小椋 康光

Objective: To evaluate the concentrations of copper and zinc in the breast milk of mothers undergoing treatment for Wilson's disease (WD) and clarify whether they can safely breast feed their infants. Design: This was an observational and prospective study in an individual-based case series. Setting: Breast milk samples were collected from participants across Japan from 2007 to 2018 at the Department of Pediatrics, Teikyo University in Tokyo. This was a primary-care level study. Clinical data were collected from the participants' physicians. Patients: Eighteen Japanese mothers with WD who were treated with trientine, penicillamine or zinc, and 25 healthy mothers as controls, were enrolled. Main outcome measures: Whey exacted from the milk was used to evaluate the distribution of copper by high-performance liquid chromatography-inductively coupled plasma mass spectrometry. Copper and zinc concentrations in the breast milk samples were analysed by atomic absorption spectrometry. Results: Copper distribution was normal in the breast milk of mothers with WD treated with trientine, penicillamine or zinc. No peak was detected for trientine-bound or penicillamine-bound copper. The mean copper concentrations in the mature breast milk of patients treated with trientine, penicillamine and zinc were 29.6, 26 and 38 µg/dL, respectively, and were within the normal range compared with the value in healthy controls (33 µg/dL). Likewise, mean zinc concentrations were normal in the mature breast milk of patients treated with trientine and penicillamine (153 and 134 µg/dL, respectively vs 160 µg/dL in healthy controls). Zinc concentrations in the breast milk of mothers treated with zinc were significantly higher than those in control milk. All infants were born normally, breast fed by mothers undergoing treatment and exhibited normal development. Conclusions: Our results suggest that mothers with WD can safely breast feed their infants, even if they are receiving treatment for WD.

日本においては、かつて薬学科あるいは薬学部に裁判化学講座があり、法医解剖事例の薬物検査を実施していた時期があったが、現在は法医学教室と同様に衰退し、法医学における薬物検査の実施状況は諸外国に比べ手薄となっている。法医解剖において薬物検査は死因判定上重要な位置を占め、また傷害や殺人未遂などの事件では、生体の薬物検査も必要とされるので、現状を放置することは問題であろう。一方で、薬物検査を警察の科学捜査研究所に依存してしまっては、医学診断上の妥当性や客観性を維持することは困難であると考えられる。今後は、薬学部出身者などの薬物分析の専門家が捜査の都合などに左右されず、死因や傷害度を判定する検案医、法医専門医などと密接に連携して薬物検査を実施できる体制を整備したうえで、検出法や致死濃度が未知である新規薬物による死亡や傷害事例に対応するために研究を行ったり、後継者を育成するために大学薬学部と連携をはかるべきである。(著者抄録)

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-β-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.

Selenium (Se) shows biologically ambivalent characteristics in animals. It is an essential element but becomes severely toxic when the amount ingested exceeds the adequate intake level. Animals must be able to metabolize the various selenocompounds in meat, fish and vegetables to utilize Se for selenoprotein synthesis. It is known that the biological, nutritional, and toxicological effects of Se are strongly dependent on its chemical form. First, we evaluated the nutritional availability of nine naturally occurring Se compounds, or the so-called bioselenocompounds, in vivo. Second, we evaluated that gut microflora might contributes to the Se nutritional availability. Se is mainly excreted into urine. However, a substantial amount of Se was secreted into bile although Se was hardly detected in feces. Third, we evaluated the biological significance of biliary secretion of Se in terms of mineral nutrition. Finally, we discussed the entire Se metabolism in gut contributing to Se homeostasis in animal.

Laser ablation-inductively coupled plasma mass spectrometry (LA-ICP-MS) is capable of metal imaging by acquiring local spatial information. However, the preparation of an appropriate standard for quantitative analysis is difficult because the matrices between the standard and the sample should match, and homogeneity of metal concentration in the standard is required. Hence, the aim of this study was to establish a highly quantitative mercury imaging method that utilizes LA-ICP-MS and an appropriate mercury standard consisting of rat tissue. Our standard showed homogeneous mercury concentration and good linearity between concentration and signal intensity, and met the qualifications for quantitative imaging by LA-ICP-MS. Mercury concentration in MeHg-exposed rat kidneys obtained by LA-ICP-MS measurement of the standard (7.84 ± 0.57 µg/g) was comparable to that obtained by cold vapor atomic absorption spectrophotometry (AAS, 7.27 ± 0.46 µg/g). The results indicate that LA-ICP-MS enabled quantitative imaging with the appropriate standard.

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.

Fibulin-4 is a matricellular protein required for extracellular matrix (ECM) assembly. Mice deficient in fibulin-4 (Fbln4-/- ) have disrupted collagen and elastin fibers and die shortly after birth from aortic and diaphragmatic rupture. The function of fibulin-4 in ECM assembly, however, remains elusive. Here, we show that fibulin-4 is required for the activity of lysyl oxidase (LOX), a copper-containing enzyme that catalyzes the covalent cross-linking of elastin and collagen. LOX produced by Fbln4-/- cells had lower activity than LOX produced by wild-type cells due to the absence of lysine tyrosyl quinone (LTQ), a unique cofactor required for LOX activity. Our studies showed that fibulin-4 is required for copper ion transfer from the copper transporter ATP7A to LOX in the trans-Golgi network (TGN), which is a necessary step for LTQ formation. These results uncover a pivotal role for fibulin-4 in the activation of LOX and, hence, in ECM assembly.

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.

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.

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, 1 beta-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.

Numerous zinc ectoenzymes are metalated by zinc and activated in the compartments of the early secretory pathway before reaching their destination. Zn transporter (ZNT) proteins located in these compartments are essential for ectoenzyme activation. We have previously reported that ZNT proteins, specifically ZNT5?ZNT6 heterodimers and ZNT7 homodimers, play critical roles in the activation of zinc ectoenzymes, such as alkaline phosphatases (ALPs), by mobilizing cytosolic zinc into these compartments. However, this process remains incompletely understood. Here, using genetically-engineered chicken DT40 cells, we first determined that Zrt/Irt-like protein (ZIP) transporters that are localized to the compartments of the early secretory pathway play only a minor role in the ALP activation process. These transporters included ZIP7, ZIP9, and ZIP13, performing pivotal functions in maintaining cellular homeostasis by effluxing zinc out of the compartments. Next, using purified ALP proteins, we showed that zinc metalation on ALP produced in DT40 cells lacking ZNT5?ZNT6 heterodimers and ZNT7 homodimers is impaired. Finally, by genetically disrupting both ZNT5 and ZNT7 in human HAP1 cells, we directly demonstrated that the tissue-nonspecific ALP-activating functions of both ZNT complexes are conserved in human cells. Furthermore, using mutant HAP1 cells, we uncovered a previously-unrecognized and unique spatial regulation of ZNT5?ZNT6 heterodimer formation, wherein ZNT5 recruits ZNT6 to the Golgi apparatus to form the heterodimeric complex. These findings fill in major gaps in our understanding of the molecular mechanisms underlying zinc ectoenzyme activation in the compartments of the early secretory pathway.

Although selenium (Se) is mainly excreted in urine, it has been reported that an unknown Se metabolite is excreted in bile. When we administered selenomethionine (SeMet), selenocyanate or selenite to rats, a common biliary selenometabolite was detected 10 min after administration. The amount of the selenometabolite originating from SeMet was less than that originating from the two inorganic Se compounds, selenocyanate and selenite, suggesting that the transformation from the methylated organic selenocompound, i.e., SeMet, was less efficient than that from the inorganic Se compounds. The common biliary selenometabolite was concretely identified as selenodiglutathione (GSSeSG) by two types of mass spectrometry, i.e., LC-inductively coupled mass spectrometry (ICP-MS) and LC-ESI-Q/TOF. The bile-drained rats had lower urinary Se levels than the sham-operated rats. In addition, the Se amounts in urine plus bile of the bile-drained rats were comparable to the Se amount in the urine of the sham-operated rats. These results suggest that the biliary selenometabolite, GSSeSG, was reabsorbed in the gut and finally excreted in urine. Enterohepatic circulation occurs to maintain Se status in the body.

We discovered previously that Formosan squirrels (Callosciurus erythraeus) accumulate copper (Cu) in their livers at levels averaging 1700 mu g per dry g (approximately 420 mu g per wet g). In the current study, we investigated the relationship between Cu accumulation and hepatic injury, and we determined the distribution and chemical form of Cu in the liver supernatant. In particular, we explored the role of metallothionein in the liver supernatant. We observed no significant differences in hepatic Cu concentration between squirrels that showed pathological changes in the liver and those that did not. Serum alanine aminotransferase activity did not increase with increasing hepatic Cu concentration. These results suggest that abnormal Cu accumulation in the livers of Formosan squirrels does not induce severe hepatic injury. We found that 26.7% of the Cu in the liver was distributed to the supernatant, and only 11.0% of the Cu in the liver was bound to metallothionein, suggesting that metallothionein in the hepatic supernatant does not contribute to detoxification of excess Cu in Formosan squirrels.

It is known that copper (Cu) is highly accumulated in several organs in the perinatal period, suggesting changes in Cu metabolism with development, although the precise mechanisms are still unclear. To elucidate the mechanisms underlying Cu accumulation in the organs of neonatal rats, we performed speciation analysis using high-performance liquid chromatography hyphenated to inductively coupled plasma mass spectrometry. In the neonatal rat liver immediately after birth, the Cu concentration was elevated 10-fold compared to that in the juvenile rat liver. Most of the accumulated Cu was bound to metallothionein, although Cu in Cu, zinc-superoxide dismutase (SOD) was reduced. Contrary to the hepatic Cu accumulation, the serum Cu concentrations in the neonatal rats were low due to the decreased amount of Cu bound to ceruloplasmin. The mRNA expression of antioxidant protein 1 (Atox1), a Cu chaperone that transports Cu to Atp7b, remained low up to two weeks after birth. These results suggest that Cu accumulation in the neonatal rat liver is caused by the low expression of Atox1, and the accumulation is useful to distribute Cu to Cu-containing anti-oxidative enzymes (e.g., SOD and Atox1) after respiration starts.

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.

An interaction between the Rad17-RFC2-5 and 9-1-1 complexes is essential for ATR-Chk1 signaling, which is one of the major DNA damage checkpoints. Recently, we showed that the polyanionic C-terminal tail of human Rad17 and the embedded conserved sequence iVERGE are important for the interaction with 9-1-1 complex. Here, we show that Rad17-S667 in the C-terminal tail is constitutively phosphorylated in vivo in a casein kinase 2-dependent manner, and the phosphorylation is important for 9-1-1 interaction. The serine phosphorylation of Rad17 could be seen in the absence of exogenous genotoxic stress, and was mostly abolished by S667A substitution. Rad17-S667 was also phosphorylated when the C-terminal tail was fused with EGFP, but the phosphorylation was inhibited by two casein kinase 2 inhibitors. Furthermore, interaction between Rad17 and the 9-1-1 complex was inhibited by the casein kinase 2 inhibitor CX-4945/Silmitasertib, and the effect was dependent on the Rad17-S667 residue, indicating that S667 phosphorylation is the only role of casein kinase 2 in the 9-1-1 interaction. Our data raise the possibility that the C-terminal tail of vertebrate Rad17 regulates ATR-Chk1 signaling through multi-site phosphorylation in the iVERGE.

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-β-d-galactopyranoside (SeSug1, selenosugar 1), methyl-2-acetamido-2-deoxy-1-seleno-β-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.

Selenium (Se) is an essential element in animals but becomes severely toxic when the amount ingested exceeds the adequate intake level. It is known that the toxicological effects of Se are highly dependent on its chemical form. In this study, we evaluated the mutagenicity of nine naturally occurring Se compounds or the so-called bioselenocompounds, including selenite, selenate, selenocyanate, selenomethionine, selenocystine, Se-methylselenocysteine, selenohomolanthionine, N-acetylgalactosamine-type selenosugar, and trimethylselenonium ion, by using the Ames test. Salmonella typhimurium TA98, TA100, and TA1535 were used for the mutagenicity evaluation in the presence or absence of S9 mix, a metabolic activator. Only selenate showed weak mutagenicity even in the absence of S9 mix. None of the bioselenocompounds except selenate exhibited mutagenicity in all the strains tested in the presence or absence of S9 mix. Selenomethionine and selenocystine reduced the number of colonies in all the strains although no other selenoamino acids exerted the same effect. These results indicate that selenate directly or indirectly injures genome. Among the bioselenocompounds tested, selenomethionine and selenocystine show antibacterial activity, but the mechanism is unclear.

The nutritional availability of selenium (Se) is highly dependent on its chemical form because chemical form affects absorption, distribution, metabolism, and excretion. We evaluated the effects of administration route and dose on the bioavailability of nine Se compounds found in biota, the so-called bioselenocompounds, such as selenite, selenate, selenocyanate (SeCN), Se-methylselenocysteine (MeSeCys), selenomethionine (SeMet), sele-nohomolanthionine (SeHLan), selenocystine (SeCys2), 1 beta-methylseleno-N-acetyl-o-galactosamine (SeSugi), and trimethylselenonium ion (TMSe). We determined the bioavailability of bioselenocompounds recovered as urinary selenometabolites and serum selenoproteins from urine and serum of Se-deficient rats after the administration of bioselenocompounds by speciation analysis. Urinary Se was more easily recovered than serum selenoproteins, suggesting that the speciation of urinary Se is a better tool to indicate Se status in the body. The intravenous administration of bioselenocompounds showed different Se bioavailability from the oral administration. Intestinal microflora might be involved in the bioavailability of some bioselenocompounds, such as SeCN, MeSeCys, and SeSug1.

Selenium (Se) is an interesting element for bioanalytical chemists. Se forms Se-containing compounds having Se-carbon covalent bond(s), i.e., selenometabolites in its metabolic pathway. In this chapter, the analytical techniques for the speciation and identification of unique selenometabolites in animals are highlighted. First, the instruments required for analyses are overviewed. In particular, hyphenated techniques consisting of high-performance liquid chromatography and inductively coupled plasma (tandem) mass spectrometry (ICP-MS) or electrospray ionization (tandem) mass spectrometry are focused on. Second, laser ablation hyphenated with ICP-MS for Se imaging is briefly overviewed. Then, advanced techniques of nuclear magnetic resonance (NMR) spectroscopy for Se analysis are mentioned with an application to a biological sample.

© 2017 Elsevier Ltd 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 146 ng/g.

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 degrees 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. (c) 2017 Elsevier B.V. All rights reserved.

Selenium (Se) shows biologically ambivalent characteristics in animals. It is an essential element but becomes severely toxic when the amount ingested exceeds the adequate intake level. Its biological, nutritional, and toxicological effects are strongly dependent on its chemical form. In this study, we evaluated the toxicity and bioavailability of nine naturally occurring Se compounds, or the so-called bioselenocompounds, in vivo and in vitro. Selenite and selenocystine showed higher toxicity than the other bioselenocompounds in vitro. In an in vitro membrane permeability study using Caco-2 cells, selenomethionine and Se-methylselenocysteine were more efficiently transported than the other bioselenocompounds. The effect of bioselenocompounds on nutritional availability was quantitatively determined from the recovery of serum selenoproteins in Se-deficient rats by speciation analysis. In contrast to the in vitro study, there were no significant differences in the assimilation of Se into serum selenoproteins among the bioselenocompounds, including selenoamino acids, selenosugar, and inorganic Se species, such as selenite, selenate, and selenocyanate, except trimethylselenonium ion. These results indicate that animals can equally assimilate both inorganic and organic naturally occurring selenocompounds except trimethylselenonium ion, which is the urinary metabolite of excess Se. We confirmed that the bioselenocompounds except trimethylselenonium ion had equivalent nutritional availabilities.

© 2017 American Chemical Society. Chemotherapy for ovarian cancer often causes severe side effects. As candidates for combretastatin A4 (CA4) prodrug for ovarian cancer prodrug monotherapy (PMT), we designed and synthesized two β-galactose-conjugated CA4s (CA4-βGals), CA4-βGal-1 and CA4-βGal-2. CA4 was liberated from CA4-βGals by β-galactosidase, an enzyme more strongly expressed in ovarian cancer cells than normal cells. CA4-βGal-2, which has a self-immolative benzyl linker between CA4 and the β-galactose moiety, was more cytotoxic to ovarian cancer cell lines than CA4-βGal-1 without a linker. Therefore, CA4-βGal-2 can serve as a platform for the design and manufacture of prodrugs for ovarian cancer PMT.

Nuclear magnetic resonance (NMR) spectroscopy is widely used for the determination of the chemical structure of an organic compound. NMR spectroscopy is theoretically applicable to all metallic elements except cerium. In this chapter, we demonstrate selenium (Se) speciation in animal Se metabolites (selenometabolites) by NMR spectroscopy and review hitherto performed NMR spectroscopic studies of Se detection in biological samples. 77Se, an NMR-active isotope of Se, was directly observed, but its NMR receptivity was lower than its sensitivity in mass spectrometry. However, the use of enriched stable isotope improved the receptivity. Each selenometabolite had its own chemical shift, suggesting that the chemical shift of 77Se could be used as a fingerprint. Indirect measurement by heteronuclear multiple bond correlation (HMBC) spectroscopy with 1H nuclides was also effective for the Se speciation.

Selenium (Se) and tellurium (Te) are chalcogen elements belonging to group 16 in the periodic table. These elements have unique physical, chemical, and biological properties. Se and Te form Se- or Te-containing compounds having Seor Te-carbon covalent bond(s), i.e., selenometabolites or tellurometabolites, respectively, in their metabolic pathways. In this chapter, the speciation and identification of selenometabolites and tellurometabolites in animal and plant samples are highlighted. First, the instruments required for analyses are overviewed. In particular, hyphenated techniques consisting of high-performance liquid chromatography and inductively coupled plasma mass spectrometry or electrospray ionization (tandem) mass spectrometry are focused on. Then, newly identified metabolites are introduced. The identification of selenosugars in urine, selenohomolanthionine in Se-accumulating plants, selenoneine in marine animals, trimethyltelluronium ion in urine, Te-methyltellurocysteine in Se-accumulating plants, and selenocyanate in cultured cells is discussed.

This book focuses on recent topics in metallomics, a study of the metallome, or metal-containing biomolecules. Metals can induce various physiological and toxicological effects in a very small amounts, in other words, the concentrations of biometals are very low in organisms. Thus, analytical techniques for a trace amount of metal are crucial to understand the biological and toxicological functions of metals.This volume begins with an overview of metallomics including the history and development of the field. Subsequent parts provide basic and advanced techniques for metallomics. Speciation and imaging of metals are basic approaches to reveal the function of the metallome. The applications of speciation using an HPLC hyphenated with inductively coupled plasma mass spectrometry (LC-ICP-MS) and flow cytometry ICP-MS are described. As advanced approaches, the applications using a micro-flow injection-ICP-MS, an ICP-triple quadrupole mass spectrometer, an ICP-sector field mass spectrometer, and an ICP-multi-collector mass spectrometer are mentioned. For the imaging of metals, basic principles and applications of several techniques such as scanning X-ray fluorescence microscopy and ICP-MS equipped with laser ablation (LA-ICP-MS) are presented. Speciation analyses using electrospray ionization mass spectrometry (ESI-MS), X-ray Absorption Spectroscopy (XAS), and nuclear magnetic resonance spectroscopy (NMR) are also introduced. The last part highlights the medical and pharmaceutical applications of metallomics. Molecular biological approaches to reveal the effects of toxic metals, metal functions in brain and neurodegenerative diseases, and metallodrugs are explained. The topic of metal transporters is also presented.

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. gamma-Glutamyl-Se-methylselenocysteine (gamma-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.

Antimony cytotoxicity was assessed in human embryonic kidney cells (HEK-293). Uptake, mitochondrial respiratory activity, ROS generation and diffusional kinetics were measured using fluorescence recovery after photobleaching (FRAP). Furthermore, the toxic effect induced by Sb was compared with As toxicity in regard to ROS generation and diffusional kinetics, which provides information on the protein aggregation process. Our results show a favored uptake of Sb(III) and a more severe effect, decreasing the mitochondrial activity more than in the presence of Sb(V). In comparison with As, the Sb species did not generate a significant increase in ROS generation, which was observed with As(III) and As(V). FRAP analysis yielded important information on the diffusion and binding dynamics of live cells in presence of these metalloids. The mobile fraction showed a strong decrease with the As species and Sb(III). The diffusion rate and the k(off-rate) were significantly decreased for the As and Sb species but were more strong in the presence of As(III).

It is suspected that some neurodegenerative diseases are a result of the disturbance of copper (Cu) homeostasis, although it remains unclear whether the disturbance of Cu homeostasis has aberrant effects on neurons. Herein, we investigated Cu metabolism specifically in neurons in terms of changes in the intracellular Cu concentration and the expression of Cu-regulating genes, such as Cu transporters and metallothioneins (MTs), before and after the differentiation of rat pheochromocytoma cells (PC12 cells) into neurons. After the differentiation, Cu and Zn imaging with fluorescent probes revealed an increase in intracellular Cu concentration. The concentrations of other essential metals, which were determined by an inductively coupled plasma mass spectrometer, were not altered. The mRNA expression of the Cu influx transporter, Ctr1, was decreased after the differentiation, and the differentiated cells acquired tolerance to Cu and cisplatin, another substrate of Ctr1. In addition, the expression of MT-3, a brain-specific isoform, was increased, contrary to the decreased expression of MT-1 and MT-2. Taken together, the differentiation of PC12 cells into neurons induced MT-3 expression, thereby resulting in intracellular Cu accumulation. The decrease in Ctr1 expression was assumed to be a response aimed at abolishing the physiological accumulation of Cu after the differentiation.

We aimed to establish an element array analysis that involves the simultaneous detection of all elements in cells and the display of changes in element concentration before and after a cellular event. In this study, we demonstrated changes in element concentration during the differentiation of 3T3-L1 mouse fibroblasts into adipocytes. This metallomics approach yielded unique information of cellular response to physiological and toxicological events.

セレン（Se）やテルル（Te）は硫黄と同じ第16族元素であり、類似した化学的性質を示す。生体必須元素であるSeは、化学形態によって生理活性や毒性が異なることが知られているが、非必須元素であるTeについて化学形態に着目した情報は少ない。最近我々の研究グループは、Se代謝能が高く様々なSe含有アミノ酸やその誘導体を産生するニンニクに無機Te化合物のテルル酸を曝露すると、Te含有アミノ酸を含む複数のTe代謝物が産生されることを報告した。本研究では、植物が産生するTe代謝物の動物への生体影響を評価するために、テルル酸を曝露したニンニクの葉をラットに投与し、Teの体内挙動および毒性を解析した。<br> 雄性Wistar系ラットにテルル酸を曝露したニンニクの葉懸濁液（garlic Te群）、比較としてテルル酸（tellurate群）を0.1 mg Te/kg体重/日となるよう7日間経口投与した。対照群には精製水を投与した。7日間の尿と、解剖して得られた臓器および血中のTe濃度をICP-MSで分析した。また、ALT, AST, BUNなど10項目の生化学パラメータを測定した。<br> Te濃度測定の結果、Teを投与した両群のラットにおいて腎臓で最も高く、摂取した化学形態に依らずTeは腎臓に集積しやすいことが示唆された。Garlic Te群とtellurate 群を比較すると、臓器および血液中のTe濃度はgarlic Te群で2倍以上の高値を示し、特に全血では約10倍と顕著であった。一方、尿中Te排泄量はtellrurate群で有意に多かった。血清中の生化学パラメータ測定の結果、Teを投与した両群において、いずれのパラメータも対照群との間に有意な差は見られなかった。以上より、植物Te代謝物は赤血球に取り込まれることにより生体内に保持されやすいと示唆され、従って、長期的な摂取による毒性影響発現の可能性が懸念される。

When human hepatoma HepG2 cells were exposed to sodium selenite, an unknown selenium metabolite was detected in the cytosolic fraction by HPLC-inductively coupled plasma mass spectrometry (ICP-MS). The unknown selenium metabolite was also detected in the mixture of HepG2 homogenate and sodium selenite in the presence of exogenous glutathione (GSH). The unknown selenium metabolite was identified as selenocyanate by electrospray ionization mass spectrometry (ESI-MS) and ESI quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS). Because exogenous cyanide increased the amount of selenocyanate in the mixture, selenocyanate seemed to be formed by the reaction between selenide or its equivalent, the product of the reduction of selenite, and endogenous cyanide. Rhodanase, an enzyme involved in thiocyanate synthesis, was not required for the formation of selenocyanate. Selenocyanate was less toxic to HepG2 cells than selenite or cyanide, suggesting that it was formed to reduce the toxicity of selenite. However, selenocyanate could be assimilated into selenoproteins and selenometabolites in rats in the same manner as selenite. Consequently, selenite was metabolized to selenocyanate to temporarily ameliorate its toxicity, and selenocyanate acted as an intrinsic selenium pool in cultured cells exposed to surplus selenite.

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.

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.

As selenium (Se) is a nonmetal, it is transformed into Se-containing compounds having carbon-Se covalent bond(s), i.e., selenometabolites, in the metabolic pathway. Thus, it is necessary to identify selenometabolites to completely reveal the metabolic pathway of Se and understand the physiological and toxicological effects of selenometabolites. However, as Se is an essential trace element, selenometabolites exist in extremely small amounts in animals. The difficulty of detecting and identifying selenometabolites has been overcome with the emergence of several mass spectrometers, such as an inductively coupled plasma mass spectrometer and an electrospray tandem mass spectrometer. In this chapter, mass-spectrometric techniques for the identification of selenometabolites in animal and plants are discussed.

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.

Selenohomolanthionine (SeHLan) is the common name of 4,4′-selenobis[2-aminobutanoic acid] or Se-(3-amino-3-carboxypropyl)-homocysteine. SeHLan was first identified in an extract of Japanese pungent radish (Raphanus sativus L. cv. "Yukibijin") fortified with selenate, an inorganic form of selenium (Se). Thereafter, its derivative, 2,3-dihydroxy-propionyl-selenohomolanthionine (2,3-DHP-SeHLan), was identified in selenized yeast extract. SeHLan is less toxic than inorganic Se, and the tissue distribution of SeHLan differs from that of selenomethionine (SeMet) in experimental animals. In addition, it was reported that SeHLan exhibited an antiseptic effect in mouse. In this chapter, the metabolism of SeHLan in plants and animals, and the toxicological and pharmacological effects of SeHLan are explained.

テルル（Te）は、相変化型DVDの記録層を形成する中心素材などとして使用されるレアメタルである。相変化型DVD（いわゆるDVD-RAM等）は、広く我々の生活圏に浸透し、製品の劣化とともに環境中にTeが流出する可能性が考えられる。<br> Teは典型元素と金属元素の物理化学的性質を併有したいわゆる類金属であり、生体内では炭素とTeが直接共有結合した有機金属化合物として代謝物が生成することが想定される。従って、代謝物の化学形態を同定することによって、代謝過程を明らかにすることができる。<br> 金属含有代謝物の化学形態分析は、化学形態に応じて分離する手法と金属を特異的に検出する手法とから構成される分析法である。金属含有代謝物の化学形態分析では、分離手段としてHPLCを、検出の手段として、高感度に金属を分析可能な誘導結合質量分析装置（ICP-MS）や、最近ではICP-MSの欠点を補う目的でいわゆるLC-MSが利用されるようになってきた。本研究では、金属含有代謝物の化学形態分析を利用し、Teの生体内及び環境中の動態について解析を行った。<br> 相変化型DVDに由来する無機のTe化合物を、動物に曝露した場合、尿中に排泄されたTe代謝物の99 %以上は唯一の代謝物であり、その化学形はトリメチルテルロニウム（TMTe、(CH₃)₃Te⁺）であると同定できた。同族であるセレンの主たる尿中代謝物はセレノ糖であったが、これに相当するテルロ糖は検出できなかった。DVDからのTeの曝露を想定した場合、直接動物に無機のTe化合物が曝露されるよりも、一旦、無機のTe化合物が植物に取り込まれ、植物のTe代謝物を介して動物に取り込まれることが、動物に対するより現実的な曝露経路と想定される。そこで、無機のTe化合物を植物に曝露し、植物内のTe代謝物の化学形態分析も行ったところ、ある種の植物ではTeはTe含有アミノ酸として代謝されることが明らかとなった。<br> 以上のことを含め、Teの代謝、毒性や環境中の動態について報告する。

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 [Se-77]-labeled selenite and selenomethionine (SeMet) at the nutritional dose of 10 mu g 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.