Yasumitsu Ogra

Selenosugars are key and urinary metabolites for selenium excretion within the required to low-toxic range

Essential micronutrient selenium is excreted into the urine and/or expired after being transformed to methylated metabolites. Monomethylated selenium is excreted into the urine in response to a supply within the required to low-toxic range, whereas tri- and dimethylated selenium increase with excessive supply at a toxic dose. Here we show that the major urinary selenium metabolite within the required to low-toxic range is a selenosugar. The structure of 1β-methylseleno-N-acetyl-d-galactosamine was deduced from the spectroscopic data and confirmed by chemical synthesis. This metabolite was also detected in the liver, and an additional metabolite increased with inhibition of methylation. The latter metabolite was again a selenosugar conjugated with glutathione instead of a methyl group and was assumed to be a precursor for methylation to the former metabolite. A metabolic pathway for the urinary excretion of selenium, i.e., from the glutathione-S-conjugated selenosugar to the methylated one, was proposed. Urinary monomethylated (selenosugar) and trimethylated selenium can be used as specific indices that increase within the required to low-toxic range and with a distinct toxic dose, respectively.

Speciation of arsenic in human nail and hair from arsenic-affected area by HPLC-inductively coupled argon plasma mass spectrometry.

Nail and hair are rich in fibrous proteins, i.e., alpha-keratins that contain abundant cysteine residues (up to 22% in nail and 10-14% in hair). Although they are metabolically dead materials in the epidermis, the roots are highly influenced by the health status of the living beings and their analyses are used as a tool to monitor occupational and environmental exposure to toxic elements. The aims of the present study are to speciate arsenicals in human nail and hair and also to judge whether they should be used as a biomarker to arsenic (As) exposure and/or toxicity. All human fingernail and hair samples (n = 47) were collected from the As-affected area of West Bengal, India. Speciation of arsenicals in water extracts of fingernails and hair at 90 degrees C was carried out by HPLC-inductively coupled argon plasma mass spectrometer (ICP MS). Fingernails contained iAs(III) (58.6%), iAs(V) (21.5), MMA(V) (7.7), DMA(III) (9.2), and DMA(V) (3.0), and hair contained iAs(III) (60.9%), iAs(V) (33.2), MMA(V) (2.2), and DMA(V) (3.6). Fingernails contained DMA(III), but hair did not. The higher percentage of iAs(III) both in fingernails and hair than that of iAs(V) suggests more affinity of iAs(III) to keratin. Although all arsenicals in fingernails and hair correlate to As exposure positively, As speciation in fingernails seems to be more correlated with arsenism than that in hair. Exogenous contamination is a confounding factor for hair to consider it as a biomarker, whereas this is mostly absent in fingernails, which recommends it to be a better biomarker to arsenic exposure. DMA(III) content in fingernails and DMA(V) contents in both fingernails and hair could be the biomarker to As exposure.

Identification of a novel selenium metabolite, Se-methyl-N-acetylselenohexosamine, in rat urine by high-performance liquid chromatography--inductively coupled plasma mass spectrometry and--electrospray ionization tandem mass spectrometry.

The major urinary metabolite of selenium (Se) in rats was identified by HPLC-inductively coupled argon plasma mass spectrometry (ICP-MS) and--electrospray tandem mass spectrometry (ESI-MS/MS). As the urine sample was rich in matrices such as sodium chloride and urea, it was partially purified to meet the requirements for ESI-MS. The group of signals corresponding to the Se isotope ratio was detected in both the positive and negative ion modes at m/z 300 ([M+H]+) and 358 ([M+CH3COO]-) for 80Se, respectively. These results suggested that the molecular mass of the Se metabolite was 299 Da for 80Se. The Se metabolite was deduced to contain one methylselenyl group, one acetyl group and at least two hydroxyl groups from the mass spectra of the fragment ions. The spectrum of the Se metabolite was completely identical to that of the synthetic selenosugar, 2-acetamide-1,2-dideoxy-beta-D-glucopyranosyl methylselenide. However, the chromatographic behavior of the Se metabolite was slightly different from that of the synthetic selenosugar. Thus, the major urinary Se metabolite was assigned as a diastereomer of a selenosugar, Se-methyl-N-acetyl-selenohexosamine.

Heavy metal tolerance of transgenic tobacco plants over-expressing cysteine synthase.

Cysteine synthase [O-acetyl-l-serine(thiol)lyase] catalyzes the final step for l-cysteine biosynthesis in plants. The tolerance of transgenic tobacco plants over-expressing cysteine synthase cDNA in cytosol (3F), chloroplasts (4F) and in both organelles (F1) was investigated towards heavy metals such as Cd, Se, Ni, Pb and Cu. The transgenic plants were significantly more tolerant than wild-type plants in agar medium containing Cd, Se and Ni. The F1 transgenic plants had a higher resistance than other transgenic lines towards these metals and could enhance accumulation of Cd in shoot. These results suggest that the transgenic plants over-expressing cysteine synthase both in cytosol and chloroplasts can be applicable to phyto-remediation of Cd from contaminated soils.

Speciation of arsenic in body fluids

Inorganic arsenic is metabolized by consecutive reduction and methylation reactions to dimethylated arsenic (DMA), and then excreted into the urine mostly in the form of DMA. Therefore, arsenic metabolites in the body fluids and organs/tissues are present in the form of inorganic (arsenite and arsenate) and methylated arsenics (MMA and DMA). Although pentavalent arsenics can be present mostly in the form of free ions, trivalent ones may be present more in the forms conjugated with thiol groups of glutathione (GSH) or proteins. Arsenic in the body fluids (plasma, bile and urine) is present in the soluble forms and can be speciated on ion exchange columns by HPLC with on-line detection by an inductively coupled argon plasma-mass spectrometer (ICP-MS). Free forms of arsenite, arsenate, and monomethylarsonous, monomethylarsonic, dimethylarsinous and dimethylarsinic acids in the body fluids have been demonstrated to be speciated simultaneously within 10 min or so on both anion and cation exchange columns together with arsenobetaine (AsB) and arsenocholine (AsC). Trivalent arsenics conjugated with GSH were eluted in intact forms on an anion exchange column but were liberated into free forms on a cation exchange column. Thus, free and GSH-conjugated arsenic metabolites in the bile and urine have been speciated simultaneously on ion exchange columns by HPLC-ICP-MS.

Roles of metallothionein in copper homeostasis: responses to Cu-deficient diets in mice

Metallothionein (MT) protects the body from both harmful non-essential and excessive essential metals. Copper (Cu) is an essential metal, and its concentration in the body is regulated at a constant level between excess and deficient ones. Cu accumulating in the livers of Wilson disease patients and its animal model, Long-Evans rats with a cinnamon-like coat color (LEC) rats, is in the form of Cu,Zn-MT, MT being an antioxidant. Contrary to the efficient production of MT in response to excessive accumulation of Cu in LEC rats, Cu-binding to MT only occurs marginally under normal conditions. However, the present study revealed that Cu binds to MT more with a severe Cu-deficiency. Namely, male C57BL/6J mice were fed a Cu-deficient diet (0.037 mg Cu/g) and deionized water containing trientine, and then the concentration and distribution of Cu were determined. It was suggested that the cessation of biliary excretion and limitation of the Cu supply to ceruloplasmin are the first responses on feeding of a Cu-deficient diet, followed by an increase in Cu-MT with maintenance of the Cu concentration in the liver. These results suggest that MT causes the recruitment of Cu in a Cu-deficient environment by sequestering Cu from degraded Cu-enzymes and delivering it to Cu chaperones.

Evidence for toxicity differences between inorganic arsenite and thioarsenicals in human bladder cancer cells.

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 microM) was much less cytotoxic than DMMTA(V) (LC(50)=16.7 microM) 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.

Mitochondria are the main target organelle for trivalent monomethylarsonous acid (MMA(III))-induced cytotoxicity.

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 dimethylarsinous acid (DMA(III))]. MMA(III) (IC(50) = 1 μM) was found to be the most toxic form, followed by DMA(III) (IC(50) = 2 μM) and iAs(III) (IC(50) = 18 μM). 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 iAs(III), 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.

Speciation and metabolism of selenium injected with 82Se-enriched selenite and selenate in rats

Selenate and selenite injected intravenously into rats were speciated by the HPLC-ICP MS method with use of an enriched stable isotope as the tracer. In dose-relation experiments, 82Se-enriched selenate or selenite was injected intravenously into male Wistar rats of 8 weeks of age (three rats/group) at single doses of 10, 25, 50, 100 and 200 microg/kg body weight for the selenate group, and 2, 5, 10, 25 and 50 microg/kg body weight for the selenite group. The animals were sacrificed 1 or 24 h later, and the concentrations and distributions of 82Se in the liver, kidneys, serum, and urine remaining in the bladder or 24-h urine were determined. In time-course experiments, 82Se-enriched selenate and selenite were injected at doses of 50 and 10 microg/kg body weight, respectively, and the animals were sacrificed 5, 15, 30, 60 and 180 min later. It was suggested that selenate is directly taken up by the liver with an efficiency of approximately 1/2 compared with selenite, the latter being taken up by the liver after being metabolized to selenide in red blood cells. Although selenate and selenite were metabolized differently in the bloodstream, and also a part of only selenate was excreted directly into the urine, the 82Se taken up by the liver was shown to be metabolized in a manner indistinguishable between selenate and selenite. 82Se of selenite origin but not of selenate origin was suggested to undergo redox reaction in the bloodstream. These results suggest that although parenteral selenate is utilized less efficiently by the body, it is utilized in the liver in a similar manner to selenite much more safely.

Selenometabolomics: Identification of selenometabolites and specification of their biological significance by complementary use of elemental and molecular mass spectrometry

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.