selenomethionine toxicity is mediated by the trans-sulfuration pathway amino acids selenohomocysteine and/or selenocysteine. Involvement of superoxide radicals in selenomethionine toxicity in vivo is suggested by the hypersensitivity of a Δsod1 mutant strain, increased resistance afforded by the superoxide scavenger manganese, and inactivation of aconitase. In parallel, we showed that, in vitro, the complete oxidation of the selenol function of selenocysteine or selenohomocysteine by dioxygen is achieved within a few minutes at neutral pH and produces superoxide radicals. These results establish a link between superoxide production and trans-sulfuration pathway seleno-amino acids and emphasize the importance of the selenol function in the mechanism of organic selenium toxicity

Neutralization by metal ions of the toxicity of sodium selenide.

Inert metal-selenide colloids are found in animals. They are believed to afford cross-protection against the toxicities of both metals and selenocompounds. Here, the toxicities of metal salt and sodium selenide mixtures were systematically studied using the death rate of Saccharomyces cerevisiae cells as an indicator. In parallel, the abilities of these mixtures to produce colloids were assessed. Studied metal cations could be classified in three groups: (i) metal ions that protect cells against selenium toxicity and form insoluble colloids with selenide (Ag⁺, Cd²⁺, Cu²⁺, Hg²⁺, Pb²⁺ and Zn²⁺), (ii) metal ions which protect cells by producing insoluble metal-selenide complexes and by catalyzing hydrogen selenide oxidation in the presence of dioxygen (Co²⁺ and Ni²⁺) and, finally, (iii) metal ions which do not afford protection and do not interact (Ca²⁺, Mg²⁺, Mn²⁺) or weakly interact (Fe²⁺) with selenide under the assayed conditions. When occurring, the insoluble complexes formed from divalent metal ions and selenide contained equimolar amounts of metal and selenium atoms. With the monovalent silver ion, the complex contained two silver atoms per selenium atom. Next, because selenides are compounds prone to oxidation, the stabilities of the above colloids were evaluated under oxidizing conditions. 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), the reduction of which can be optically followed, was used to promote selenide oxidation. Complexes with cadmium, copper, lead, mercury or silver resisted dissolution by DTNB treatment over several hours. With nickel and cobalt, partial oxidation by DTNB occurred. On the other hand, when starting from ZnSe or FeSe complexes, full decompositions were obtained within a few tens of minutes. The above properties possibly explain why ZnSe and FeSe nanoparticles were not detected in animals exposed to selenocompounds

Exposure to the Methylselenol Precursor Dimethyldiselenide Induces a Reductive Endoplasmic Reticulum Stress in Saccharomyces cerevisiae

International audienceMethylselenol (MeSeH) is a major cytotoxic metabolite of selenium, causing apoptosis in cancer cells through mechanisms that remain to be fully established. Previously, we demonstrated that, in Saccharomyces cerevisiae, MeSeH toxicity was mediated by its metabolization into selenomethionine by O-acetylhomoserine (OAH)-sulfhydrylase, an enzyme that is absent in higher eukaryotes. In this report, we used a mutant met17 yeast strain, devoid of OAH- sulfhydrylase activity, to identify alternative targets of MeSeH. Exposure to dimethyldiselenide (DMDSe), a direct precursor of MeSeH, caused an endoplasmic reticulum (ER) stress, as evidenced by increased expression of the ER chaperone Kar2p. Mutant strains (∆ire1 and ∆hac1) unable to activate the unfolded protein response were hypersensitive to MeSeH precursors but not to selenomethionine. In contrast, deletion of YAP1 or SKN7, required to activate the oxidative stress response, did not affect cell growth in the presence of DMDSe. ER maturation of newly synthesized carboxypeptidase Y was impaired, indicating that MeSeH/DMDSe caused protein misfolding in the ER. Exposure to DMDSe resulted in induction of the expression of the ER oxidoreductase Ero1p with concomitant reduction of its regulatory disulfide bonds. These results suggest that MeSeH disturbs protein folding in the ER by generating a reductive stress in this compartment

Recent advances in the mechanism of selenoamino acids toxicity in eukaryotic cells

International audienceSelenium is an essential trace element due to its incorporation into selenoproteins with important biological functions. However, at high doses it is toxic. Selenium toxicity is generally attributed to the induction of oxidative stress. However, it has become apparent that the mode of action of seleno-compounds varies, depending on its chemical form and speciation. Recent studies in various eukaryotic systems, in particular the model organism Saccharomyces cerevisiae, provide new insights on the cytotoxic mechanisms of selenomethionine and selenocysteine. This review first summarizes current knowledge on reactive oxygen species (ROS)-induced genotoxicity of inorganic selenium species. Then, we discuss recent advances on our understanding of the molecular mechanisms of selenocysteine and selenomethionine cytotoxicity. We present evidences indicating that both oxidative stress and ROS-independent mechanisms contribute to selenoamino acids cytotoxicity. These latter mechanisms include disruption of protein homeostasis by selenocysteine misincorporation in proteins and/or reaction of selenols with protein thiols

Formation of metal-selenide colloids.

<p>To monitor the formation of metal-selenide colloids, mixtures containing sodium selenide (50 µM) and the indicated metal salt concentrations were prepared inside an anaerobic glove box. After 10 min incubation at room temperature (180 min in the case of iron), the optical densities of the solution were measured at 340 nm. In the cases of cadmium, cobalt, copper, mercury, nickel, lead, silver and zinc, data were fitted to a one-site binding equation (least square minimization) assuming that the affinities between the constituents involved in complex formation were infinitely high. In the case of iron, data were fitted to the solubility product equation ([Fe²⁺][Se^2–] = K_sp). Obtained pK_sp value was 18.5±0.2. Manganese, magnesium and calcium did not modify the optical density of the sodium selenide solution. Data with manganese are shown as an example.</p

Colors and apparent molar turbidity coefficients of metal-selenide colloids.

<p>Indicated colors are those of samples containing 100 µM sodium selenide and 200 µM of each studied metal ion and incubated for 10 min. Apparent molar turbidity coefficients (ε₃₄₀) were deduced from the data in Fig. 2, assuming that, when the metal is in excess, all the selenium has passed from the soluble phase to the colloidal phase. Under our conditions, zinc selenide colloid formation could be evidenced at 340 nm (Fig. 2) but remained invisible to the eye. Nevertheless, after centrifugation, a light yellow precipitate could be recovered. ^(a) pK_sp values of the metal selenide complexes, taken from Séby <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054353#pone.0054353-Sby1" target="_blank">[12]</a>, correspond to the equilibriums M²⁺+Se^2– ⇔ MSe (divalent cations) or 2 Ag⁺+Se^2– ⇔ Ag₂Se (silver ion). Of note, in many cases, different pK_sp values are available in the literature for a same complex. ^(b) In the case of Fe²⁺, the pK_sp value that we determined in this study from data in Fig. 2 is also shown. ^(c) Sodium selenide (50 µM) was added to a mixture of metal ion (100 µM) and of either EDTA or NTA (1 mM). The sample was incubated anaerobically in a 50 mM MES buffer (pH 6.0) for 3 h. Metals for which colloid formation was no longer observed in the presence of the chelator are labeled with “<b>–</b>” (minus). For the metals which still produced colloids, t₅₀ was measured. ^(d) Effective equilibrium constants of the chelator-divalent metal complexes at pH 6.0 (K_eq^{pH 6}) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054353#pone.0054353-Chaberek1" target="_blank">[64]</a> were calculated using absolute equilibrium constants of these complexes (M.EDTA²⁻ or M.NTA⁻) and protonation pK values of 0.0, 1.5, 2.0, 2.68, 6.11 and 10.17 for EDTA and of 0.8, 1.8, 2.48 and 9.65 for NTA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054353#pone.0054353-Martell1" target="_blank">[65]</a>. In the case of the monovalent Ag⁺ ion, effective pK_eq^{pH 6} values were drawn from equilibrium constants of the Ag.EDTA³⁻, Ag.H-EDTA²⁻ and Ag.NTA²⁻ complexes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054353#pone.0054353-Martell1" target="_blank">[65]</a>.</p

Reaction of metal-selenide colloids with DTNB.

<p>Mixtures containing 50 µM sodium selenide and 100 µM of the metal ion under study were prepared in an anaerobic glove box and left to incubate in this box for 10 min at room temperature (180 min in the case of Fe²⁺). At time zero, 100 µM DTNB was added to the samples in the glove box. (<b>A</b>) Data with Co²⁺, Cd²⁺, Fe²⁺, Mn²⁺, Ni²⁺, Pb²⁺ and Zn²⁺ ions. TNB absorbance was followed at 412 nm. Data with Ca²⁺ or Mg²⁺, which are similar to those with Mn²⁺ or without metal, are not displayed in the figure. (<b>B</b>) Data with Ag⁺, Cu²⁺ and Hg²⁺ ions. TNB absorbance was followed at 390 nm instead of 412 nm (see Materials and Methods). In all cases, turbidities of the metal-selenide mixtures were measured in the glove box before DTNB addition. Corresponding values are indicated on the left side of the figure. Next, turbidities were measured just after DTNB addition. Because DTNB slightly absorbs light at 390 and 412 nm, values measured immediately after DTNB addition (0.25 min on the figure) systematically exceeded the values before DTNB addition (−1 min on the figure). With Ag⁺, Cd²⁺, Cu²⁺, Hg²⁺, Pb²⁺ and Zn²⁺, the OD increments upon DTNB addition (0.065±0.005 and 0.24±0.01 at 412 and 390 nm, respectively) were close to those obtained in a control experiment without metal and selenide (0.060 and 0.24 at 412 and 390 nm, respectively). With Co²⁺ and Ni²⁺, the increments at 412 nm were slightly higher (0.14 and 0.16, respectively). With Fe²⁺, the increment was far higher (0.45 at 412 nm, see Results).</p

Effect of metal ions on dioxygen consumption during oxidation of sodium selenide.

<p>Dissolved dioxygen (O₂ saturation percentage) was measured as a function of time in samples initially containing 1.28 ml of the indicated metal (125 µM). At time 10 s (as indicated by the arrow), 320 µl of an anaerobically prepared solution of sodium selenide was added to give final concentrations of 100 µM metal and 100 µM selenide. This mixing caused instantaneous drop in the dioxygen saturation level, from 100% (234 µM) to 80% (187 µM). After this drop, dissolved oxygen concentration varied depending on the assayed metal. At longer times, slight increases in dioxygen concentration were observed in the samples containing mercury, cobalt or nickel ions. These increases originate from slow diffusion of ambient air in the open vials used for the experiments. These slight increases are expected to also occur in the experiments with manganese or iron or without metal.</p