Selenium metabolism in Escherichia coli

Escherichia coli will reduce selenite (SeO 3 2- ) andselenate (SeO 4 2- ) to elemental selenium Se 0 . Seleniumwill also become incorporated intoproteins as part of the amino acids selenocysteine or selenomethionine.The reaction of selenitewith glutathione produces selenodiglutathione (GS-Se-GS). Selenodiglutathioneand itssubsequent reduction to glutathioselenol (GS-SeH) are likely the key intermediatesin the possiblemetabolic fates of selenium. This review presents the possible pathwaysinvolving selenium in E. coli. Identification of intermediates and potentialprocesses from uptake of the toxic oxyanions through to theirdetoxification will assist us inunderstanding the complexities of metalloid oxyanion metabolism in thesebacteria.     

Interaction of glutathione and sodium selenite in vitro investigated by electrospray ionization tandem mass spectrometry

Selenite has been found to be an active catalyst for the oxidation of sulphhydryl compounds, such as glutathione (GSH). Considering the biological importance of GSH oxidation and the implication of sulphhydryl compounds in selenium poisoning and other biological activities, more information on selenite oxidation of GSH in enzyme-free conditions is desirable. Herein, we describe glutathione and sodium selenite simply mixed in aqueous solutions. The interaction products and transient intermediate are identified and characterized using electrospray ionization (ESI) tandem mass spectrometry. In the first step, GSH directly reacts to form diglutathione (GSSG) and unstable selenodiglutathione (GS-Se-SG). Then selenodiglutathione further reacted with remaining GSH to form diglutathione and elemental selenium, Se(0). As the amount of GSSG significantly increased or acidity of the solution increased, the redox potential of glutathione [E(0')(GSSG/2GSH) approximately -250 mV (NHE)] significantly shifted to the positive direction. This makes the GSSG react with elemental selenium formed in the solution, which can be demonstrated by another unstable intermediate ion identified at m/z 418 by mass spectrometry with the elemental composition of [GSS-Se](-). The reaction mechanism between GSH and sodium selenite has been proposed according to the ESI-MS, NMR and UV-vis spectrometric measurements.     

还原亚硒酸盐产生红色单质硒光合细菌菌株的筛选与鉴定

从实验室保藏的光合细菌中筛选出一株对亚硒酸钠还原效率较高的菌株S3,其亚硒酸钠还原产物通过透射电子显微镜及EDX(Electron-Dispersive X-ray)分析确定为红色单质硒。菌株S3的形态学特征、生理生化特征及光合色素扫描结果与固氮红细菌(Rhodobacter azotoformans)的特征基本一致;16S rDNA序列(GenBank登录号为DQ402051)在系统发育树中与固氮红细菌同属一个类群,序列同源性为99%。根据上述结果将菌株S3鉴定为固氮红细菌。初步研究了该菌株还原亚硒酸钠的特性,首次报道固氮红细菌具有还原亚硒酸盐产生红色单质硒的能力,为今后利用微生物方法治理环境中硒污染、利用微生物方法获得活性红色单质硒以及对微生物还原亚硒酸盐产生红色单质硒的机理研究奠定了良好的基础。     

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.     

Glutathione contributes to the efflux of selenium from hepatoma cells

Selenite is a selenium source for selenoprotein biosynthesis in mammalian cells. Although previous studies have suggested the involvement of glutathione (GSH) and/or thioredoxin reductase in selenite metabolism, intracellular selenite metabolism remains largely unknown. Here, we report that GSH depletion did not affect the amount of selenoprotein in Hepa 1–6 cells, suggesting that GSH does not play a central role in the reduction of selenite in selenoprotein biosynthesis. On the other hand, we found that GSH is involved in the efflux of low-molecular-weight selenium compounds from cells, presumably via the formation of selenodiglutathione. Moreover, selenite inhibited the efflux of a fluorescent bimane-GS conjugate that is mediated by ATP-dependent multidrug-resistant proteins, implying the existence of an active transporter for selenodiglutathione. This is the first report demonstrating that GSH plays a role in selenium excretion from cells by forming a GSH-conjugate, which may contribute to the distribution, detoxification, and homeostasis of selenium in the body.     

Products of the reaction of selenite with intracellular sulfhydryl compounds

The usual first step in the intracellular metabolism of exogenous selenite is its chemical reaction with glutathione to form selenodiglutathione (1). We have investigated whether selenite also reacts intracellularly with other SH compounds. HeLa cells were exposed to [75Se]selenite and lysed with SDS. Cellular proteins and nucleic acids were precipitated with trichloroacetic acid, and the acid-soluble fraction was analyzed by ion-exchange thin-layer chromatography (ion-exchange TLC) and autoradiography. In control cells, the major [75Se]-containing species detected can be identified by its mobility as selenodiglutathione. Two other species were detected, which can be identified as selenodimercaptoethylamine and the mixed selenotrisulfide of mercaptoethylamine and glutathione. In contrast, in cells that were depleted of glutathione (by treatment with buthionine sulfoximine), very little, if any, selenodiglutathione was detected. However, new [75Se]-containing species were detected, which can be identified as selenodicysteine and the mixed selenotrisulfide of cysteine and glutathione. The same species were detected when [75Se]selenite was added to the acid-soluble fraction of a cell extract (as opposed to living cells), confirming that these compounds can be formed by nonenzymatic reactions.     

Reduction of selenite and detoxification of elemental selenium by the phototrophic bacterium Rhodospirillum rubrum.

The effect of selenite on growth kinetics, the ability of cultures to reduce selenite, and the mechanism of detoxification of selenium were investigated by using Rhodospirillum rubrum. Anoxic photosynthetic cultures were able to completely reduce as much as 1. 5 mM selenite, whereas in aerobic cultures a 0.5 mM selenite concentration was only reduced to about 0.375 mM. The presence of selenite in the culture medium strongly affected cell division. In the presence of a selenite concentration of 1.5 mM cultures reached final cell densities that were only about 15% of the control final cell density. The cell density remained nearly constant during the stationary phase for all of the selenite concentrations tested, showing that the cells were not severely damaged by the presence of selenite or elemental selenium. Particles containing elemental selenium were observed in the cytoplasm, which led to an increase in the buoyant density of the cells. Interestingly, the change in the buoyant density was reversed after selenite reduction was complete; the buoyant density of the cells returned to the buoyant density of the control cells. This demonstrated that R. rubrum expels elemental selenium across the plasma membrane and the cell wall. Accordingly, electron-dense particles were more numerous in the cells during the reduction phase than after the reduction phase.     

细菌还原氧化态硒产生红色单质硒的研究进展

硒是一种生命必需的微量元素,但高浓度时毒性较强且会造成环境污染。许多细菌可以将亚硒酸盐(SeO32-)或硒酸盐(SeO42-)等毒性较高的氧化态硒还原为毒性较小的红色单质硒(Se°),形成硒-蛋白复合物,它们对于获得最佳补硒方式和治理硒环境污染具有应用潜力。近年来,关于这一生物还原过程,人们进行了大量的研究,包括碳源、氧气、元素硫、谷胱甘肽以及一些氧化还原酶和膜转运蛋白等在内的多种物质都被发现可能影响或参与了细菌对硒的代谢。综述了细菌进行生物还原氧化态硒的影响因素及不同细菌产生红色单质硒机理的研究进展。     

Uptake of selenotrisulfides of glutathione and cysteine by brush border membranes from rat intestines.

The uptake of selenodiglutathione and selenodicysteine was compared to that of selenite by brush border membrane vesicles (BBMV) prepared from rat intestinal tracts. It was found that it is critical to maintain a pH of 6.0 or below to prevent the spontaneous breakdown of these compounds. When conducted at pH 6.0, the uptake of selenodiglutathione and selenodicysteine was more than ten times faster than for selenite selenium. Ligated intestinal loop studies were conducted to determine if similar results would be obtained in vivo. In comparison to selenite, selenium absorption was enhanced 68% and the transfer to the body increased 2.4-fold when selenium as selenodiglutathione was placed in the ileum. The absorption of selenium as selenodicysteine was increased by 57% and the transfer doubled in comparison to selenite when placed in the ileum. Thus, the stimulated absorption of selenite by glutathione or cysteine appears to be through the formation of complexes with these compounds.     

Oxidation of glutathione and superoxide generation by inorganic and organic selenium compounds

The carcinostatic activities of selenium (Se) compounds have been shown to be composition and concentration dependent. Several studies have indicated that the ratios between glutathione (GSH) and Se may play an important role in Se catalysis and toxicity. The present study examined the catalytic effect of three selenium compounds on GSH oxidation using lucigenin-dependent chemiluminescence (CL) as an indirect measure of superoxide generation. Various GSH:Se ratios were assayed for the glutathione oxidase activity of selenite, selenocystamine and diselenodipropionic acid. CL emitted from the reaction of selenite with GSH increased more rapidly and was greater than those from the diselenides, but the diselenide CL reactions were sustainable. Both selenite- and diselenide-induced CL were markedly suppressed by superoxide dismutase (SOD). Iodoacetic acid (IAc) effectively inhibited CL generated from selenite-, selenocystamine- and diselenodipropionic acid-catalyzed GSH oxidation. These results suggest that GSH oxidation catalyzed by selenite, and the diselenides selenocystamine and diselenodipropionic acid, generated the superoxide radical in which the CL was inhibited by SOD. Furthermore, CL inhibition by IAc suggests that the catalytic species producing superoxide were the GSSe(-) or RSe(-) anion. This redox chemistry may be responsible for selenite and organoselenium toxicity and apoptosis, making possible the design and synthesis of organoselenium-containing pharmaceuticals.     

Induction of apoptosis by selenite and selenodiglutathione in HL-60 cells: correlation with cytotoxicity.

Effects of selenite and selenodiglutathione, an initial metabolite of selenite, on the induction of apoptosis and cytotoxicity were investigated in human promyelocytic leukemia HL-60 cells. Treatment of selenite or selenodiglutathione resulted in concentration-dependent cytotoxicity, measured by lactate dehydrogenase leakage assay, and by tetrazolium salt reduction assay. Selenodiglutathione has been shown to exert more cytotoxic effect than selenite in both assay systems. Time-course study of cellular selenium uptake suggests that the higher cytotoxicity of selenodiglutathione be largely due to faster and greater selenium uptake rate. Treatment with selenite or selenodiglutathione also induced apoptosis in a dose-dependent manner, as detected by enzyme-linked immunosorbent assay and by DNA fragmentation assay. The dose-response data of apoptosis induced by selenite or selenodiglutathione were similar to those of cytotoxicity, implicating a relationship between the induction of apoptosis and cytotoxicity. Zn, which is a well-known inhibitor of apoptosis, dose-dependently blocked not only the induction of apoptosis, but also the membrane damage induced by selenium, corroborating this hypothesis. It was noted that the inhibition of apoptosis by Zn exerted little protective effect on cytotoxicity at higher concentrations of selenium, compared with a perfect protective effect at low concentration of selenium. These results suggest that cytotoxicity induced by selenium may be partially correlated with apoptosis.     

Comparison of short-term toxicity between Nano-Se and selenite in mice

We previously reported that, as compared with selenite, nano red elemental selenium (Nano-Se) had lower acute toxicity in mice and similar bioavailability in terms of up-regulating seleno-enzymes. The short-term toxicity of both selenite and Nano-Se in mice was further compared in this study. At an oral dose of 6 mg/kg bw per day administered for consecutive 12 days, selenite and Nano-Se completely and partially suppressed mice growth respectively. Abnormal liver function was more pronounced with selenite treatment than Nano-Se as indicated by the increase of both alanine aminotransferase and aspartate aminotransferase in serum. Selenite inhibited liver catalase and superoxide dismutase activities, whereas, Nano-Se did not affect these two antioxidant enzymes. Selenite increased the malondialdehyde content of liver, but Nano-Se decreased it. Both Se forms had similar effects on depletion of reduced glutathione and up-regulated glutathione peroxidase. Nano-Se was more potent than selenite in the induction of glutathione S-transferase. At oral doses of 2 or 4 mg/kg bw per day for consecutive 15 days, selenite was more active than Nano-Se in supressing growth, deleting reduced glutathione, and inhibiting superoxide dismutase activities. Taken together, these results indicate that over a short-term, a high-dose of selenite caused more pronounced oxidative stress, greater liver injury, and prominent retardation of growth as compared to Nano-Se.     

Biological effects of a nano red elemental selenium.

A novel selenium form, nano red elemental selenium (Nano-Se) was prepared by adding bovine serum albumin to the redox system of selenite and glutathione. Nano-Se has a 7-fold lower acute toxicity than sodium selenite in mice (LD(50) 113 and 15 mg Se/kg body weight respectively). In Se-deficient rat, both Nano-Se and selenite can increase tissue selenium and GPx activity. The biological activities of Nano-Se and selenite were compared in terms of cell proliferation, enzyme induction and protection against free racial-mediated damage in human hepatoma HepG2 cells. Nano-Se and selenite are similarly cell growth inhibited and stimulated synthesis of glutathione peroxidase (GPx), phospholipid hydroperoxide glutathione peroxidase (PHGPx) and thioredoxin reductase (TR). When HepG2 cells were co-treated with selenium and glutathione, Nano-Se showed less pro-oxidative effects than selenite, as measured by cell growth. These results demonstrate that Nano-Se has a similar bioavailability in the rat and antioxidant effects on cells.     

Tissue distribution of S-adenosylmethionine and S-adenosylhomocysteine in the rat. Effect of age, sex and methionine administration on the metabolism of S-adenosylmethionine, S-adenosylhomocysteine and polyamines

In the presence of ATP and Mg2+, ATP sulphurylase from Saccharomyces cerevisiae catalysed the conversion of selenate into a compound with the electrophoretic and acid-lability properties of adenosine 5'-sulphatophosphate. Structural characterization, involving extensive purification of adenosine 5'-selenophosphate, proved impossible. However, we showed ATP-, Mg2+- and ATP sulphurylase-dependent, and inorganic pyrophosphatase-stimulated, production of elemental selenium from selenate in the presence of GSH (reduced glutathione). Since selenate was not reduced by GSH, this reaction proved that ATP sulphurylase had formed an active selenate. The enzyme catalysed formation of elemental selenium had the same kinetics and GSH-dependency as the non-enzymic reduction of selenite to elemental selenium by GSH. In the presence of inorganic pyrophosphatase, 2 mol of Pi was released for each mol of 'active selenate' formed. This was shown by a spectrophotometric assay for elemental selenium. The observed reactivity with thiols and the instability of the enzymic product were those predicted for selenium anhydrides. By analogy with the chemistry of sulphur, the product of the thiolytic cleavage of a selenium anhydride would be converted into selenite. The selenite would then be reduced by the thiol to elemental selenium. We conclude that ATP sulphurylase can catalyse the formation of adenosine 5'-selenophosphate. The anhydride can be reduced by thiols in a manner similar to the reduction of selenite. These results probably explain the ability of mammals, lacking a sulphate reductase system, to incorporate selenium from selenate into seleno-amino acids.     

Chemopreventive activity of selenocysteine prodrugs against tobacco-derived nitrosamine (NNK) induced lung tumors in the A/J mouse

Prodrugs of L-selenocysteine have potential utility in cancer chemoprevention. This study reports the efficacy of three selenazolidine-4(R)-carboxylic acids, (2-unsubstituted, 2-oxo, and 2-methyl derivatives; SCA, OSCA, and MSCA, respectively) against tobacco-related lung tumorigenesis in a mouse model. Seven days after initiation of an AIN-76A diet supplemented with sodium selenite (5 ppm Se), L-selenomethionine (3.75 ppm Se), Se-methyl-L-selenocysteine (3 ppm Se), L-selenocystine (15 ppm Se), SCA (15 ppm Se), OSCA (15 ppm Se), or MSCA (15 ppm Se), mice received 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK; 10 micromol, i.p.). After an additional 16 weeks on the diets, two compounds, OSCA and selenocystine, significantly reduced lung adenoma multiplicity from 7.2 tumors per mouse in the NNK group to 4.5 and 4.6 tumors per mouse, respectively. Neither selenium concentration nor glutathione peroxidase activity in either RBCs or liver served as surrogate indicators of tumor reduction. Hepatic selenium levels were significantly elevated by all selenium-containing compounds except Se-methyl-L-selenocysteine and SCA; RBC selenium levels by all except sodium selenite and MSCA. With the exception of L-selenomethionine, RBC glutathione peroxidase activity was increased along with the elevated selenium levels. Hepatic glutathione peroxidase activity was elevated by all Se-compounds except SCA. The two compounds showing significant tumor reduction (OSCA and selenocystine) were the only two compounds that showed ubiquity of changes, elevating both selenium levels and GPx activity in both liver and RBC.     

Mode of in vitro interaction of mercuric mercury with selenite to form high-molecular weight substance in rabbit blood

Mode of interaction of mercuric mercury and selenite in rabbit blood was investigated in vitro. After the incubation of rabbit blood with 10^?5 M each of ²⁰³HgCl₂ and Na₂⁷⁵SeO₃, the amounts of both ²⁰³Hg and ⁷⁵Se incorporated into erythrocytes were markedly larger than the case where the blood was treated separately with one of these compounds. Most of ²⁰³Hg and ⁷⁵Se distributed into plasma and erythrocytes were found in high-molecular weight substance(s) (HMWS) fractionated by gel filtration at a molar ratio of 1:1. The ²⁰³Hg and ⁷⁵Se in HMWS found in plasma and erythrocytes were hardly diffusable through the erythrocytes membrane. The formation of the HMWS containing mercury and selenium was observed in stroma-free hemolysate incubated with mercuric chloride and selenite, but not in plasma. Addition of reduced glutathione (GSH) to the plasma, however, gave the HMWS as reaction products containing equimolar amounts of mercury and selenium. Further the binding properties of selenium to proteins were studied in the plasma incubated with selenodiglutathione (GSSeSG) or with selenite in the presence of GSH. The results indicated that GSH, a cellular component, is essential for the formation of an active selenium compound from selenite and that the interaction of mercuric mercury and selenite in plasma in the presence of GSH may occur through the other mechanism than the formation of GSSeSG.     

螺旋藻转化纳米元素硒的制备及其体外清除自由基活性的初步研究

研究利用高密度富硒螺旋藻(Se-SP)细胞通过生物转化制备纳米元素硒(Nano-Se)的可行性,观察Nano-Se在体外对氧自由基的清除作用。用梯度离心分选Nano-Se,原子力显微镜(AFM)、透射电镜(TEM)及X-射线能谱(EDX)联用表征纳米粒中的元素硒形态,电感耦合等离子质谱仪(ICP-MS)测定Nano-Se中的硒含量,化学发光方法检测Nano-Se在体外对超氧自由基和羟自由基的清除作用。结果发现,Nano-Se主要由元素硒构成,形态呈球形,73%的纳米粒子直径大小分布在(61±17)nm范围。Nano-Se在体外对两种氧自由基的最大清除率分别为:30.1%和27.6%,相应的EC50分别为:0.8 μg/ml和2.2 μg/ml。相同剂量时,Nano-Se对氧自由基的清除作用比硒代蛋氨酸(Se-Met)及Se-SP中其它含硒活性成分更强。结果提示,利用高密度Se-SP可诱导Nano-Se的大量生成,Se-SP转化的Nano-Se可能是一种新的抗氧化硒形态,其作用机制和体内生物活性有待深入研究。     

Selenium induces a multi‐targeted cell death process in addition to ROS formation

Selenium compounds inhibit neoplastic growth. Redox active selenium compounds are evolving as promising chemotherapeutic agents through tumour selectivity and multi‐target response, which are of great benefit in preventing development of drug resistance. Generation of reactive oxygen species is implicated in selenium‐mediated cytotoxic effects on cancer cells. Recent findings indicate that activation of diverse intracellular signalling leading to cell death depends on the chemical form of selenium applied and/or cell line investigated. In the present study, we aimed at deciphering different modes of cell death in a single cell line (HeLa) upon treatment with three redox active selenium compounds (selenite, selenodiglutathione and seleno‐DL‐cystine). Both selenite and selenodiglutathione exhibited equipotent toxicity (IC₅₀ 5 μM) in these cells with striking differences in toxicity mechanisms. Morphological and molecular alterations provided evidence of necroptosis‐like cell death in selenite treatment, whereas selenodiglutathione induced apoptosis‐like cell death. We demonstrate that selenodiglutathione efficiently glutathionylated free protein thiols, which might explain the early differences in cytotoxic effects induced by selenite and selenodiglutathione. In contrast, seleno‐DL‐cystine treatment at an IC₅₀ concentration of 100 μM induced morphologically two distinct different types of cell death, one with apoptosis‐like phenotype, while the other was reminiscent of paraptosis‐like cell death, characterized by induction of unfolded protein response, ER‐stress and occurrence of large cytoplasmic vacuoles. Collectively, the current results underline the diverse cytotoxic effects and variable potential of redox active selenium compounds on the survival of HeLa cells and thereby substantiate the potential of chemical species‐specific usage of selenium in the treatment of cancers.     

Subcellular distribution of selenium-containing proteins in the rat

The subcellular distribution of selenium in rat tissues was studied by measuring 75Se in animals provided for 5 months with [75Se]selenite as the main dietary source of selenium. Equilibration of the animals to a constant specific activity allowed the measurement of 75Se to be used as a specific elemental assay for selenium. Of the whole-body selenium, 51% was in the soluble fractions and 48% was bound to the particulate fractions as follows: 21% in plasma membranes, 11% in microsomes, and 16% in mitochondria. Glutathione peroxidase was primarily a soluble enzyme, but part of the activity was associated with plasma membrane in liver, mitochondria in liver and kidney, and microsomes in testes. Selenium in glutathione peroxidase accounted for about one-third of the particulate-associated selenium. These results indicate that other selenium-containing proteins besides glutathione peroxidase are present in membranes.     

Abundance and tissue distribution of selenocysteine-containing proteins in the rat

The form and distribution of selenium (Se) in proteins from selected tissues of the rat were studied by measuring 75Se radioactivity in animals provided for 5 months with [75Se]selenite as the main dietary source of Se. Equilibration of the animals to a constant specific activity of 75Se allowed the measurement of 75Se to be used as a specific elemental assay for Se. Skeletal muscle, liver and blood accounted for 73% of the whole-body Se and 95% of the total Se-dependent glutathione peroxidase activity. Over 80% of the whole-body Se was in protein in the form of the selenoamino acid, selenocysteine. All other forms of Se that were measured accounted for less than 3% of the whole-body Se. The Se in protein was distributed in seven subunit sizes and nine chromatographic forms. The Se in glutathione peroxidase accounted for one-third of the whole-body Se. These results show that the main use of dietary Se, as selenite, in rats is for the synthesis of selenocysteine-containing proteins. Furthermore, the presence of two-thirds of the whole-body Se in nonglutathione peroxidase, selenocysteine-containing proteins suggests that there may be other important mammalian selenoenzymes besides glutathione peroxidase.