Differential molecular mechanisms of substrate recognition by selenium methyltransferases, INMT and TPMT, in selenium detoxification and excretion.

It is known that the recommended dietary allowance of selenium (Se) is dangerously close to its tolerable upper intake level. Se is detoxified and excreted in urine as trimethylselenonium ion (TMSe) when the amount ingested exceeds the nutritional level. Recently, we demonstrated that the production of TMSe requires two methyltransferases: thiopurine S-methyltransferase (TPMT) and indolethylamine N-methyltransferase (INMT). In this study, we investigated the substrate recognition mechanisms of INMT and TPMT in the Se-methylation reaction. Examination of the Se-methyltransferase activities of two paralogs of INMT, namely, nicotinamide N-methyltransferase and phenylethanolamine N-methyltransferase, revealed that only INMT exhibited Se-methyltransferase activity. Consistently, molecular dynamics simulations demonstrated that dimethylselenide was preferentially associated with the active center of INMT. Using the fragment molecular orbital method, we identified hydrophobic residues involved in the binding of dimethylselenide to the active center of INMT. The INMT-L164R mutation resulted in a deficiency in Se- and N-methyltransferase activities. Similarly, TPMT-R152, which occupies the same position as INMT-L164, played a crucial role in the Se-methyltransferase activity of TPMT. Our findings suggest that TPMT recognizes negatively charged substrates, whereas INMT recognizes electrically neutral substrates in the hydrophobic active center embedded within the protein. These observations explain the sequential requirement of the two methyltransferases in producing TMSe.

Comparison of Nutritional Availability of Biogenic Selenium Nanoparticles and Chemically Synthesized Selenium Nanoparticles.

Selenium (Se) is an essential micronutrient, and animals biosynthesize selenoproteins from various selenocompounds such as inorganic salts and organic selenocompounds as a Se source. In addition to the inorganic and organic forms of Se, it is also known that elemental Se is biologically synthesized at the nanoscale in nature. Biologically synthesized Se nanoparticles (Se-NPs), i.e., biogenic Se-NPs (Se-BgNPs), have not been fully investigated as a Se source compared with the other forms of Se. In this study, we evaluated the nutritional availability of Se-BgNPs biosynthesized in E. coli and revealed that Se-BgNPs were less assimilated into selenoproteins in rats as a Se source than inorganic Se salt or chemically synthesized Se-NPs. Se-BgNPs showed tolerance toward digestion and low absorbability in gut, which resulted in the low nutritional availability. Se-BgNPs seem to be coated with a biomaterial that functions to reduce their toxicity toward E. coli and at the same time lowers their availability to animals.

Band 3/anion exchanger 1/solute carrier family 4 member 1 expression as determinant of cellular sensitivity to selenite exposure.

Selenium is a chalcogen element that is essential in animals, but is highly toxic when ingested above the nutritional requirement. Selenite is used as a supplement in patients receiving total parenteral nutrition. However, the therapeutic and toxic doses of selenite are separated by a narrow range. This ambivalent character of selenite implies the presence of cellular mechanisms that precisely control selenite homeostasis. Here, we investigated mechanisms that determine cellular susceptibility to selenite exposure. The resistance to selenite exposure was significantly different among cell lines. We determined the expression levels of TPMT (thiopurine S-methyltransferase) and SLC4A1 (solute carrier family 4 member 1), which encode selenium methyltransferase and selenite transporter, respectively. We also examined the effect of inhibition of Band 3 protein activity, which is encoded by SLC4A1, on the cellular sensitivity to selenite. The data suggest that the expression level of SLC4A1 is the determinant of cellular sensitivity to selenite.

Formation Mechanism and Toxicological Significance of Biogenic Mercury Selenide Nanoparticles in Human Hepatoma HepG2 Cells.

It is widely recognized that the toxicity of mercury (Hg) is attenuated by the simultaneous administration of selenium (Se) compounds in various organisms. In this study, we revealed the mechanisms underlying the antagonistic effect of sodium selenite (Na2SeO3) on inorganic Hg (Hg2+) toxicity in human hepatoma HepG2 cells. Observations by transmission electron microscopy indicated that HgSe (tiemannite) granules of up to 100 nm in diameter were accumulated in lysosomal-like structures in the cells. The HgSe granules were composed of a number of HgSe nanoparticles, each measuring less than 10 nm in diameter. No accumulation of HgSe nanoparticles in lysosomes was observed in the cells exposed to chemically synthesized HgSe nanoparticles. This suggests that intracellular HgSe nanoparticles were biologically generated from Na2SeO3 and Hg2+ ions transported into the cells and were not derived from HgSe nanoparticles formed in the extracellular fluid. Approximately 85% of biogenic HgSe remained in the cells at 72 h post culturing, indicating that biogenic HgSe was hardly excreted from the cells. Moreover, the cytotoxicity of Hg2+ was ameliorated by the simultaneous exposure to Na2SeO3 even before the formation of insoluble HgSe nanoparticles. Our data confirmed for the first time that HepG2 cells can circumvent the toxicity of Hg2+ through the direct interaction of Hg2+ with a reduced form of Se (selenide) to form HgSe nanoparticles via a Hg-Se soluble complex in the cells. Biogenic HgSe nanoparticles are considered the ultimate metabolite in the Hg detoxification process.

Hyperthermia Selectively Destabilizes Oncogenic Fusion Proteins.

The PML/RARα fusion protein is the oncogenic driver in acute promyelocytic leukemia (APL). Although most APL cases are cured by PML/RARα-targeting therapy, relapse and resistance can occur due to drug-resistant mutations. Here we report that thermal stress destabilizes the PML/RARα protein, including clinically identified drug-resistant mutants. AML1/ETO and TEL/AML1 oncofusions show similar heat shock susceptibility. Mechanistically, mild hyperthermia stimulates aggregation of PML/RARα in complex with nuclear receptor corepressors leading to ubiquitin-mediated degradation via the SIAH2 E3 ligase. Hyperthermia and arsenic therapy destabilize PML/RARα via distinct mechanisms and are synergistic in primary patient samples and in vivo, including three refractory APL cases. Collectively, our results suggest that by taking advantage of a biophysical vulnerability of PML/RARα, thermal therapy may improve prognosis in drug-resistant or otherwise refractory APL. These findings serve as a paradigm for therapeutic targeting of fusion oncoprotein-associated cancers by hyperthermia. SIGNIFICANCE: Hyperthermia destabilizes oncofusion proteins including PML/RARα and acts synergistically with standard arsenic therapy in relapsed and refractory APL. The results open up the possibility that heat shock sensitivity may be an easily targetable vulnerability of oncofusion-driven cancers.See related commentary by Wu et al., p. 300.

Distributions of mercury and selenium in rats ingesting mercury selenide nanoparticles.

Mercury (Hg) is one of the most toxic environmental pollutants, and is biocondensed via the food chain. Selenium (Se) is an essential element that possesses an antagonistic property towards Hg in vivo. The antagonistic property is explained by the assumption that Hg and Se directly interact to form HgSe nanoparticles (HgSe NPs) in organs. It is presumed that the toxic effects of HgSe NPs are lower than that of ionic Hg; however, no precise evaluation has been conducted so far. In the present study, we evaluated the distribution of HgSe NPs ingested in Se-deficient rats. The recovery of serum selenoproteins from a deficient level was not observed in rats orally administered HgSe NPs. In addition, the excretion of Hg and Se via urine was not observed. Interestingly, the biosynthesis of selenoproteins and urinary selenometabolites would have required the production of selenide through the degradation of HgSe NPs. Therefore, it seems that selenide and Hg are not released from HgSe NPs in vivo. The administration of HgSe NPs did not increase Hg and Se concentrations in organs, and almost all HgSe NPs were recovered in feces, indicating no or low bioaccessibility of HgSe NPs even in Se-deficient rats. These results suggest that HgSe NPs are biologically inert and do not become a secondary environmental pollutant of Hg.

Systematic study of the selenium fractionation in human plasma from a cancer prevention trial using HPLC hyphenated to ICP-MS and ESI-MS/MS.

This work represents the first systematic speciation study of selenium (Se) in plasma from subjects participating in a pilot study for a cancer prevention trial (PRECISE). This involved supplementation of elderly British and Danish individuals with selenised yeast for 6 months and 5 years, respectively, at 100, 200, and 300 µg Se/day or placebo. Speciation data was obtained for male plasma using HPLC-ICP-MS and HPLC-ESI-MS/MS. With the proposed strategy, approximately 1.5 mL of plasma was needed to determine total Se concentration and the fractionation of Se in high molecular weight (HMW) and low molecular weight (LMW) pools, and for quantification and identification of small Se species. For the first time, Se-methyl-selenocysteine (MSC) and methyl-2-acetamido-2deoxy1-seleno-ß-D-galactopyranoside (Selenosugar-1) were structurally confirmed in plasma after supplementation with selenised yeast within the studied range. Determination of selenomethionine (SeMet) incorporated non-specifically into albumin (SeALB) was achieved by HPLC-ICP-MS after hydrolysis. By subtracting this SeMet concentration from the total Se in the HMW pool, the concentration of Se incorporated into selenoproteins was calculated. Results from the speciation analysis of the free Se metabolite fraction (5% of total plasma Se) suggest a significant increase in the percentage of Se (as SeMet plus Selenosugar-1) of up to 80% of the total Se in the LMW fraction after 6 months of supplementation. The Se distribution in the HMW fraction reflects a significant increase in SeALB with Se depletion from selenoproteins, which occurs most significantly at doses of over 100 µg Se/day after 5 years. The results of this work will inform future trial design. Graphical abstract.

Production of a Urinary Selenium Metabolite, Trimethylselenonium, by Thiopurine S-Methyltransferase and Indolethylamine N-Methyltransferase.

Selenium (Se) is an essential trace element in animals; however, the element can become highly toxic in excess amounts beyond the nutritional level. Although Se is mainly excreted into urine as a selenosugar within the nutritional level, excess amounts of Se are transformed as an alternative urinary metabolite, trimethylselenonium ion (TMSe). Se methylation appears to be an important metabolic process for the detoxification of excess Se; however, the biochemical mechanisms underlying the Se methylation have not been elucidated. In this study, we evaluated biochemical characteristics of two human methyltransferases for Se methylation, thiopurine S-methyltransferase (TPMT) and indolethylamine N-methyltransferase (INMT). The first methylation of Se, i.e., a nonmethylated to a monomethylated form, was specifically driven by TPMT, and INMT specifically mediated the third methylation, i.e., dimethylated to trimethylated form. The second methylation, i.e., a monomethylated to dimethylated form, was driven by either TPMT or INMT. Exogenous expression of TPMT, but not INMT, ameliorated the cytotoxicity of inorganic nonmethylated selenium salt, suggesting that only TPMT gave the cellular resistance against selenite exposure. TPMT was ubiquitously expressed in most mouse tissues and preferably expressed in the liver and kidneys, while INMT was specifically expressed in the lung and supplementally expressed in the liver and kidneys. Our results revealed that both TPMT and INMT cooperatively contributed to the TMSe production, enabling urinary excretion of Se and maintenance of homeostasis of this essential yet highly toxic trace element. Thus, TPMT and INMT can be recognized as selenium methyltransferases as a synonym.

Elucidation of tellurium biogenic nanoparticles in garlic, Allium sativum, by inductively coupled plasma-mass spectrometry.

BACKGROUND: Biosynthesis of Te nanoparticles may occur in higher plants exposed to Te, as reported on microorganisms. However, unambiguous observations of the biogenic nanoparticles (BgNPs) of Te in plants are lacking. Hence, in this study, we investigated the formation of insoluble BgNPs of Te in garlic (Allium sativum) as a model plant. METHOD: We performed elemental analysis based on inductively coupled plasma-mass spectrometry (ICP-MS) technique, and obtained Te concentration and distribution in various parts of garlic. In addition, insoluble Te particles were detected by fast time-resolved ICP-MS. Direct observation of the insoluble Te particle was also conducted by scanning electron microscope (SEM) and transmission electron microscope (TEM). RESULTS: A part of the roots and clove from Te-exposed garlic showed black coloration. Te concentrations in the black-colored parts were significantly increased compared with the non-colored parts. Transient signals of Te unique to nanoparticles were detected from the insoluble fractions of the black-colored parts. Finally, rod-shaped biogenic Te nanoparticles consisting of highly crystalline elemental Te was observed by SEM and TEM. CONCLUSION: Our data provide new insights to the metabolic pathway of Te in higher plants for the formation of insoluble biogenic nanoparticles, which is extremely important for the detoxification of Te.

Quantitative Elemental Analysis of a Single Cell by Using Inductively Coupled Plasma-Mass Spectrometry in Fast Time-Resolved Analysis Mode.

The elemental composition of a single yeast, green alga, or red blood cell (RBC) was precisely determined by using inductively coupled plasma-mass spectrometry (ICP-MS) operating in fast time-resolved analysis (TRA) mode. The technique is known as single-cell (SC)-ICP-MS. Phosphorus, sulfur, magnesium, zinc, and iron were detected in the three types of cell. The elemental composition of yeast and green alga obtained by SC-ICP-MS was consistent with results obtained from conventional ICP-MS measurements following acid digestion of the cells. Slight differences were found in the measured values between SC-ICP-MS and the conventional ICP-MS results for RBC. However, the SC-ICP-MS results for S and Fe in RBC were closer to the estimated values for these elements that were calculated from the level of hemoglobin in RBCs. The data suggest that SC-ICP-MS is suitable for the analysis of various cell types, namely, fungus, plant, and animal cells.

Effect of gut microflora on nutritional availability of selenium.

Since selenium (Se) is an essential mineral, animals must be able to metabolize the various selenocompounds in meat, fish and vegetables. It is unclear how animals, including humans, utilize selenocompound efficiently, but we hypothesized that gut microflora might contribute to these processes. In this study, we revealed that Se-methylselenocysteine and selenocyanate were metabolized to selenomethionine (SeMet) by intestinal microflora, suggesting selenocompounds might be metabolized to SeMet, which can be used by the host organism. The major urinary selenosugar, 1ß-methylseleno-N-acetyl-d-galactosamine, was utilized less in microflora-suppressed than healthy rats, suggesting that this sugar can be transformed to a nutritionally available form by gut microflora in animals with a healthy microbiota. We concluded that, in rats at least, gut microflora has a role in the metabolism of Se in the host animal, and this finding might be worth investigating in humans.

Speciation of Selenium in Brown Rice Fertilized with Selenite and Effects of Selenium Fertilization on Rice Proteins.

Foliar Selenium (Se) fertilizer has been widely used to accumulate Se in rice to a level that meets the adequate intake level. The Se content in brown rice (Oryza sativa L.) was increased in a dose-dependent manner by the foliar application of sodium selenite as a fertilizer at concentrations of 25, 50, 75, and 100 g Se/ha. Selenite was mainly transformed to organic Se, that is, selenomethionine in rice. Beyond the metabolic capacity of Se in rice, inorganic Se also appeared. In addition, four extractable protein fractions in brown rice were analyzed for Se concentration. The Se concentrations in the glutelin and albumin fractions saturated with increasing Se concentration in the fertilizer compared with those in the globulin and prolamin fractions. The structural analyses by fluorescence spectroscopy, Fourier transform infrared spectrometry, and differential scanning calorimetry suggest that the secondary structure and thermostability of glutelin were altered by the Se treatments. These alterations could be due to the replacements of cysteine and methionine to selenocysteine and selenomethionine, respectively. These findings indicate that foliar fertilization of Se was effective in not only transforming inorganic Se to low-molecular-weight selenometabolites such as selenoamino acids, but also incorporating Se into general rice proteins, such as albumin, globulin glutelin, and prolamin, as selenocysteine and selenomethionine in place of cysteine and methionine, respectively.

Biotransformation of organic selenium compounds in budding yeast, Saccharomyces cerevisiae.

Selenium (Se) is not essential for yeast growth, but it has a metabolic capacity to transform inorganic Se species to organic Se compounds such as selenomethionine (SeMet). Although the metabolism of inorganic Se species has been well discussed, there are no studies revealing how organic Se compounds are metabolized in yeast. The aim of this study was to show the specific metabolic pathway of organic Se species in yeast. We performed the speciation analysis of selenometabolites in budding yeast, Saccharomyces cerevisiae, exposed to selenometabolites produced by animals, plants, and microorganisms, such as methyl-2-acetamido-2-deoxy-1-seleno-ß-d-galactopyranoside (SeSug1, selenosugar 1), methyl-2-acetamido-2-deoxy-1-seleno-ß-d-glucopyranoside (SeSug2, selenosugar 2), trimethylselenonium ions (TMSe), Se-methylselenocysteine (MeSeCys), and SeMet. Four selenometabolites, SeSug1, SeSug2, SeMet, and MeSeCys, were commonly metabolized into SeMet in yeast. Yeast was able to incorporate TMSe but could not metabolize it. Since MeSeCys and selenosugars are the major selenometabolites in plants and animals, respectively, yeast is useful for recovering Se as SeMet from the selenometabolites produced by other organisms in the ecosystem.

Preparation of envelope-type lipid nanoparticles containing gold nanorods for photothermal cancer therapy.

The use of gold nanorods (AuNRs) that produce heat in response to near infrared (NIR) irradiation is an attractive approach to cancer photothermal therapy. AuNRs are usually prepared by using a highly toxic detergent: cetyltrimethylammonium bromide (CTAB). Thus, the removal of CTAB from the reaction mixture, and further stabilization of the surface of the AuNRs is required. In the present study, AuNRs were encapsulated in a multifunctional envelope-type nano device (AuNR-MEND) formed with an SS-cleavable and pH-activated lipid-like material. In the process of encapsulation, AuNRs were first stabilized with bovine serum albumin (AuNR-BSA), and then further encapsulated in the lipid envelope by the ethanol dilution method. The in vitro photothermal cytotoxicity of AuNR-MEND was further demonstrated on 4T1 breast cancer cells. After NIR radiation, the temperature of the medium was increased to approximately 60°C, and cell viability was drastically decreased to approximately 11%. However, this cytotoxic effect cannot simply be explained by medium heating. It therefore appears that intracellular delivery of the AuNRs is a key factor for achieving a high degree of cytotoxicity. Dose dependent cytotoxicity data revealed that a higher dose of AuNR-MEND resulted in the complete destruction of the cells when they were subjected to NIR irradiation, while the cell survival rate reached a plateau at 30% in the case of AuNR-BSA. Apoptosis was induced after treatment with the nanoparticles. AuNR-MEND showed superior cellular uptake activity over AuNR-BSA. Thus, delivering AuNR by means of functionalized lipid nanoparticles represents a promising approach to induce NIR-triggered apoptosis.

Identification and determination of selenohomolanthionine - The major selenium compound in Torula yeast.

Torula yeast (Candida utilis) was found to metabolize selenium in a totally different way to Brewer's yeast (S. cerevisiae) leading to the biosynthesis of selenohomolanthionine (SeHLan), a major selenium compound accounting for 60-80% of the total selenium. The identity of SeHLan was confirmed by retention time matching in hydrophilic ion interaction chromatography (HILIC) with inductively coupled plasma mass spectrometric detection (ICP MS) using a custom synthesized standard molecule and by HILIC - Orbitrap MS and MS-MS fragmentation. Selenohomolanthionine escapes the current assays for the organic character of Se-rich yeast based on the protein-bound selenomethionine determination. A HILIC - ICP MS method was developed for the quantitative determination of selenohomolanthionine in yeast supplements with a detection limit of 146ng/g.

Comparison of the metabolism of inorganic and organic selenium species between two selenium accumulator plants, garlic and Indian mustard.

The metabolism of selenomethionine (SeMet) in two major selenium (Se) accumulator plants, garlic and Indian mustard, was compared to that of stable isotope labeled selenate. Indian mustard more efficiently transported Se from roots to leaves than garlic. In addition, Indian mustard accumulated larger amounts of Se than garlic. γ-Glutamyl-Se-methylselenocysteine (γ-GluMeSeCys) and Se-methylselenocysteine (MeSeCys) were the common metabolites of selenate and SeMet in garlic and Indian mustard. Indian mustard had a specific metabolic pathway to selenohomolanthionine (SeHLan) from both inorganic and organic Se species. SeMet was a more effective fertilizer for cultivating Se-enriched plants than selenate in terms of the production of selenoamino acids.

Complementary Use of LC-ICP-MS and LC-ESI-Q-TOF-MS for Selenium Speciation.

We demonstrated the complementary use of inductively coupled plasma-mass spectrometry (ICP-MS) and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) for the analysis of Se-containing compounds, such as selenate, selenomethionine (SeMet), and trimethylselenonium ion (TMSe), found in biological samples. The sensitivity of ESI-Q-TOF-MS for Se-containing compounds was strongly dependent on the chemical species. ICP-MS exhibited higher sensitivity than ESI-Q-TOF-MS, and had no species dependency. On the other hand, ESI-Q-TOF-MS enabled easy and robust identification of Se-containing compounds.

[Development of metallomics research on environmental toxicology].

Metallomics is newly coined terms and defined as a comprehensive analysis of the entirety of metal and metalloid species within a cell or tissue type. Then, metallome is defined as the entire category of metalloproteins and any other metal-containing biomolecules. Metallomics and research on metallome require analytical techniques that can provide information on the identification and quantification of metal/metalloid-containing biomolecules. This concept has been called speciation, and the acquisition of data according to the concept is performed using hyphenated techniques involving both separation and detection methods. In this review, the author intends to present several applications of complementary use of HPLC-inductively coupled plasma mass spectrometry (ICP-MS) and HPLC-electrospray ionization tandem mass spectrometry for identification of unknown selenium-containing metabolites, and also to present a newly developed technique, capillary LC-ICP-MS to be used for the analysis of metal-binding proteins.

Comparison of accumulation of four metalloids in Allium sativum.

In this study, we evaluated the accumulation and metabolism of four metalloids: arsenic (As), selenium (Se), antimony (Sb), and tellurium (Te) in garlic to determine whether garlic can be used for the phytoremediation of those metalloids. Garlic was able to efficiently accumulate As and Se, the two-fourth-period metalloids. However, it was not able to accumulate Sb and Te, the two-fifth-period metalloids, because their bioaccumulation factors were below one. Speciation analyses revealed that four metalloids could be metabolized in garlic, although their metabolites could not be identified yet. Results also suggested that garlic was able to distinguish the metalloids in groups 15 and 16 and the fourth and fifth periods, i.e., As, Se, Sb, and Te. Therefore, garlic is one of the potential plants for the phytoremediation of the fourth-period metalloids.

Metabolic pathway of inorganic and organic selenocompounds labeled with stable isotope in Japanese quail.

The distribution and metabolism of an inorganic selenium (Se) compound and a selenoamino acid in quails were evaluated by speciation with inductively coupled plasma mass spectrometry (ICP-MS) and a stable isotope. Quails were orally administered stable isotope [(77)Se]-labeled selenite and selenomethionine (SeMet) at the nutritional dose of 10 µg Se/bird. Then, the quails were dissected 3, 9, and 24 h after the administration to examine the metabolic pathway and the time-dependent change of Se. The concentrations of exogenous Se in all the organs and tissues of the SeMet-administered group were significantly higher than those of the selenite-administered group 3 h after the administration. This suggested that SeMet was more rapidly and/or efficiently incorporated into the quail body than selenite. A Se-containing protein in the serum was detected only in the SeMet-administered quails, but not in the selenite-administered quails. The major urinary Se metabolite, i.e., Se-methylseleno-N-acetyl-galactosamine (selenosugar), was detected in the quail serum after the administration of both selenite and SeMet. The endogenous amount of Se-methylated selenosugar (MeSeSug) in the serum of quails seemed to be larger than that of the rodents. We conclude that the metabolic pathway of Se in quails was the same as that in rodents, but the metabolic capacity for Se seemed to be larger in quails than in rodents.