Cytotoxicity comparison of 35 developmental neurotoxicants in human induced pluripotent stem cells (iPSC), iPSC-derived neural progenitor cells, and transformed cell lines.

The Organization for Economic Co-operation and Development (OECD) test guideline 426 for developmental neurotoxicity (DNT) of industrial/environmental chemicals depends primarily on animal experimentation. This requirement raises various critical issues, such as high cost, long duration, the sacrifice of large numbers of animals, and interspecies differences. This study demonstrates an alternative protocol that is simple, quick, less expensive, and standardized to evaluate DNT of many chemicals using human induced pluripotent stem cells (iPSC) and their differentiation to neural progenitor cells (NPC). Initially, concentration-dependent cytotoxicity of 35 DNT chemicals, including industrial materials, insecticides, and clinical drugs, were compared among iPSC, NPC, and two transformed cells, Cos-7 and HepG2, using tetrazolium dye (MTS)-reducing colorimetric and ATP luciferase assays, and IC50 values were calculated. Next, inhibitory effects of the 14 representative chemicals (mainly insecticides) on iPSC differentiation to NPC were evaluated by measuring altered expression of neural differentiation and undifferentiation marker genes. Results show that both iPSC and NPC were much more sensitive to most DNT chemicals than the transformed cells, and 14 chemicals induced differential patterns of marker gene expression, highlighting the validity and utility of the protocol for evaluation and classification of DNT chemicals and preclinical DNT tests for safety assessment.

Dietary selenium deficiency or selenomethionine excess drastically alters organ selenium contents without altering the expression of most selenoproteins in mice.

Selenium is an essential trace element, and its deficiency can cause cardiomyopathy, arrhythmias and increased susceptibility to infection. Such clinical symptoms are considered primarily attributed to decreased expression of some of the 25 selenocysteine-containing selenoproteins in humans. Conversely, a selenium-excessive diet can cause acute poisoning and chronic symptoms with unknown mechanisms. To reveal the impact of selenium deficiency and excess on selenoprotein expression in vivo, mice (that possess 24 selenoproteins) were fed with selenium-deficient or selenomethionine-excessive diets for up to 4 weeks, and the expression levels of nine representative selenoproteins [glutathione peroxidase (Gpx) 1/2/3/4, thioredoxin reductase 1/2, deiodinase 1, and selenoprotein P/S] were measured in 10 organs (brain, heart, liver, lung, kidney, pancreas, spleen, testis, skeletal muscle and thymus). We observed a time-dependent decrease in the selenium content of most organs (except testis) of selenium-deficient mice but not in the expression levels of the nine selenoproteins, with the exceptions of Gpx1/2 in the heart/liver/kidney/pancreas/spleen and Gpx3 in the pancreas/spleen. Serum lipid peroxidation levels were up-regulated in response to Se deficiency because of the decreased expression/activity of Gpx3, a plasma-type Gpx. In contrast, a time-dependent increase was observed in the selenium content of all organs but not the expression levels of the nine selenoproteins in most organs of selenomethionine-excessive mice; however, markedly elevated protein-bound selenium levels were observed in the liver/kidney. These results suggest that the systemic response to selenium deficiency and selenomethionine excess involves the down-regulation of some selenoproteins such as Gpx1/Gpx3 and up-regulation of selenium-containing proteins (not selenoproteins), respectively.

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

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

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

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

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

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

Comparison of accumulation of four metalloids in Allium sativum.

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

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

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

Cooperative functions of ZnT1, metallothionein and ZnT4 in the cytoplasm are required for full activation of TNAP in the early secretory pathway.

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.

Speciation and identification of tellurium-containing metabolites in garlic, Allium sativum.

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.

Analysis of animal and plant selenometabolites in roots of a selenium accumulator, Brassica rapa var. peruviridis, by speciation.

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-ß-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.

Selenometabolomics explored by speciation.

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.

Comparison of selenoneine found in marine organisms with selenite in the interaction with mercury compounds in vitro.

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.

Distribution and metabolism of selenohomolanthionine labeled with a stable isotope.

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 77Se-labeled SeHLan at a dose of 25 µg/kg body weight in rats. Exogenous 77Se 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.

Comparison of selenohomolanthionine and selenomethionine in terms of selenium distribution and toxicity in rats by bolus administration.

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.

Body distribution of trace elements in black-tailed gulls from Rishiri Island, Japan: age-dependent accumulation and transfer to feathers and eggs.

Body distribution and maternal transfer of 18 trace elements (V, Cr, Mn, Co, Cu, Zn, Se, Rb, Sr, Mo, Ag, Cd, Sb, Cs, Ba, Hg, Tl, and Pb) to eggs were examined in black-tailed gulls (Larus crassirostris), which were culled in Rishiri Island, Hokkaido Prefecture, Japan. Manganese, Cu, Rb, Mo, and Cd showed the highest levels in liver and kidney, Ag, Sb, and Hg in feather, and V, Sr, and Pb in bone. Maternal transfer rates of trace elements ranged from 0.8% (Cd) to as much as 65% (Tl) of maternal body burden. Large amounts of Sr, Ba, and Tl were transferred to the eggs, though maternal transfer rates of V, Cd, Hg, and Pb were substantially low. It also was observed that Rb, Sr, Cd, Cs, and Ba hardly were excreted into feathers. Concentrations of Co in liver, Ba in liver and kidney, and Mo in liver increased significantly with age, whereas Se in bone and kidney, Hg in kidney, and Cr in feather decreased with age in the known-aged black-tailed gulls (2-20 years old). It also was suggested that feathers might be useful to estimate contamination status of trace elements in birds, especially for Hg on a population basis, although the utility is limited on an individual basis for the black-tailed gulls. To our knowledge, this is the first report on the maternal transfer rate of multielements and also on the usefulness of feathers to estimate contamination status of Hg in birds on a population basis.