SYNTHESIS OF SELENIUM-DERIVATIZED NUCLEOSIDES AND OLIGONUCLEOTIDES FOR X-RAY CRYSTALLOGRAPHY

We report here the synthesis of nucleoside and oligonucleotide analogs containing selenium, which serves as an anomalous scattering center to enable MAD phase determination in nucleotide X-ray crystallography. We have developed a phase transfer approach to introduce the selenium functionality in A, C, G, T, and U nucleosides at 5′-positions. In the incorporation of the selenium functionality, the leaving groups (bromide, mesyl, and tosyl) were readily displaced by sodium selenide, sodium diselenide, and sodium methyl selenide with yields higher than 90%. Selenium-derivatized oligonucleotides have been synthesized via phosphoramidite chemistry.     

Extracellular production of hydrogen selenide accounts for thiol-assisted toxicity of selenite against Saccharomyces cerevisiae

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.     

Selenium detoxification of heavy metals: a possible mechanism for the blood plasma

Among the activities of the essential trace element selenium is the ability to reduce the toxicity of heavy metal ions like cadmium(II) and mercury(II). Detoxification often depends on the metabolic reduction of selenium to hydrogen selenide; the mechanism generally advanced to explain such selenium/metal interactions is that selenide combines with heavy metal ions to give a metal selenide which is metabolically inert. However, this hypothesis does not consider circumstances where selenide is quickly removed by other reactions. Given the ease with which selenide is oxidized, such conditions are likely to occur in the blood plasma, an environmental rich in oxidizing agents and a site for many selenium/metal interactions. Using polarography to monitor both selenide and cadmium, we have found that selenide reacts rapidly in vitro with the disulfide bonds present in bovine serum albumin in preference to forming cadmium selenide. We hypothesize that a similar reaction occurs in the blood plasma with the disulfide bonds of plasma proteins to generate thiol groups on the protein involved, and that these newly formed thiols are responsible for the observed reduction of metal toxicity through the ability to chelate heavy metal ions.     

Redox reactions of hydrogen selenide ion

Hydrogen selenide ion (HSe^-) is an important product in the metabolism of the essential trace element selenium. Although its role in selenium metabolism is recognized, aspects of the basic chemistry of selenide have been ignored, particularly the tendency of selenide to undergo rapid redox reactions with biological oxidants. Using polarography, we have found that selenide reacts in vitro with a variety of compounds including dehydroascorbic acid, quinones like vitamin K₁ and FAD (flavin adenine dinucleotide), and disulfides such as oxidized glutathione and lipoic acid. The fact selenide reacts readily in vitro suggests similar reactions may also occur in vivo with important biological consequences. Contrary to expectations, selenide was found not to reduce the disulfide bond of oxidized dithiothreitol (trans-4,5-dihydroxyl-1,2-dithiane), indicating the commonly published value for the standard electrode potential of the selenium/hydrogen selenide ion couple is in error. The electrode potential is an important parameter to aid in anticipating possible redox reactions of selenide in vivo.     

High affinity selenium uptake in a keratinocyte model

The distribution of selenium in mammals has been recently shown to be mediated primarily by selenoprotein P. Even in the absence of selenoprotein P, selenium is distributed from the liver into all organs and tissues when supplemented in the diet. The form of selenium that is actively taken up by mammalian cells at trace concentrations has yet to be determined. We used a human keratinocyte model to determine whether reduction of the oxyanion selenite (SeO(3)(2-)) to the more reduced form of selenide (HSe(-)) would affect uptake. Indeed a reduced form of selenium, presumably selenide, was actively transported into keratinocytes and displayed saturation kinetics with an apparent K(m) of 279 nM. ATPase inhibitors blocked the uptake of selenide, as did the competing anions molybdate and chromate, but not sulfate. These results suggest that the small molecule form of selenium that is distributed in tissues is hydrogen selenide, despite its sensitivity to oxygen and reactivity to thiols.     

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 (H(2)Se/HSe(-/)Se(2-)). Among the genes whose deletion caused hypersensitivity, 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 O(2)-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 O(2). Finally we showed that, in vivo, toxicity strictly depended on the presence of O(2). Therefore, by combining genome-wide and biochemical approaches, we demonstrated that, in yeast cells, hydrogen selenide induces toxic DNA breaks through an O(2)-dependent radical-based mechanism.     

Hydrogen selenide ion adsorption to colloidal elemental selenium

Hydrogen selenide ion (HSe^?) has an important role in the metabolism of the essential trace element selenium. Several redox reactions of selenide were found to be dominated by the amount of colloidal elemental selenium (Se°) generated during the reaction. The following reaction of selenide with the disulfide, oxidized glutathione (GSSG), was used as an example: HSe^? + GSSG + H⁺ → Se° + 2 GSH. The resulting thiol is reduced glutathione (GSH; γ-glutamylcysteinylglycine). By following this reaction with polarography, it was seen that the ratio of colloidal selenium produced to selenide unreacted was a constant 2.1 ± 0.1, and was the only factor found to determine the extent of oxidation. This is best explained by the hypothesis that freshly generated colloidal selenium adsorbs selenide readily; no evidence for polyselenide formation was found. Adsorption of selenide should be considered in any reaction involving the oxidation of selenide to colloidal selenium.     

The effect of vitamin E on the oxidation state of selenium in rat liver

1. (75)Se as Na(2) (75)SeO(3) was administered orally to rats under different nutritional conditions. 2. The selenium found in the liver subcellular organelle fractions was present in at least three oxidation states: acid-volatile selenium, assumed to be selenide, zinc-hydrochloric acid-reducible selenium, assumed to be selenite, and higher oxidation states of selenium and organic derivatives, called selenate for convenience. 3. The proportion of the total selenium present as selenide present as selenide is susceptible to oxidation in vitro, which can be prevented by the addition of antioxidants in vitro. 4. The proportion of selenide is also directly related to the vitamin E status of the rats, and treatment of vitamin E-deficient rats with vitamin E results in an increase in the proportion of selenide. 5. Freezing the liver in situ before preparation of the organelle fractions did not alter the susceptibility of the selenide proportion to dietary vitamin E, indicating that the observed effects occur in vivo and not as a result of oxidation post mortem. 6. Intravenous administration of Na(2) (75)SeO(3), to rats whose alimentary tract was partially sterilized by neomycin treatment, gave a similar result to that in paragraph 4, indicating that the reduction of selenite to selenide probably occurs in vivo, and that intestinal micro-organisms are not responsible. 7. Treatment of vitamin E-deficient rats with silver produced a fall in the total (75)Se content of the liver, an effect only partially reversed by vitamin E administration. The proportion of the total selenium present as selenide was also lowered by the treatments with silver, and vitamin E significantly reversed this trend in most cases. 8. These results are consistent with the hypothesis that the active form of Se may be selenide and that the selenide may form part of the active centre of an uncharacterized class of catalytically active non-haem-iron proteins that are protected from oxidation in vivo by vitamin E.     

Exploring the structural basis for selenium/mercury antagonism in Allium fistulosum

While continuing efforts are devoted to studying the mutually protective effect of mercury and selenium in mammals, few studies have investigated the mercury-selenium antagonism in plants. In this study, we report the metabolic fate of mercury and selenium in Allium fistulosum (green onion) after supplementation with sodium selenite and mercuric chloride. Analysis of homogenized root extracts via capillary reversed phase chromatography coupled with inductively coupled plasma mass spectrometry (capRPLC-ICP-MS) suggests the formation of a mercury-selenium containing compound. Micro-focused synchrotron X-ray fluorescence mapping of freshly excised roots show Hg sequestered on the root surface and outlining individual root cells, while Se is more evenly distributed throughout the root. There are also discrete Hg-only, Se-only regions and an overall strong correlation between Hg and Se throughout the root. Analysis of the X-ray absorption near edge structure (XANES) spectra show a "background" of methylselenocysteine within the root with discrete spots of SeO(3)(2-), Se(0) and solid HgSe on the root surface. Mercury outlining individual root cells is possibly binding to sulfhydryl groups or plasma membrane or cell wall proteins, and in some places reacting with reduced selenium in the rhizosphere to form a mercury(ii) selenide species. Together with the formation of the root-bound mercury(ii) selenide species, we also report on the formation of cinnabar (HgS) and Hg(0) in the rhizosphere. The results presented herein shed light on the intricate chemical and biological processes occurring within the rhizosphere that influence Hg and Se bioavailability and will be instrumental in predicting the fate and assisting in the remediation of these metals in the environment and informing whether or not fruit and vegetable food selection from aerial plant compartments or roots from plants grown in Hg contaminated soils, are safe for consumption.     

Zinc-specific autometallographic in vivo selenium methods: tracing of zinc-enriched (ZEN) terminals, ZEN pathways, and pools of zinc ions in a multitude of other ZEN cells.

In vivo-applied sodium selenide or sodium selenite causes the appearance of zinc-selenium nanocrystals in places where free or loosely bound zinc ions are present. These nanocrystals can in turn be silver enhanced by autometallographic (AMG) development. The selenium method was introduced in 1982 as a tool for zinc-ion tracing, e.g., in vesicular compartments such as synaptic vesicles of zinc-enriched (ZEN) terminals in the central nervous system, and for visualization of zinc ions in ZEN secretory vesicles of, e.g., somatotrophic cells in the pituitary, zymogene granules in pancreatic acinar cells, beta-cells of the islets of Langerhans, Paneth cells of the crypts of Lieberkühn, secretory cells of the tubuloacinar glands of prostate, epithelium of parts of ductus epididymidis, and osteoblasts. If sodium selenide/selenite is injected into brain, spinal cord, spinal nerves containing sympathetic axons, or intraperitoneally, retrograde axonal transport of zinc-selenium nanocrystals takes place in ZEN neurons, resulting in accumulation of zinc-selenium nanocrystals in lysosomes of the neuronal somata. The technique is, therefore, also a highly specific tool for tracing ZEN pathways. The present review includes an update of the 1982 paper and presents evidence that only zinc ions are traced with the AMG selenium techniques if the protocols are followed to the letter.     

Escherichia coli NifS-like proteins provide selenium in the pathway for the biosynthesis of selenophosphate

Selenophosphate synthetase (SPS), the selD gene product from Escherichia coli, catalyzes the biosynthesis of monoselenophosphate, AMP, and orthophosphate in a 1:1:1 ratio from selenide and ATP. Kinetic characterization revealed the K(m) value for selenide approached levels that are toxic to the cell. Our previous demonstration that a Se(0)-generating system consisting of l-selenocysteine and the Azotobacter vinelandii NifS protein can replace selenide for selenophosphate biosynthesis in vitro suggested a mechanism whereby cells can overcome selenide toxicity. Recently, three E. coli NifS-like proteins, CsdB, CSD, and IscS, have been overexpressed and characterized. All three enzymes act on selenocysteine and cysteine to produce Se(0) and S(0), respectively. In the present study, we demonstrate the ability of each E. coli NifS-like protein to function as a selenium delivery protein for the in vitro biosynthesis of selenophosphate by E. coli wild-type SPS. Significantly, the SPS (C17S) mutant, which is inactive in the standard in vitro assay with selenide as substrate, was found to exhibit detectable activity in the presence of CsdB, CSD, or IscS and l-selenocysteine. Taken together the ability of the NifS-like proteins to generate a selenium substrate for SPS and the activation of the SPS (C17S) mutant suggest a selenium delivery function for the proteins in vivo.     

Microbial Transformations of Selenium

Resting cell suspensions of a strain of Corynebacterium isolated from soil formed dimethyl selenide from selenate, selenite, elemental selenium, selenomethionine, selenocystine, and methaneseleninate. Extracts of the bacterium catalyzed the production of dimethyl selenide from selenite, elemental selenium, and methaneseleninate, and methylation of the inorganic Se compounds was enhanced by S-adenosylmethionine. Neither trimethylselenonium nor methaneselenonate was metabolized by the Corynebacterium. Resting cell suspensions of a methionine-utilizing pseudomonad converted selenomethionine to dimethyl diselenide. Six of 10 microorganisms able to grow on cystine used selenocystine as a sole source of carbon and formed elemental selenium, and one of the isolates, a pseudomonad, was found also to produce selenide. Soil enrichments converted trimethylselenonium to dimethyl selenide. Bacteria capable of utilizing trimethylselenonium, dimethyl selenide, and dimethyl diselenide as carbon sources were isolated from soil.     

S-adenosyl-L-methionine:thioether S-methyltransferase, a new enzyme in sulfur and selenium metabolism

The final urinary excretion product of selenium detoxification is trimethylselenonium ion. An assay has been developed for the enzyme, S-adenosylmethionine:thioether S-methyltransferase, responsible for this final methylation reaction. This assay employed high pressure liquid chromatography separation and quantitation of the trimethylselenonium ion produced by thioether methyltransferase acting on S-adenosylmethionine and dimethyl selenide. The enzyme was shown to reside primarily in the cytosol of mouse lung (30 pmol/mg protein/min) and liver (7 pmol/mg protein/min). Purification from mouse lung to a preparation that exhibited a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was achieved by DEAE, gel filtration, and chromatofocusing chromatographies. Thioether methyltransferase is monomeric with a molecular weight of 28,000 and has a pI of 5.3. The pH optimum was 6.3, and Km values for dimethyl selenide and S-adenosylmethionine were 0.4 and 1.0 microM, respectively. The enzyme was inhibited 50% by 25 microM sinefungin, an analog of S-adenosylmethionine, or 40 microM S-adenosylhomocysteine, the reaction product. Pure thioether methyltransferase methylated selenium in dimethyl selenide, tellurium in dimethyl telluride, and S in dimethyl sulfide and many other thioethers. These data suggest a general role for this novel enzyme in the synthesis of onium compounds with increased aqueous solubility helpful in their excretion.     

Kinetics of the action of three selenides on Staphylococcus aureus growth as studied by microcalorimetry

The effect of three kinds of selenide on Staphylococcus aureus growth was studied by means of microcalorimetry. Differences in their capacities to inhibit the metabolism of this bacterium were observed. The rate constant k (in the log phase) in the presence of the compounds decreased with increasing concentrations of the compounds. The relationship of k and c is nearly linear for the selenium compounds. Judged from the rate constant, k, and the half-inhibitory concentration IC50, the experimental results reveal that the sequence of antibiotic activity of the three tested selenides compounds is (2-hydroxy benzyl imino)ethyl n-hexyl selenide> n-butyl(2- hydroxy benzyl imino)ethyl selenide > bis[(2,4-dihydroxy benzyl imino)ethyl] selenide.     

Effects of growth conditions on production of methyl selenides in cultures of Rhodobacter sphaeroides

Rhodobacter sphaeroides 2.4.1 exposed to selenate or selenite produced volatile selenium compounds. Total amounts of dimethyl selenide, dimethyl diselenide, dimethyl sulfide and dimethyl disulfide in culture medium and headspace were determined. The highest selenate volatilization occurred in the late stationary phase of growth. However, cultures deprived of light in the stationary phase of growth produced much less of the volatile organo-selenium compounds. Lower culture pHs increased the rate of selenium volatilization. Low sulfate concentration limited biomass production and selenium volatilization; high sulfate concentrations had an enhancing effect on the release of organo-selenium compounds. Cultures of R. sphaeroides reacted very differently to amendments with increasing amounts of selenate and selenite. Only small amounts of selenite were volatilized; meanwhile high amounts of methylated selenides were found in selenate-poisoned cultures. Received 03 February 1997/ Accepted in revised form 16 May 1997     

Utilization of selenocysteine as a source of selenium for selenophosphate biosynthesis

Selenophosphate synthetase (SPS), the selD gene product from Escherichia coli, catalyzes the biosynthesis of monoselenophosphate from selenide and ATP. Characterization of selenophosphate synthetase revealed the determined K(m) value for selenide is far above the optimal concentration needed for growth and approached levels which are toxic. Selenocysteine lyase enzymes, which decompose selenocysteine to elemental selenium (Se(0)) and alanine, were considered as candidates for the control of free selenium levels in vivo. The ability of a lyase protein to generate Se(0) in the proximity of SPS maybe an attractive solution to selenium toxicity as well as the high K(m) value for selenide. Recently, three E. coli NifS-like proteins, CsdB, CSD, and IscS, were characterized. All three proteins exhibit lyase activity on L-cysteine and L-selenocysteine and produce sulfane sulfur, S(0), or Se(0) respectively. Each lyase can effectively mobilize Se(0) from L-selenocysteine for selenophosphate biosynthesis.     

Methylselenol Formed by Spontaneous Methylation of Selenide Is a Superior Selenium Substrate to the Thioredoxin and Glutaredoxin Systems

Naturally occurring selenium compounds like selenite and selenodiglutathione are metabolized to selenide in plants and animals. This highly reactive form of selenium can undergo methylation and form monomethylated and multimethylated species. These redox active selenium metabolites are of particular biological and pharmacological interest since they are potent inducers of apoptosis in cancer cells. The mammalian thioredoxin and glutaredoxin systems efficiently reduce selenite and selenodiglutathione to selenide. The reactions are non-stoichiometric aerobically due to redox cycling of selenide with oxygen and thiols. Using LDI-MS, we identified that the addition of S-adenosylmethionine (SAM) to the reactions formed methylselenol. This metabolite was a superior substrate to both the thioredoxin and glutaredoxin systems increasing the velocities of the nonstoichiometric redox cycles three-fold. In vitro cell experiments demonstrated that the presence of SAM increased the cytotoxicity of selenite and selenodiglutathione, which could neither be explained by altered selenium uptake nor impaired extra-cellular redox environment, previously shown to be highly important to selenite uptake and cytotoxicity. Our data suggest that selenide and SAM react spontaneously forming methylselenol, a highly nucleophilic and cytotoxic agent, with important physiological and pharmacological implications for the highly interesting anticancer effects of selenium.     

Selenium incorporation into Saccharomyces cerevisiae cells: a study of different incorporation methods

AIMS: To study the effects of the selenium enrichment protocols in yeast at various points in the cell cycle, total selenium accumulation and the forms of selenium incorporated. METHODS AND RESULTS: The use of selenized yeast as enriched selenium supplements in human nutrition has become a topic of increasing interest over the last decade. Four enrichment procedures have been evaluated using sodium selenite as the selenium source: enrichment during the growth phase; enrichment at the non-growth phase, both of these at different selenium levels; enrichment by seeding in a fermentable carbon source (glucose); Se-enrichment with a non-fermentable carbon source (glycerol). A nitric acid digestion of the yeast samples prepared under different conditions has been performed in order to evaluate the total selenium incorporated into the yeast cells. Also, an enzymatic digestion of the yeast samples with pepsin has been carried out as an initial step to begin the process of determining which of the different possible selenium species are formed. The cell count evaluations of the selenium-enriched yeast showed that the growth phase, seeding and the use of YEPG media is influenced by the addition of Se, while the non-growth phase is not. Total selenium incorporation studies showed that seeding the yeast permits more accumulation of selenium. Speciation studies of the enriched yeast showed that the growth phase increases the formation of L-Se-methionine. CONCLUSIONS: When the aim of enriching yeast with selenium is the formation of L-Se-methionine, the best enrichment procedure is using the growth phase with small concentrations of sodium selenite. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of selenium supplements is widespread and most of the supplements use selenium-enriched yeast in their formulation. Studies made on supplements do not have the appropriate Se-species for optimal absorption in the human body. This study presents and compares methods for the best selenium yeast enrichment that could ultimately be used in selenium supplement formulations.     

Incorporation of selenium from selenite and selenocystine into glutathione peroxidase in the isolated perfused rat liver

The erythrocyte-free, isolated perfused rat liver was used to study the incorporation of selenium into glutathione peroxidase. Gel filtration and ion exchange chromatography of liver supernatant demonstrated ⁷⁵Se incorporation into glutathione peroxidase. A 9-fold excess of unlabelled selenium as selenite or selenide very effectively reduced ⁷⁵Se incorporation from L[⁷⁵Se]-selenocystine, but a 100-fold excess of unlabelled selenium as selenocystine was relatively ineffective as compared to selenite or selenide in diluting ⁷⁵Se incorporation from [⁷⁵Se]selenite. These results indicate that selenide and selenite are more readily metabolized than is selenocysteine to the immediate selenium precursor used for glutathione peroxidase synthesis, and suggest a posttranslational modification at another amino acid residue, rather than direct incorporation of selenocysteine, as the mechanism for formation of the presumed selenocysteine moiety of the enzyme.     

Kinetics of the reaction between hydrogen selenide ion and oxygen

Hydrogen selenide ion (HSe^?) reacts with oxygen in the following manner: HSe^? + 1/2O₂ → Se^o + OH^?. Interest in the kinetics of this reaction comes from the fact that selenide is an important product in the metabolism of the essential trace element selenium. Using polarography to monitor both selenide and oxygen, we have found the reaction exhibits complex kinetics, including autoaccelerating behavior and the generation of reactive intermediates capable of inducing reactions in other substances present. Probable intermediate species include superoxide, peroxide and polyselenides. The reaction is slow with respect to diffusion controlled reactions, but fast with respect to the time required to prepare solutions for biological study. Selenide concentrations greater than 10^?6 M decay to give solutions of predominantly colloidal elemental selenium less than 3 minutes after exposure to atmospheric levels of oxygen.