小椋 康光

Inorganic metalloids, such as arsenic (As), antimony (Sb), selenium (Se), and tellurium (Te), are methylated in biota. In particular, As, Se, and Te are methylated and excreted in urine. The biomethylation is thought to be a means to detoxify the metalloids. The methylation of As is catalyzed by arsenic (+3 oxidation state) methyltransferase (AS3MT). However, it is still unclear whether AS3MT catalyzes the methylation of the other metalloids. It is also unclear whether other factors catalyze the As methylation instead of AS3MT. Recombinant human AS3MT (rhAS3MT) was prepared and used in the in vitro methylation of As, Se, and Te. As, but not Se and Te, was specifically methylated in the presence of rhAS3MT. Then, siRNA targeting AS3MT was introduced into human hepatocarcinoma (HepG2) cells. Although AS3MT protein expression was completely silenced by the gene knockdown, no increase in As toxicity was found in the HepG2 cells transfected with AS3MT-targeting siRNA. We conclude that AS3MT catalyzes the methylation of As and not other biomethylatable metalloids, such as Se and Te. We speculate that other methylation enzyme(s) also catalyze the methylation of As in HepG2 cells.

The activation process of secretory or membrane-bound zinc enzymes is thought to be a highly coordinated process involving zinc transport, trafficking, transfer and coordination. We have previously shown that secretory and membrane-bound zinc enzymes are activated in the early secretory pathway (ESP) via zinc-loading by the zinc transporter 5 (ZnT5)-ZnT6 hetero-complex and ZnT7 homo-complex (zinc transport complexes). However, how other proteins conducting zinc metabolism affect the activation of these enzymes remains unknown. Here, we investigated this issue by disruption and re-expression of genes known to be involved in cytoplasmic zinc metabolism, using a zinc enzyme, tissue non-specific alkaline phosphatase (TNAP), as a reporter. We found that TNAP activity was significantly reduced in cells deficient in ZnT1, Metallothionein (MT) and ZnT4 genes (ZnT1-/-MT-/-ZnT4-/- cells), in spite of increased cytosolic zinc levels. The reduced TNAP activity in ZnT1-/-MT-/-ZnT4-/- cells was not restored when cytosolic zinc levels were normalized to levels comparable with those of wild-type cells, but was reversely restored by extreme zinc supplementation via zinc-loading by the zinc transport complexes. Moreover, the reduced TNAP activity was adequately restored by re-expression of mammalian counterparts of ZnT1, MT and ZnT4, but not by zinc transport-incompetent mutants of ZnT1 and ZnT4. In ZnT1-/-MT-/-ZnT4-/- cells, the secretory pathway normally operates. These findings suggest that cooperative zinc handling of ZnT1, MT and ZnT4 in the cytoplasm is required for full activation of TNAP in the ESP, and present clear evidence that the activation process of zinc enzymes is elaborately controlled. © 2013 Fujimoto et al.

Tellurium (Te) is a widely used metalloid in industry because of its unique chemical and physical properties. However, information about the biological and toxicological activities of Te in plants and animals is limited. Although Te is expected to be metabolized in organisms via the same pathway as sulfur and selenium (Se), no precise metabolic pathways are known in organisms, particularly in plants. To reveal the metabolic pathway of Te in plants, garlic, a well-known Se accumulator, was chosen as the model plant. Garlic was hydroponically cultivated and exposed to sodium tellurate, and Te-containing metabolites in the water extract of garlic leaves were identified using HPLC coupled with inductively coupled plasma mass spectrometry (ICP-MS) or electrospray tandem mass spectrometry (ESI-MS-MS). At least three Te-containing metabolites were detected using HPLC-ICP-MS, and two of them were subjected to HPLC-ESI-MS-MS for identification. The MS spectra obtained by ESI-MS-MS indicated that the metabolite was Te-methyltellurocysteine oxide (MeTeCysO). Then, MeTeCysO was chemically synthesized and its chromatographic behavior matched with that of the Te-containing metabolite in garlic. The other was assigned as cysteine S-methyltellurosulfide. These results suggest that garlic can assimilate tellurate, an inorganic Te compound, and tellurate is transformed into a Te-containing amino acid, the so-called telluroamino acid. This is the first report addressing that telluroamino acid is de novo synthesized in a higher plant. © 2013 The Royal Society of Chemistry.

Two major selenoproteins are present in mammalian serum: extracellular glutathione peroxidase (eGPx) and selenoprotein P (Sel P). The chromatographic behaviors of the two serum selenoproteins were compared in four rodent species, and the selenoproteins in rat serum were identified by measuring enzyme activity and Western blotting. The selenoproteins in rat serum showed a specific chromatographic behavior. In particular, rat eGPx was eluted faster than eGPxs of the other rodent species, although the amino-acid sequences of the rodent species were identical. The elution profiles of Se in rat serum obtained by inductively coupled plasma tandem mass spectrometry (ICP-MS-MS) and ICP-MS were compared. The tandem quadrupoles and the O-2 reaction/collision gas completely removed severe interferences with the Se speciation originating from the plasma source and the biological sample matrix. ICP-MS-MS under the O-2 mass shift mode gave us more accurate abundance ratios of Se than ICP-MS.

The toxic and therapeutic effects of selenohomolanthionine (4,4'-selenobis[2-aminobutanoic acid], SeHLan), a newly identified selenoamino acid found in selenized Japanese pungent radish, were compared with those of selenomethionine (SeMet) and selenite in mice. Each Se compound was injected intravenously at the dose of 0.1, 1.0, and 10 mg Se kg(-1) body weight. SeHLan and selenite were equally distributed to the liver and the kidneys, whereas SeMet was more preferably distributed to the liver than to the kidneys. Although a part of SeHLan was assimilated and transformed into the urinary Se metabolite, Se-methylseleno-N-acetyl-galactosamine (MeSeGalNAc), the major part of the urinary Se species was intact SeHLan. The results suggest that the metabolic capacity of SeHLan to MeSeGalNAc reached a plateau even at the lowest dose. SeMet at the highest dose significantly increased ALT and amylase activities in mice. On the other hand, treatment with SeHLan at the dose of 10 mg Se kg(-1) body weight significantly increased BUN and creatinine levels in mouse sera, that is, nephrotoxicity was noted. SeHLan more effectively improved the survival rate reduced by LPS treatment than selenite and SeMet at the non-toxic dose of Se. The results indicate that SeHLan may be safely and effectively used to treat sepsis in place of selenite that is currently used in the clinical setting.

Many studies have examined the metabolic pathway of selenium (Se) compounds in Se-accumulating plants (hereafter "Se accumulators") when the plants are exposed to inorganic Se, such as selenite and selenate. However, if we were to consider Se circulation in the biosphere, the metabolism of organic Se, in particular, selenometabolites of animals and plants, in plants should be elucidated. In this study, Brassica rapa var. peruviridis, a known Se accumulator, was hydroponically cultivated and then exposed to selenometabolites of animals and plants, such as methyl-2-acetamido-2-deoxy-1-seleno-b-D-galactopyranoside (selenosugar, SeSug), trimethylselenonium (TMSe), selenomethionine (SeMet), and Se-methylselenocysteine (MeSeCys). Then, the metabolic pathway of the organic Se compounds/selenometabolites in B. rapa var. peruviridis was investigated by speciation analysis. Two selenometabolites were detected in the roots when the plant was exposed to SeMet, MeSeCys, and SeSug. They were assigned to S-(methylseleno)-glutathione and MeSeCys using electrospray tandem mass spectrometry (ESI-MS-MS) and HPLC-inductively coupled plasma mass spectrometry (ICP-MS). Contrary to SeMet, MeSeCys, and SeSug, TMSe was not metabolized even if it was more efficiently incorporated into the roots than the other Se compounds. The identified metabolites enabled us to propose a metabolic pathway for the organic Se metabolites except TMSe in the plant roots: a monomethylseleno moiety (CH3Se-) commonly existing in SeMet, MeSeCys, and SeSug was cleaved off and conjugated with GSH, and then the CH3Se group was transferred to O-acetylserine to form MeSeCys.

Selenium (Se) belongs to the same group as sulfur in the periodic table but possesses certain chemical properties characteristic of a metal. It is an essential element in animals but becomes severely toxic when the amount ingested exceeds the required level. On the other hand, Se is not essential in plants although some plants are Se hyperaccumulators. Se changes into several chemical forms when metabolized. Thus, the identification of selenometabolites would enable us to formulate a metabolic chart of Se. Recently, speciation analysis by hyphenated techniques has contributed immensely to the study of selenometabolomes, i.e., the entirety of selenometabolites. Indeed, speciation has unveiled some unique selenometabolites in biological samples. The aim of this review is to present newly identified selenometabolites in animals and plants by speciation using hyphenated techniques and to delineate the perspectives of Se biology and toxicology from the viewpoint of speciation.

Three kinds of sprouts in the Brassicaceae family of plants, namely, pink kale, radish and mustard were evaluated for the possibility of phytoremediation of lanthanides. The mustard sprout more efficiently accumulated lanthanides (e.g. 0.26 nmol La/g) than other Brassicaceae family plant sprouts (0.16 nmol La/g in the radish), however the radish sprout showed the fastest growth among three sprouts. Faster growth compensated for less efficiency in lanthanide accumulation (28 pmol La in the radish vs. 12 pmol La in the mustard) indicating that the radish is the most preferable sprout for the phytoremediation of lanthanides.

Metallomics and metallome are newly coined terms. Metallomics and research on metallome require analytical techniques that can provide information for the identification and quantification of metal species. This concept has been named speciation, and the acquisition of data according to this concept is performed with hyphenated techniques involving both separation and detection methods. In this paper, the author intended to present a newly developed technique multi-mode gel filtration capillary HPLC was evaluated regarding applications to nano level speciation, which is named nano-speciation. In addition, several applications of a complementary use of HPLC-inductively coupled plasma mass spectrometry and HPLC-electrospray ionization tandem mass spectrometry for selenometabolomics were presented. © 2012 The Japan Society for Analytical Chemistry.

Compared to the many studies on the physiological and toxicological effects of selenium (Se) in mammals, avian Se metabolism is still an unexplored topic. Some birds are useful as poultry for human nutrition. Moreover, birds belong to higher trophic levels in the biosphere and thus may play an important role in Se circulation in the ecosystem in the same way as mammals do. In this study, we analyzed the distribution and metabolism of Se in an experimental bird, the Japanese quail, which was fed drinking water containing sodium selenite or selenomethionine (SeMet). The highest concentration of Se was detected in the pancreas, followed by down feathers, liver, and kidneys. SeMet was more efficiently incorporated into the quail than selenite. The specific and preferable distribution of Se to the high molecular weight fraction in the serum of the quail was observed only in the SeMet-ingestion group. As in mammals, selenosugar and trimethylselenonium were the major metabolites in quail excreta. Three unknown Se metabolites were detected by HPLC-ICP-MS. Although part of the metabolic pathway of Se in the Japanese quail fed selenite and SeMet was the same as that observed in mammals, the bird also showed certain avian-specific metabolic process for Se.

セレンは動物にとって生体必須元素の一つであり、その生体内利用、代謝あるいは毒性はヒトや実験動物を用いて明らかにされてきた。近年では、植物や微生物のセレン代謝についても報告されているが、哺乳類以外の動物綱における生体内セレン代謝に関する情報は少ない。鳥類は哺乳類と同様に生態系の高次に位置し、環境中でのセレンの循環において重要な位置を占めると考えられる。そこで本研究ではニホンウズラに無機および有機セレン化合物を投与し、セレンの体内分布および化学形態を解析した。 WE系ニホンウズラ（オス、５週齢）を一週間馴化後、5 µg Se/mLの亜セレン酸ナトリウム（selenite）またはセレノメチオニン（SeMet）を含む水を１週間自由摂取させた。対照群には精製水を与えた。各臓器・組織および排泄物を採取し、硝酸湿式灰化後、ICP-MSでセレン濃度を定量した。また、肝臓と腎臓の上清、および排泄物抽出液について、HPLC-ICP-MSによりSeの化学形態を分析した。 セレン化合物を投与したウズラでは多くの組織で有意にSe濃度が増加した。また、２つの投与群を比べると、SeMet群の組織中セレン濃度はselenite群の2-5倍の高値を示した。従って、有機セレン化合物であるSeMetはseleniteに比べ生体内利用されやすいと示唆された。HPLC-ICP-MSによるセレン化学形態別分析の結果、肝臓および腎臓の上清においてセレンタンパク質に加え、セレン糖（selenosugars; SeSugs）とトリメチルセレノニウム（trimethylselenonium; TMSe）が検出された。これらは哺乳類において尿中代謝物として知られているが、鳥類でも検出されたことから、SeSugsおよびTMSeの生成は高等動物に共通したセレン代謝経路であると考えられた。また、これらの代謝物はセレン化合物投与により顕著に増加した。排泄物抽出液ではSeSugs、TMSeに加え、複数の未知代謝物が検出された。従って、鳥類は哺乳類と共通の経路の他に、特異的なセレン代謝機構を有することが示唆された。

セレン（Se）は動物においてグルタチオンペルオキシダーゼなどの酵素に要求される必須微量元素であり、生体内で代謝され最終的にセレン糖（SeSug）として尿中に排泄される。一方植物では必須元素であるとは認められていないが、Seの存在が間接的に植物の成長を促すことが知られており､有用な元素と考えられている。植物ではSeは主にセレノメチオニン（SeMet）やメチルセレノシステイン（MeSeCys）などの有機代謝物として存在する。環境中には多様な化学形態のSe化合物が存在するが､これまでの植物におけるSe代謝の研究の多くは無機セレン塩を用いたものであった。しかしながら、環境中におけるSeの循環を考える上で、動物由来のSeSugや、植物由来のSeMetやMeSeCysなど有機Se代謝物の植物における代謝機構の解明が必要であると考えられる。本研究では､Se蓄積性を有するアブラナ科植物にSeSug、SeMet及びMeSeCysを曝露し、各種質量分析を用いたスペシエーションを行った。有機Se代謝物曝露後に植物内のSeの化学形態をHPLC－誘導結合プラズマ質量分析計（ICP-MS）で分析したところ、3つの有機Se代謝物に共通して、未知のSe代謝物とMeSeCysの存在が確認できた。未知のSe代謝物についてエレクトロスプレータンデム質量分析計（ESI-MS-MS）により同定を試みたところ、その構造はメチルセレノグルタチオン（GSSeMe）であると想定できた。従って、植物体内で動植物由来の有機Se代謝物は共通して、一旦GSSeMeを経て、MeSeCysが新たに生合成されるという新たな代謝経路が存在すると想定された。

Selenium (Se) is an essential micronutrient because it forms the active center of selenoenzymes/selenoproteins in the form of selenocysteine. Another biological significance of Se is that it detoxifies inorganic mercury (iHg) by directly interacting with it. Recently, a novel selenometabolite, selenoneine (2-selenyl-N,N,N-trimethyl-L-histidine), was identified in several marine animals. However, its biological significance is still unclear. In this study, the ability of selenoneine to form a complex with iHg and methyl Hg (MeHg) was evaluated in vitro. Whereas selenite serving as the positive control reacted with iHg by direct interaction after being converted into selenide by endogenous reductants, such as glutathione (GSH), selenoneine did not interact with iHg or MeHg in the liver homogenate of marine turtle. This indicates that selenoneine may not play a role in the detoxification of Hg.

Copper (Cu) is an essential component of biological redox reactions and its deficiency is fatal to the body. At the same time, Cu is extremely toxic when present in excess. In this regard, several groups of Cu-regulating proteins in the body act to regulate the concentration of Cu within a certain range. However, the overall mechanism underlying the maintenance of Cu homeostasis in the body and cells remains poorly understood. In this review, recent research tools, such as animal models and gene-modified animals, and techniques, such as speciation and imaging of Cu, are highlighted.

Inorganic arsenic (iAs) is an established human carcinogen. Although methylation of iAs was considered as a detoxification mechanism, recently it is considered as an intoxification pathway in mammals. Our study population consisted of four groups A-D with drinking water iAs concentrations 33 ± 7, 148 ± 34, 210 ± 2.6, and 248 ± 59 μg As/l (mean ± SE), respectively in West Bengal, India. The ratios (monomethylated arsenicals)/(inorganic As metabolites - arsenate) = (MMA + DMA)/(iAs Met - iAsV), (dimethylated As)/(mono- and dimethylated As) = (DMA)/(MMA + DMA), and (dimethylated As)/(inorganic As metabolites - arsenate) = (DMA)/(iAs Met - iAsV) were used to assess methylation efficiency in the present study. High performance liquid chromatography-inductively coupled argon plasma mass spectrometry (HPLC-ICP MS) was used to determine As species in spot urine samples. The detection limit of As compounds was 0.14-0.33 mg As/l. All trivalent arsenicals were stable for up to 2 months when arsenic spiked urine samples were stored at -28° C without any preservatives. Although females appeared to be better methylators than males and children (considering first and total methylations), they were statistically not significant (p>0.05). Only second methylation capacity was statistically significant between different age groups (p<0.05). Although methylation capacity is not statistically conclusive in the present study, this is the first study, which documents the results on reduction capacity of the As-affected population in the endemic areas. Another outcome of this study suggests that researchers must consider the intake route of As via food-chain during the preparation of toxicity model of As.

Cimicifugoside is a triterpenoid originating from the rhizomes of Cimicifuga simplex. and acts to inhibit the subcellular transport of nucleosides. Cimicifugoside. when used in combination with methotrexate, showed a cell-specific synergic effect on the promonocytic leukemia cell line U937, but not on the chronic myelogenetic leukemia cell line K562. Thymidine uptake was more severely inhibited by cimicifugoside in a dose-dependent fashion in U937 than in K562. The mRNA expression of one of the equilibrative Na(+)-independent nucleoside transporters. ENT2, was lower in U937 than in K562. This suggests that the thymidine uptake by ENT2 of U937 is more severely affected by cimicifugoside than that of K562, resulting in a decrease in DNA synthesis by methotrexate. In addition, cimicifugoside more efficiently stimulated the activity of thymidine kinase (TK) in K562 than in U937. suggesting that K562 resisted the decrease in DNA synthesis caused by the inhibition of nucleoside transporters. Cimicifugoside bifunctionally potentiated the cell-specific cytotoxicity of methotrexate by inhibiting ENT2 and activating TK.

Excessive generation of reactive oxygen species (ROS) is considered to play an important role in arsenic-induced carcinogenicity in the liver, lungs, and urinary bladder. However, little is known about the mechanism of ROS-based carcinogenicity, including where the ROS are generated, and which arsenic species are the most effective ROS inducers. In order to better understand the mechanism of arsenic toxicity, rat liver RLC-16 cells were exposed to arsenite (iAs(III)) and its intermediate metabolites [i.e., monomethylarsonous acid (MMA(III)) and dimethyl-arsinous acid (DMA(III))]. MMA(III) (IC50 = 1 mu M) was found to be the most toxic form, followed by DMA(III) (IC50 = 2 mu M) and iAs(III) (IC50 = 18 mu M). Following exposure to MMA(III), ROS were found to be generated primarily in the mitochondria. DMA(III) exposure resulted in ROS generation in other organelles, while no ROS generation was seen following exposures to low levels of iAs(III). This suggests the mechanisms of induction of ROS are different among the three arsenicals. The effects of iAsIII, MMA(III), and DMA(III) on activities of complexes I-IV in the electron transport chain (ETC) of rat liver submitochondrial particles and on the stimulation of ROS production in intact mitochondria were also studied. Activities of complexes II and IV were significantly inhibited by MMA(III), but only the activity of complexes II was inhibited by DMA(III). Incubation with iAs(III) had no inhibitory effects on any of the four complexes. Generation of ROS in intact mitochondria was significantly increased following incubation with MMA(III), while low levels of ROS generation were observed following incubation with DMA(III). ROS was not produced in mitochondria following exposure to iAs(III). The mechanism underlying cell death is different among As-III MMA(III), and DMA(III), with mitochondria being one of the primary target organelles for MMA(III)-induced cytotoxicity.

The distribution and metabolism of selenohomolanthionine (4,4'-selenobis[2-aminobutanoic acid], SeHLan), a newly identified selenoamino acid in selenized Japanese pungent radish, were evaluated by administering (77)Se-labeled SeHLan at a dose of 25 mu g/kg body weight in rats. Exogenous (77)Se of SeHLan was preferably distributed to the kidneys and remained in the intact form for up to 6 h after dosing. The accumulation in the kidneys is one of the specific characteristics of SeHLan, differing from other selenoamino acids, such as selenomethionine and Se-methylselenocysteine, which preferably accumulate in the pancreas. The intact form of SeHLan was detected in the serum and kidney supernatant but not in the urine, suggesting that the amount of exogenous Se that was distributed to the kidneys was within metabolic capacity. Indeed, the exogenous Se was converted into two urinary metabolites, Se-methylseleno-N-acetyl-galactosamine and trimethylselenonium. Exogenous Se was also detected in several selenoproteins, including selenoprotein P and extracellular glutathione peroxidase. SeHLan is expected to be a potential supplemental source of Se because its distribution differs from that of selenomethionine and Se-methylselenocysteine.

It is known that marine mammals and seabirds co-accumulate selenium (Se) and mercury (Hg) in their organs in the insoluble form called mercury selenide. In this study, we found that two sea turtles, hawksbill turtle (Eretmochelys imbricata) and green turtle (Chelonia mydas), accumulated Se but not Hg in their livers. Se speciation by HPLC-ICP-MS demonstrated that the livers contained low molecular weight selenometabolites in addition to selenoproteins. Two of the selenometabolites existed in relatively small amounts and were identified as selenosugar (1 beta-methylseleno-N-acetyl-D-galactosamine) and trimethylselenonium (TMSe) based on their chromatographic behavior. This suggests that selenosugar and TMSe are Se metabolites common to marine and terrestrial animals. The chromatographic behavior of the major hepatic selenometabolite in sea turtles was unique and did not match that of any authentic Se standards. Further analysis using HPLC-ESI-MS-MS revealed it to be selenoneine (2-selenyl-N,N,N-trimethyl-L-histidine), a metabolite that was recently identified in the blood of bluefin tuna. The results suggest that sea turtles possess specific mechanisms for Se metabolism to result in the sole accumulation of Se.

Copper chaperone for SOD1 (CCS) specifically delivers copper (Cu) to copper, zinc superoxide dismutase (SOD1) in cytoplasm of mammalian cells. In the present study, small interfering RNA (siRNA) targeting CCS was introduced into metallothionein-knockout mouse fibroblasts (MT-KO cells) and their wild type cells (MT-WT cells) to reveal the interactive role of CCS with other Cu-regulating proteins, in particular, MT. CCS knockdown significantly decreased Ctr1, a Cu influx transporter, mRNA expression. On the other hand, Atp7a, a Cu efflux transporter, mRNA expression was increased 3.0 and 2.5 times higher than those of the control in MT-WT and MT-KO cells. These responses of Cu-regulating genes to the CCS knockdown reflected the presence of excess Cu in the cells. To evaluate the Atp7a function in the Cu-replete cells, siRNA of Atp7a and the other Cu transporter, Atp7b were introduced into MT-WT and MT-KO cells. The Atp7a knockdown significantly increased the intracellular Cu concentration, whereas the Atp7b knockdown had no affect. Although two MT isoforms were induced by the CCS knockdown in MT-WT cells, the expression and activity of SOD1 were maintained in both MT-WT and MT-KO cells even when CCS protein expression was reduced to 0.30-0.35 of control. This suggests that the amount of CCS protein exceeds that required to supply Cu to SOD1 in the cells. Further, the CCS knockdown induces Cu accumulation in cells, however, the Cu accumulation is ameliorated by the MT induction, the decrease of Ctr1 expression and the increase of Atp7a expression to maintain Cu homeostasis.

Three minor sulfur-containing arsenic metabolites: monomethylmonothioarsonic acid (MMMTA(V)), dimethylmonothioarsinic acid (DMMTA(V)), and dimethyldithioarsinic acid (DMDTA(V)) were recently found in human and animal urine after exposure to inorganic arsenic. However, it remains unclear how the thioarsenicals are formed in the body and then excreted into the urine. It is hypothesized that the generation of thioarsenicals occurs during enterohepatic circulation. To address this hypothesis, male Sprague Dawley (SD) rats and Eisai hyperbilirubinuric (EHB) rats (with deficiency of multidrug resistance-associated protein 2) were orally administered a single dose of inorganic arsenite (iAs(III)) at 3.0 mg kg(-1) of body weight. qFive hours after dosing, less than 1.0% of the dose was recovered in the bile of EHB rats, while more than 27% of the dose was recovered in the bile of SD rats, with the majority being monomethylarsinodiglutathione [MMA(SG)(2)] with a small amount of arsenic triglutathione [iAs(SG)(3)]. During the early time periods (3 h and 6 h) the arsenic levels in the liver, red blood cells (RBCs) and plasma of EHB rats were higher than those of SD rats, and approximately 76% and 87% of the dose was recovered in the RBCs of SD and EHB rats, respectively, at day 5 after dosing. However, there were no significant differences in arsenic concentration in urine between the two types of animal. Regarding the arsenic species in the urine of both types of rat, significant levels of thiolated arsenicals MMMTA(V) and DMMTA(V) were detected in SD rat urine, however in EHB rat urine only low levels of DMMTA(V) were detected. The present result of the metabolic balance and speciation study suggests that the formation of MMMTA(V) and DMMTA(V) in rats is dependent on enterohepatic circulation. In addition, in vitro experiments indicated that arsenicals excreted from bile may be transformed by gastrointestinal microbiota into MMMTA(V) and DMMTA(V), which are then absorbed into the bloodstream and finally excreted into the urine.

Although selenium (Se) is not an essential element in plants, Se metabolism in plants is remarkable, mainly for the purpose of phytoremediation and nutritional supplementation of Se. Brassica juncea (Indian mustard) is known as an Se accumulator. In addition to Indian mustard, unique Brassicaceae plants are consumed in Japan. Brassica rapa var. hakabura, commonly known as nozawana, and Brassica rapa var. peruviridis, commonly known as komatsuna, are typical Brassicaceae plants. In this study, we evaluated the Se-accumulating ability of these unique Brassicaceae plants. There were no significant differences in the Se concentrations in the roots and the leaves of the three Brassicaceae plants. However, nozawana most efficiently accumulated Se among the three Brassicaceae plants because it showed the most rapid growth, resulting in the highest biomass. Speciation revealed that the Se species accumulated in the three plants were identical. In addition, nozawana contained easily extractable essential minerals, such as iron, copper, and zinc. Potentialities of nozawana as a source of minerals and the phytoremediation of soil and water contaminated with Se were discussed.

Quite a few new thioarsenicals have recently been found in urine of arsenic-exposed humans and animals, and some of them have been shown to be highly toxic to cells. However, little is known about their toxic effects and metabolism in the body. In order to elucidate the toxic mechanism of thioarsenicals, we further focused on the distribution and metabolism of monomethylmonothioarsonic acid (MMMTA(v)) in rats. MMMTA(v) was synthesized chemically and injected intravenously into rats at the dose of 0.5 mg As/kg, followed by speciation analysis of selected organs and body fluids at 10 min and 12 h after the injection. MMMTA(v) was excreted into urine in its intact form, and approximately 35% of the dose was recovered in urine at 12 h after the injection, suggesting that MMMTA(v) was taken up more effectively by organs/tissues than non-thiolated, monomethylarsonous acid (MMA(v)) previously studied. On the other hand, the liver and kidneys contained arsenic that was in a protein-binding form with free forms of DMA(v) or DMDTA(v) at 10 min, and disappeared at 12 h after the injection. Moreover, these bound arsenic species in kidneys were converted back to MMA(v) after oxidation with H(2)O(2), suggesting that the arsenic bound to proteins had been reduced within the body and was in a trivalent oxidation state. In red blood cells (RBCs), most of the arsenic was in the form of DMA(III) bound to hemoglobin (Hb), and approximately 40% of the dose was recovered in RBCs at 12 h after injection. These results indicate that arsenic accumulated preferentially in RBCs after being transformed to DMA(III). In addition, we have also discussed the effect of MMMTA(v) on viability of human bladder cancer T24 cells in comparison with MMA(v). Consequently, MMMTA(v) was assumed to be a more toxic arsenic metabolite than non-thiolated MMA(v). (C) 2010 Elsevier Ltd. All rights reserved.

Tetrathiomolybdate (TTM) is a powerful and selective copper (Cu) chelator that is used as a therapeutic agent for Wilson disease. TTM is the sole agent that can remove Cu bound to metallothionein (MT) in the livers of Long-Evans rats with a cinnamon-like coat color (LEC rats). However, the administration of excess TTM causes the deposition of Cu and molybdenum (Mo) in the liver. In the present study, the effect of hepatic glutathione (GSH) depletion on the removal of Cu from the livers of LEC rats was evaluated to establish an effective therapy by TTM. Pretreatment with L-buthionine sulfoximine (BSO), a depletor of GSH in vivo, reduced the amounts of Cu and Mo excreted into both the bile and the bloodstream, and increased the amounts of Cu and Mo deposited in the livers of LEC rats in the form of an insoluble complex 4 h after the TTM injection. The results suggest that GSH depletion creates an oxidative environment in the livers of LEC rats, and the oxidative environment facilitates the insolubilization of Cu and Mo in the livers of LEC rats after the TTM injection. Therefore, the effect of TTM on the removal of Cu from the liver was reduced in the oxidized condition. Wilson disease patients and LEC rats develop liver injury caused by oxidative damage. From a clinical viewpoint, increasing in the GSH concentration is expected to enhance the effect of TTM. (C) 2010 Elsevier Inc. All rights reserved.

Tetrathiomolybdate (TTM) is a powerful and selective copper (Cu) chelator that is used as a therapeutic agent for Wilson disease. TTM is the sole agent that can remove Cu bound to metallothionein (MT) in the livers of Long-Evans rats with a cinnamon-like coat color (LEC rats). However, the administration of excess TTM causes the deposition of Cu and molybdenum (Mo) in the liver. In the present study, the effect of hepatic glutathione (GSH) depletion on the removal of Cu from the livers of LEC rats was evaluated to establish an effective therapy by TTM. Pretreatment with l-buthionine sulfoximine (BSO), a depletor of GSH in vivo, reduced the amounts of Cu and Mo excreted into both the bile and the bloodstream, and increased the amounts of Cu and Mo deposited in the livers of LEC rats in the form of an insoluble complex 4h after the TTM injection. The results suggest that GSH depletion creates an oxidative environment in the livers of LEC rats, and the oxidative environment facilitates the insolubilization of Cu and Mo in the livers of LEC rats after the TTM injection. Therefore, the effect of TTM on the removal of Cu from the liver was reduced in the oxidized condition. Wilson disease patients and LEC rats develop liver injury caused by oxidative damage. From a clinical viewpoint, increasing in the GSH concentration is expected to enhance the effect of TTM.

A novel function of COMMD1 {COMM [copper metabolism MURR1 (mouse U2af1-rs1 region 1)]-domain-containing 1}, a protein relevant to canine copper toxicosis, was examined in the mouse hepatoma cell line Hepa 1-6 with multi-disciplinary techniques consisting of molecular and cellular biological techniques, speciation and elemental imaging. To clarify the function of COMMD1, COMMD1-knockdown was accomplished by introducing siRNA (small interfering RNA) into the cells. Although COMMD1-knockdown did not affect copper incorporation, it inhibited copper excretion, resulting in copper accumulation, which predominantly existed in the form bound to MT (metallothionein). It is known that the liver copper transporter Atp7b (ATP-dependent copper transporter 7 beta), localizes on the trans-Golgi network membrane under basal copper conditions and translocates to cytoplasmic vesicles to excrete copper when its concentration exceeds a certain threshold, with the vesicles dispersing in the periphery of the cell. COMMD1-knockdown reduced the expression of Atp7b, and abolished the relocation of Atp7b back from the periphery to the trans-Golgi network membrane when the copper concentration was reduced by treatment with a Cu(I) chelator. The same phenomena were observed during COMMD1-knockdown when another Atp7b substrate, cis-diamminedichloroplatinum, and its sequestrator, glutathione ethylester, were applied. These results suggest that COMMD1 maintains the amount of Atp7b and facilitates recruitment of Atp7b from cytoplasmic vesicles to the trans-Golgi network membrane, i.e. COMMD1 is required to shuttle Atp7b when the intracellular copper level returns below the threshold.

Arsenic toxicity and distribution are highly dependent on animal species and its chemical species. Recently, thioarsenical has been recognized in highly toxic arsenic metabolites, which was commonly found in human and animal urine. In the present study, we revealed the mechanism underlying the distribution and metabolism of non-thiolated and thiolated dimethylarsenic compounds such as dimethylarsinic acid (DMA(V)), dimethylarsinous acid (DMA(III)), dimethylmonothioarsinic acid (DMMTA(V)), and dimethyldithioarsinic acid (DMDTA(V)) after the administration of them into femoral vein of hamsters. DMA(V) and DMDTA(V) distributed in organs and body fluids were in their unmodified form, while DMA(III) and DMMTA(V) were bound to proteins and transformed to DMA(V) in organs. On the other hand, DMA(V) and DMDTA(V) were mostly excreted into urine as their intact form 1 h after post-injection, and more than 70% of the doses were recovered in urine as their intact form. By contrast, less than 8-14% of doses were recovered in urine as DMA(V), while more than 60% of doses were distributed in muscles and target organs (liver, kidney, and lung) of hamsters after the injection of DMMTA(V) and DMA(III). However, in red blood cells (RBCs), only a small amount of the arsenicals was distributed (less than 4% of the doses) after the injection of DMA(III) and DMMTA(V), suggesting that the DMA(III) and DMMTA(V) were hardly accumulated in hamster RBCs. Based on these observations, we suggest that although DMMTA(V) and DMDTA(V) are thioarsenicals, DMMTA(V) is taken up efficiently by organs, in a manner different from that of DMDTA(V). In addition, the distribution and metabolism of DMMTA(V) are like in manner similar to DMA(III) in hamsters, while DMDTA(V) is in a manner similar to DMA(V). (C) 2010 Elsevier Inc. All rights reserved.

Tellurium (Te) is widely used in industry because of its unique chemical and physical properties, and has recently become a part of everyday life as a component of phase-change optical magnetic disks. However, the recovery of Te from the environment has not been discussed yet. In this regard, we evaluated the potential use of Indian mustard (Brassica juncea), a selenium (Se) accumulator, for the phytoremediation of Te. The Indian mustard plant was exposed to selenate and tellurate and the concentrations of Se and Te and the chemical species in the plant were determined. The Indian mustard plant accumulated less Te than Se, and the amount of Te accumulated in the plant was approximately 1/69 of that of Se. Although the incorporation of selenate was reduced by increasing sulfate concentration in the medium, the incorporation of Te was not affected by it, suggesting that this plant was able to discriminate tellurate from selenate in the roots. Three Te species were detected in the plant. The major species was tellurate. The other two species were not identical to available Te standards and thus could not be identified. Consequently, the Indian mustard plant is inappropriate for the phytoremediation of Te because it can strictly distinguish tellurate from selenate.

The distribution and metabolism of selenohomolanthionine (4,4'-selenobis[2-aminobutanoic acid], SeHLan), a newly identified selenoamino acid in selenized Japanese pungent radish, were compared with those of selenomethionine (SeMet) in rats. Either selenoamino acid was injected intravenously at a bolus dose of 1.0 mg Se/kg body weight. SeMet was preferably accumulated in the pancreas, increasing the serum amylase level, an index of pancreatic damage. SeHLan was preferably accumulated in the kidneys, raising the serum creatinine level, an index of kidney damage. On the other hand, the levels of two major urinary selenometabolites, i.e., Se-methylseleno-N-acetyl-galactosamine and trimethylselenonium, were comparable between SeHLan- and SeMet-administered rats, suggesting that there may be no differences in the efficiency of metabolism of these two selenoamino acids to the urinary selenometabolites despite the difference in distribution. SeHLan is expected to be a potential supplemental source of Se without inducing the onset of pancreatic damage. The specific toxicity of SeHLan to the kidneys may be avoided if its dose is lower than the one used in the present study.

Tellurium and antimony are widely used in industry because of their unique chemical and physical properties. Although these metalloids, which belong to period 5 of the periodic table of elements, are known to be non-essential and harmful, or the so-called "exotic" elements, little is known about their toxic effects and metabolism. The present review describes the role of speciation in considering the metabolism of tellurium and antimony from the viewpoint of toxicometallomics. Inorganic tellurium in the form of tellurite is reduced and simply methylated in the body. Rat red blood cells accumulate tellurium in the form of dimethylated tellurium, and tellurium is excreted into urine as trimethyltelluronium. Although selenium, which belongs to the same group as tellurium, is known to be excreted in the form of selenosugar as the major urinary metabolite, tellurosugar was not detected by an inductively coupled plasma-mass spectrometer hyphenated with an HPLC. Speciation studies revealed that the major metabolic pathway of antimony is oxidation in human and rat, and methylation also occurs as a minor metabolic pathway in humans.

Arsenic toxicity is dependent on its chemical species. In humans, the bladder is one of the primary target organs for arsenic-induced carcinogenicity. However, little is known about the mechanisms underlying arsenic-induced carcinogenicity, and what arsenic species are responsible for this carcinogenicity. The present study aimed at comparing the toxic effect of DMMTA(V) with that of inorganic arsenite (iAs(III)) on cell viability, uptake efficiency and production of reactive oxygen species (ROS) toward human bladder cancer EJ-1 cells. The results were compared with those of a previous study using human epidermoid carcinoma A431 cells. Although iAs(III) was known to be toxic to most cells, here we show that iAs(III) (LC(50) = 112 mu M) was much less cytotoxic than DMMTA(V) (LC(50) = 16.7 mu M) in human bladder EJ-1 cells. Interestingly, pentavalent sulfur-containing DMMTA(V) generated a high level of intracellular ROS in EJ-1 cells. However, this was not observed in the cells exposed to trivalent inorganic iAs(III) at their respective LC(50) dose. Furthermore, the presence of N-acetyl-cysteine completely inhibited the cytotoxicity of DMMTA(V) but not iAs(III), suggesting that production of ROS was the main cause of cell death from exposure to DMMTA(V), but not iAs(III). Because the cellular uptake of iAs(III) is mediated by aquaporin proteins, and because the resistance of cells to arsenite can be influenced by lower arsenic uptake due to lower expression of aquaporin proteins (AQP 3, 7 and 9), the expression of several members of the aquaporin family was also examined. In human bladder EJ-1 cells, mRNA/proteins of AQP3, 7 and 9 were not detected by reverse transcription polymerase chain reaction (RT-PCR)/western blotting. In A431 cells, only mRNA and protein of AQP3 were detected. The large difference in toxicity between the two cell lines could be related to their differences in uptake of arsenic species. (C) 2009 Elsevier Inc. All rights reserved.

Recently, tellurium (Te), antimony (Sb) and germanium (Ge) have been used as an alloy in phase-change optical magnetic disks, such as digital versatile disk-random access memory (DVD-RAM) and DVD-recordable disk (DVD-RW). Although these metalloids, the so-called "exotic" elements, are known to be non-essential and harmful, little is known about their toxic effects and metabolism. Metalloid compounds, tellurite, antimonite and germanium dioxide, were simultaneously administered to rats. Their distributions metabolites were determined and identified by speciation. Te and Sb accumulated in red blood cells (RBCs): Te accumulated in RBCs in the dimethylated form, while Sb accumulated in the inorganic/non-methylated form. In addition, trimethyltelluronium (TMTe) was the urinary metabolite of Te, whereas Sb in urine was not methylated but oxidized. Ge was also not methylated in rats. These results suggest that each metalloid is metabolized via a unique pathway.

Copper (Cu) is the active center of some enzymes because of its redox-active property, although that property could have harmful effects. Because of this, cells have strict regulation/detoxification systems for this metal. In this Study, multi-disciplinary approaches, such as speciation and elemental imaging of Cu, were applied to reveal the detoxification mechanisms for Cu in cells bearing a defect in Cu-regulating genes. Although Cu concentration in metallothionein (MT)-knockout cells was increased by the knockdown of the Cu chaperone, Atoxi1, the concentrations of the Cu influx pump, Ctr1, and another Cu chaperone, Ccs, were paradoxically increased; namely, the cells responded to the Cu deficiency despite the fact that cellular Cu concentration was actually increased. Cu imaging showed that the elevated Cu was compartmentalized in cytoplasmic vesicles. Together, the results point to the novel roles of MT and cytoplasmic vesicles in the detoxification of Cu in mammalian cells. (C) 2009 Elsevier Inc. All rights reserved.

Diphenylarsinic acid (DPAA) is an environmental degradation product of diphenylarsine chloride or diphenylarsine cyanide, which were chemical warfare agents produced by Japan during the World War II. DPAA is now considered a dangerous environmental pollutant in Kamisu, Japan, where it is suspected of inducing health effects that include articulation disorders (cerebellar ataxia of the extremities and trunk), involuntary movements (myoclonus and tremor), and sleep disorders. In order to elucidate the toxic mechanism of DPAA, we focused on the distribution and metabolism of DPAA in Fats. Systemic distribution of DPAA was determined by administering DPAA orally to rats at a single dose of 5.0 mg As/kg body weight, followed by speciation analysis of selected organs and body fluids. Most of the total arsenic burden was recovered in the urine (23% of the dose) and feces (27%), with the distribution in most other organs/tissues being less than 1%. However, compared with the typical distribution of inorganic dietary arsenic, DPAA administration resulted in elevated levels in the brain, testes and pancreas. In contrast to urine, in which DPAA was found mostly in its unmodified form, the tissues and organs contained arsenic that was mostly bound to non-soluble and soluble high molecular weight proteins. These bound arsenic species could be converted back to DPAA after oxidation with H(2)O(2), suggesting that the DPAA bound to proteins had been reduced within the body and was in a trivalent oxidation state. Furthermore, we also detected two unknown arsenic metabolites in rat urine, which were assumed to be hydroxylated arsenic metabolites. (C) 2009 Elsevier Inc. All rights reserved.

Aims: This study compares the transfer from mother to fetuses and pups of selenium (Se) in the form of selenite, selenate, and selenomethionine (SeMet) labeled with different homo-elemental isotopes. Main methods: To completely substitute endogenous Se with natural abundance with Se enriched with a single stable isotope ((82)Se), female Wistar rats delivered by mother fed (82)Se-selenite were fed Se-deficient diet and drinking water containing 82Se-selenite immediately after weaning, and then mated with male Wistar rat at the age of 15-17 weeks. The pregnant rats were divided into two groups. One group was fed Se-deficient diet and drinking water containing (76)Se-selenite, (78)Se-selenate, and (77)Se-SeMet from gestation days 11 to 20. The other group was fed the same diet and drinking water containing the three Se species after delivery for 10 days of lactation. Non-pregnant rats were also fed Se mixture and Se-deficient diet for 10 days. Key finding: Tissue and plasma Se concentrations showed significant changes among non-pregnant, pregnant, and lactating rats. The peak corresponding to selenoprotein P (Sel P) in serum of pregnant rats was reduced. The concentration of (77)Se originating from SeMet was higher than those of (76)Se from selenite and (78)Se from selenate in the stomach content of pups. Significance: Inorganic Se species are more preferably transformed into Sel P than SeMet, and Sel P is effectively incorporated into placenta during pregnancy. On the other hand, SeMet is a more efficient Se source than inorganic Se species during lactation. (C) 2009 Elsevier Inc. All rights reserved.

Se-Methylselenocysteine (MeSeCys) is not only a selenium (Se) supplement but also a more promising precursor of an anti-tumor drug containing Se than selenomethionine, which is Currently used as Se supplement. In this study, the metabolism of MeSeCys labeled with an Se isotope, Se-82, in rats depleted of endogenous natural abundance isotopes with another Se isotope, Se-78, was traced for 21 days when MeSeCys was continuously and perorally ingested at a supplemental dose. The tracer experiment was performed with our improved method that utilized an inductively coupled plasma-deuterium reaction-mass spectrometer. The substitution of endogenous Se with a single isotope, 78Se, facilitated the detection of exogenous labeled Se. Exogenous Se in the form of MeSeCys preferably accumulated and/or assimilated in the liver, kidneys and testes with long-term ingestion of MeSeCys and was utilized for the synthesis of selenoproteins, i.e., extracellular and cellular glutathione peroxidases and selenoprotein P. Meanwhile, intact MeSeCys was not excreted into urine although trimethylselenonium was detected in addition to selenosugar. The results suggest that MeSeCys was transformed into selenide via methylselenol by beta-lyase. Consequently, it is surmised that MeSeCys is a precursor of methylselenol under long-term ingestion.

Selenometabolites transformed from selenomethionine (SeMet) in wheat germ extract (WGE) were identified by complementary use of HPLC-ICP-MS and HPLC-ESI-MS/MS. Three selenium (Se)-containing peaks tentatively named WGE1, WGE2, and WGE3 were detected by HPLC-ICP-MS. WGE1 had [M](+) at m/z 212 on HPLC-ESI-MS analysis, and its fragment ions indicated that WGE1 is selenomethionine methylselenonium (MeSeMet). WGE2 and WGE3 exhibited absorption at 254 nm and molecular ions at m/z 433 and 447, respectively. Their fragment ions revealed that WGE2 and WGE3 are Se-adenosylselenohomocysteine (AdoSeHcy) and Se-adenosylselenomethionine (AdoSeMet), respectively. Their structures were coincident with the absorption of WGE2 and WGE3 at 254 nm. In addition, a trace amount of AdoSeMet was suggested to also exist in rabbit reticulocyte lysate, a mammalian in vitro translation system, because the transformation of AdoSeMet from SeMet was completely inhibited by (2S)-2-amino-4,5-epoxypentanoic acid (AEPA), a potent inhibitor of AdoMet synthetase. These results suggest that SeMet and methionine (Met) share a common metabolic pathway, i.e., SeMet is not only incorporated into proteins in place of Met but also metabolized to AdoSeMet in higher eukaryotes and MeSeMet in plants.

Selenium (Se) shows ambivalent characteristics in animals and plants. It is an essential element in animals but becomes severely toxic when the amount ingested exceeds the required level. Meanwhile, Se is not essential in plants although some plants are Se hyperaccumulators. As Se changes into several chemical forms when metabolized, the identification of selenometabolites is considered to correspond to the depiction of the metabolic chart of Se. Hence, we are very much interested in unveiling selenometabolomes, i.e., the entirety of selenometabolites. Speciation with elemental and molecular mass spectrometry coupled with separation techniques has significantly contributed to the discovery of selenometabolomes. At the same time, speciation has highlighted some metabolites required for solid verification of the metabolic chart. The aim of this article is to review currently proposed Se metabolic pathways in animals and plants and to point out ambiguous selenometabolites from the viewpoint of speciation.

Buckwheat (Fagopyrum esculentum Moench) and quinoa (Chenopodium quinoa Willdenow) are widely used as food ingredients. The nutritional characteristics of these plants, i.e., high contents of proteins and amino acids suggest that selenium (Se) is preserved as selenoamino acid derivatives, in particular, selenomethionine (SeMet) in proteins, similar to selenized yeast. Therefore, buckwheat and quinoa are expected to be a good nutritional source of Se. Selenized buckwheat and quinoa were cultivated on Se-fortified soil using sparingly soluble Se salts, such as barium selenate and barium selenite. Se concentration in the edible parts of these plants was determined, and Se extraction efficiency with enzyme or alkali was evaluated. In addition, the chemical species of Se in the low molecular weight fraction of these plants were determined by HPLC-ICP-MS. Total Se concentrations in the edible parts of selenized buckwheat and quinoa were 170.4 +/- 2.9 mu g/g and 102.7 +/- 2.4 mu g/g wet weight, respectively. Thus, these selenized seeds were found to be Se accumulators. The results indicate that Se in selenized buckwheat exists mainly as SeMet, while Se in selenized quinoa exists not only as SeMet but also as selenate (Se(VI)) and non-protein forms.

Tellurium (Te) has shown recent increase in use as a component of optical magnetic disks having phase-change property, such as digital versatile disk-random access memory (DVD-RAM) and DVD-rewritable (DVD-RW). However, the toxicity and metabolic pathway of Te remain unclear despite its being known as a non-essential and harmful metalloid. This study was performed to gain an insight into Te metabolism in the body. The mechanism for the distinction of Te from selenium (Se), an essential metalloid belonging to the same group as Te, was also clarified. Rats were given drinking water containing tellurite and Se-82-labeled selenite at the same concentration, and the concentrations of these metalloids in organs, body fluid and excreta were determined 2 days later. The results demonstrate that urinary and fecal excretion of Te was, respectively, lower and higher than that of exogenous (labeled) Se, suggesting that Te was less absorbed than Se. The ingested Te was transformed, i.e., methylated in organs and effluxed into bloodstream, and the effluxed Te was highly accumulated in rat red blood cells (RBCs) in the form of dimethylated Te. In contrast, Se was not accumulated in RBCs. Finally, Te was excreted in urine as trimethyltelluroniurn and might be exhaled as dimethyltelluride. The results suggest that the metabolism of Te was distinct from that of Se in rats. (c) 2008 Elsevier Inc. All rights reserved.

Nitric oxide (NO) enhanced the nuclear localization of metallothionein (MT) in digitonin-permeabilized semi-intact HeLa cells. Although 1-Hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-1-triazene (NOC5), an NO donor, enhanced the nuclear localization of MT in a manner similar to hydrogen peroxide (H2O2), a tenfold higher concentration of NOC5 was required to achieve the same effect as H2O2, suggesting that an endogenous NO scavenger existed in the reaction mixture of the nuclear import assay system that we used. We also evaluated the effect of NO on the nuclear localization of MT in intact HeLa cells. Treatment with NOC5 induced the nuclear localization of MT pre-induced with zinc, and its effect was greater than that of H2O2. The induced MT was localized in the nucleus and the cytoplasm. The results suggest that MT can scavenge NO using the sulfhydryl groups of cysteines in its molecule to form nitrosothiol, thereby reducing nuclear and cytoplasmic damage by NO.

Minute amounts of tissue supernatants from mouse neonates bearing a mutation in the copper (Cu)-transporter gene, Atp7a, were injected into narrow-bore HPLC coupled with an inductively coupled plasma-mass spectrometer (ICP-MS) to examine Cu metabolism. In the 14-day-old mutant neonates, Cu accumulated in the intestine in the metallothionein (MT)-bound form, and mRNA expression of the two MT isoforms was increased. Meanwhile, Cu in the MT-bound form (Cu-MT) was depleted in the liver and mRNA expression decreased in comparison with wild-type mice. These results suggest that Cu is not secreted by intestinal microvillus cells into bloodstream due to the defect of Atp7a, and systemic depletion of Cu occurred. On the other hand, in the kidneys of mutant mice, Cu accumulated in the MT-bound form despite the fact that mRNA expression of the two MT isoforms was low. Part of Cu-MT in microvillus cells may be released into bloodstream at turnover and be preferably taken up by the kidneys. Consequently, the mRNA expression of MT isoforms was not always coincident with the amounts of MT proteins binding Cu, and narrow bore HPLC-ICP-MS used for MT protein determination is a complementary technique to real-time RT-PCR used for MT mRNA determination in Cu speciation.

Unknown selenometabolites in animal and plant samples are identified by techniques such as inductively coupled plasma-mass spectroscopy (ICP-MS), electrospray ionization-tandem mass spectrometry (ESI-MS-MS), and nuclear magnetic resonance (NMR). The studies have found that major urinary selenometabolites in humans are the same as the selenosugar detected in rat urine, while the extraction efficiency is important to detect selenometabolites in plants. The enantiomeric separation of naturally occurring selenoamino acids consist of the L enantiomer of selenoamino acids. It is also reported that some selenometabolites are detected in liver by HPLC-ICP-MS whereas the most abundant hepatic selenometabolite is identified by ESI-MS-MS without the sue of NMR.

Heteroisotope and heteroatom tagging with [S-34]-enriched methionine (Met), selenomethionine (SeMet), and telluromethionine (TeMet) was applied to in vitro translation. Green fluorescent protein (GFP) and JNK stimulatory phosphatase-1 (JSP-1) genes were translated with wheat germ extract (WGE) in the presence of Met derivatives. GFPs containing Met derivatives were subjected to HPLC coupled with treble detection, i.e., a photodiode array detector, a fluorescence detector, and an inductively coupled plasma mass spectrometer (ICP-MS). The activities of JSP-1-containing Met derivatives were also measured. GFP and JSP-1 containing [S-34]-Met and SeMet showed comparable fluorescence intensities and enzyme activities to those containing naturally occurring Met. TeMet was unstable and decomposed in WGE, whereas SeMet was stable throughout the experimental period. Thus, although Te was the most sensitive to ICP-MS detection among S, Se, and Te, TeMet was less incorporated into the proteins than Met and SeMet. Finally, the potential of heteroisotope and heteroatom tagging of desired proteins in in vitro translation followed by ICP-MS detection was discussed.

A two-dimensional (2D) micro HPLC-inductively coupled plasma mass spectrometry (ICP-MS) system consisting of a gel filtration column and an anion-exchange column was constructed to separate two metallothionein (MT) isoforms. MT-I and MT-II genes were specifically knocked down with the RNA interference (RNAi) technique, and the effects of the knockdown were evaluated by the reverse transcription-polymerase chain reaction (RT-PCR) and the 2D micro HPLC-ICP-MS system. The minimum cell number required by our 2D micro HPLC-ICP-MS system was calculated to be 2.0 x 10(3), and the detection limit of MT protein was estimated to be 22.2 mg L-1.

Tellurium (Te) is one of the important metalloids used in industry, and is ubiquitously found in the environment. However, the biological and toxicological effects of Te are still unclear despite its being recognized as a hazardous element. In this study, we attempted to identify urinary Te metabolites (UTMs) in rats by HPLC-ICP-MS and electrospray ionization (ESI)-MS. To unambiguously identify UTMs, two different chromatographic mechanisms, i.e., multi-mode gel filtration and cation exchange columns, were employed. The major UTM detected after ingestion of tellurite was trimethyltelluronium, and no urinary sugar metabolites containing Te were detected despite the fact that the major urinary selenometabolite was a selenosugar (methyl-2-acetamido-2-deoxy-1-seleno-beta-D-galactopyranoside). Interestingly, the ingestion of tellurite enhanced the excretion of selenometabolites in urine. These results suggest that Te is discretely metabolized from selenium (Se), an essential element belonging to the same group, although it affects the metabolism of Se in rats. Thus, the disturbance of Se metabolism, i.e., the induction of Se defficiency, may be one of the potential toxic effects of Te.

Sulfur (S) and selenium (Se) are same-group elements that share, in part, a common metabolic pathway. Because a radish variant having pungent flavor (Raphanus sativus L. cv. `Yukibijin') has a unique S metabolic pathway, we expected that it would also produce unique Se compounds through that metabolic pathway. Se metabolites in the radish were assigned by speciation techniques, i. e., HPLC-inductively coupled plasma mass spectrometry (ICP-MS) and HPLC-electrospray ionization tandem mass spectrometry (ESI-MS-MS). 68.5% of total Se in the selenized Japanese pungent radish was water-extractable, and the water extract contained selenate (around 45% of total Se), Se-methylselenocysteine (around 11%), an unknown Se compound tentatively named KD-1 (around 5%), and others (around 1.2%). KD-1 was assigned by ESI-MS-MS as 4,4'-selenobis[2-aminobutanoic acid] or Se-(3-amino-3-carboxypropyl)homocysteine, commonly known as selenohomolanthionine (SeHLan). This Se compound was first identified in nature. The metabolic pathway of SeHLan was suggested to involve the phosphorylation of homoserine to produce O-phosphohomoserine, and the subsequent reaction of O-phosphohomoserine with selenohomocysteine (SeHCys).