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Ferroptosis is a form of regulated cell death induced by iron-dependent accumulation of lipid hydroperoxides. Selenoprotein glutathione peroxidase 4 (GPX4) suppresses ferroptosis by detoxifying lipid hydroperoxides via a catalytic selenocysteine (Sec) residue. Sec, the genetically encoded 21 st amino acid, is biosynthesized from a reactive selenium donor on its cognate tRNA [Ser]Sec . It is thought that intracellular selenium must be delivered 'safely' and 'efficiently' by a carrier protein owing to its high reactivity and very low concentrations. Here, we identified peroxiredoxin 6 (PRDX6) as a novel selenoprotein synthesis factor. Loss of PRDX6 decreases the expression of selenoproteins and induces ferroptosis via a reduction in GPX4. Mechanistically, PRDX6 increases the efficiency of intracellular selenium utilization by transferring selenium between proteins within the selenocysteyl-tRNA [Ser]Sec synthesis machinery, leading to efficient synthesis of selenocysteyl-tRNA [Ser]Sec . These findings highlight previously unidentified selenium metabolic systems and provide new insights into ferroptosis., (© 2024. The Author(s).)

While research has identified several inhibitors of the main protease (Mpro) of SARS-CoV-2, a significant portion of these compounds exhibit reduced activity in the presence of reducing agents, raising concerns about their effectiveness in vivo. Furthermore, the conventional biosafety level 3 (BSL-3) for cellular assays using viral particles poses a limitation for the widespread evaluation of Mpro inhibitor efficacy in a cell-based assay. Here, we established a BSL-1 compatible cellular assay to evaluate the in vivo potential of Mpro inhibitors. This assay utilizes mammalian cells expressing a tagged Mpro construct containing N-terminal glutathione S -transferase (GST) and C-terminal hemagglutinin (HA) tags and monitors Mpro autodigestion. Using this method, GC376 and boceprevir effectively inhibited Mpro autodigestion, suggesting their potential in vivo activity. Conversely, carmofur and ebselen did not exhibit significant inhibitory effects in this assay. We further investigated the inhibitory potential of selenoneine on Mpro using this approach. Computational analyses of binding energies suggest that noncovalent interactions play a critical role in facilitating the covalent modification of the C145 residue, leading to Mpro inhibition. Our method is straightforward, cost-effective, and readily applicable in standard laboratories, making it accessible to researchers with varying levels of expertise in infectious diseases.

It is known that the recommended dietary allowance of selenium (Se) is dangerously close to its tolerable upper intake level. Se is detoxified and excreted in urine as trimethylselenonium ion (TMSe) when the amount ingested exceeds the nutritional level. Recently, we demonstrated that the production of TMSe requires two methyltransferases: thiopurine S-methyltransferase (TPMT) and indolethylamine N-methyltransferase (INMT). In this study, we investigated the substrate recognition mechanisms of INMT and TPMT in the Se-methylation reaction. Examination of the Se-methyltransferase activities of two paralogs of INMT, namely, nicotinamide N-methyltransferase and phenylethanolamine N-methyltransferase, revealed that only INMT exhibited Se-methyltransferase activity. Consistently, molecular dynamics simulations demonstrated that dimethylselenide was preferentially associated with the active center of INMT. Using the fragment molecular orbital method, we identified hydrophobic residues involved in the binding of dimethylselenide to the active center of INMT. The INMT-L164R mutation resulted in a deficiency in Se- and N-methyltransferase activities. Similarly, TPMT-R152, which occupies the same position as INMT-L164, played a crucial role in the Se-methyltransferase activity of TPMT. Our findings suggest that TPMT recognizes negatively charged substrates, whereas INMT recognizes electrically neutral substrates in the hydrophobic active center embedded within the protein. These observations explain the sequential requirement of the two methyltransferases in producing TMSe., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: We aimed to analyze the subchronic toxicity and tissue distribution of indium after the intratracheal administration of indium-tin oxide nanoparticles (ITO NPs) to the lungs of rats., Methods: Male Wistar rats were administered a single intratracheal dose of 10 or 20 mg In/kg body weight (BW) of ITO NPs. The control rats received only an intratracheal dose of distilled water. A subset of rats was periodically euthanized throughout the study from 1 to 20 weeks after administration. Indium concentrations in the serum, lungs, mediastinal lymph nodes, kidneys, liver, and spleen as well as pathological changes in the lungs and kidneys were determined. Additionally, the distribution of ionic indium and indium NPs in the kidneys was analyzed using laser ablation-inductively coupled plasma mass spectrometry., Results: Indium concentrations in the lungs of the 2 ITO NP groups gradually decreased over the 20-week observation period. Conversely, the indium concentrations in the mediastinal lymph nodes of the 2 ITO groups increased and were several hundred times higher than those in the kidneys, spleen, and liver. Pulmonary and renal toxicities were observed histopathologically in both the ITO groups. Both indium NPs and ionic indium were detected in the kidneys, and their distributions were similar to the strong indium signals detected at the sites of inflammatory cell infiltration and tubular epithelial cells., Conclusions: Our results demonstrate that intratracheal administration of 10 or 20 mg In/kg BW of ITO NPs in male rats produces pulmonary and renal toxicities., (© The Author(s) [2024]. Published by Oxford University Press on behalf of Journal of Occupational Health.)

Zolpidem, N,N-dimethyl-2-[6-methyl-2-(4-methylphenyl)imidazo[1,2-a]pyridin-3-yl]acetamide, is a hypnotic agent widely used in clinical practice but is detected in many clinical cases of fatal intoxication and suicide. In forensic toxicology, the precise determination of zolpidem concentration in blood is a must to provide concrete evidence of death by zolpidem poisoning. However, the concentrations of zolpidem in blood at autopsy often differ from those at the estimated time of death. In the present study, we found that zolpidem was degraded by hemoglobin (Hb) via the Fenton reaction at various temperatures. The mechanism underlying zolpidem degradation involved the oxidation of its linker moiety. The MS and MS/MS spectra obtained by liquid chromatography quadrupole-Orbitrap mass spectrometry (LC-Q-Orbitrap-MS) showed the formation of 2-hydroxy-N,N-dimethyl-2-(6-methyl-2-(p-tolyl)imidazo[1,2-a]pyridin-3-yl)acetamide (2-OH ZOL) in Hb/H 2 O 2 solution incubated with zolpidem and in the blood of several individuals who died from ingestion of zolpidem. These results suggest that 2-OH ZOL is the post-mortem product of zolpidem degradation by Hb via the Fenton reaction.

Paliperidone is a widely used antipsychotic agent detected in many fatal intoxications and suicide cases. In forensic toxicology, the accurate determination of blood paliperidone concentrations is required to prove death by paliperidone poisoning. However, the lethal concentration of paliperidone in blood at autopsy differs from that at the time of death. In this study, we found that paliperidone was decomposed by hemoglobin (Hb) through the Fenton reaction in a temperature-dependent fashion. The mechanism underlying paliperidone decomposition involves the cleavage of its C-N bond linker moiety. The mass spectra obtained by liquid chromatography-quadrupole orbitrap mass spectrometry revealed the formation of 6-fluoro-3-(4-piperidinyl)benzisoxazole (PM1) in Hb/H2O2 solution incubated with paliperidone, as well as in the blood of individuals who died from intentional ingestion of paliperidone. These results suggest that PM1 is the only metabolite produced from paliperidone as a result of temperature-dependent, postmortem changes induced by Hb via the Fenton reaction and may be useful as a biomarker to correct for the concentration of paliperidone in blood at the time of death in clinical cases., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

This work studied the distribution, reactivity, and biological effects of pentavalent or trivalent antimony (Sb(V), Sb(III)) and N-methylglucamine antimonate (NMG-Sb(V)) in Wistar Rats. The expression of fibrosis genes such as α - SMA, PAI-1, and CTGF were determined in Liver, and Kidney tissues. Wistar rats were treated with different concentrations of Sb(V), Sb(III), As(V) and As(III), and MA via intra-peritoneal injections. The results indicated a noteworthy elevation in mRNA levels of plasminogen activator 1 (PAI-1) in the kidneys of rats that were injected. The main accumulation site for Sb(V) was observed to be the liver, from which it is primarily excreted in its reduced form (Sb(III)) through the urine. The generation of Sb(III) in the kidneys has been found to induce damage through the expression of α-SMA and CTGF, and also lead to a higher creatinine clearance compared to As(III)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Itai-itai disease is caused by environmental cadmium (Cd) pollution in the Jinzu River basin in Japan. To reduce the Cd contamination of rice, soil restoration of paddy fields was carried out. We evaluated the effect of soil restoration on the health status of residents of the former Cd-polluted area., Methods: Participants were 1,030 men and 944 women who lived in the area of restoration of Cd-polluted rice paddies. First morning urine was collected and urinary Cd, β2-microglobulin (β2MG), and N-acetyl-β-D-glucosaminidase (NAG) levels were measured. Associations among age, years of residence before and after soil restoration, and urinary Cd, β2MG, and NAG levels were evaluated by multiple regression analysis., Results: The geometric mean (interquartile range) of urinary Cd (µg/g Cr) was 1.00 (0.58-1.68) in men and 1.67 (1.02-2.91) in women. The geometric means of urinary β2MG (µg/g Cr) and NAG (U/g Cr) were 174.6 (92.6-234.2) and 1.47 (0.72-3.14) in men, and 217.6 (115.3-28.7) and 1.48 (0.73-2.96) in women, respectively. Urinary Cd, β2MG, and NAG were significantly positively correlated (p < 0.01 all). Age and duration of residence in the Cd-polluted area before soil restoration were independently associated with urinary Cd, β2MG, and NAG. Among the 916 participants who had resided in the area before the soil restoration, urinary Cd concentrations were significantly higher, thus by 1.03-fold (95% CI, 1.01-1.04) in men and 1.03-fold (95% CI, 1.01-1.05) in women, when the years of residence before soil restoration by each 5-years increment. By contrast, urinary Cd concentrations were significantly lower, thus 0.97-fold (95% CI, 0.96-0.99) lower in men and 0.97-fold (95% CI, 0.95-0.99) lower in women, by each 5-year increment of residence after soil restoration. A similar association was observed for urinary β2MG concentration, and no significant association was observed for urinary NAG levels in men or women., Conclusions: Cd exposure and associated renal tubular dysfunction in residents of a former Cd-polluted area were influenced by Cd exposure from the environment prior to soil restoration. Soil restoration in Cd-polluted areas reduced the Cd exposure of local residents.

The nucleolus is a non-membranous structure in the nucleus and forms around ribosomal DNA repeats. It plays a major role in ribosomal biogenesis through the transcription of ribosomal DNA and regulates mRNA translation in response to cellular stress including DNA damage. Rad17 is one of the proteins that initiate and maintain the activation of the ATR pathway, one of the major DNA damage checkpoints. We have recently reported that the central basic domain of Rad17 contains a nuclear localization signal and that the nuclear translocation of Rad17 promotes its proteasomal degradation. Here, we show that the central basic domain contains the nucleolar localization signal as well as the nuclear localization signal. The nucleolar localization signal overlaps with the nuclear localization signal and is capable of transporting an exogenous protein into the nucleolus. Phosphomimetic mutations of the central basic domain inhibit nucleolar accumulation, suggesting that the post-translational modification sites regulate the nucleolar localization. Nucleolar accumulation of Rad17 is promoted by proteasome inhibition and UV irradiation. Our data show the nucleolar localization of Rad17 and suggest a possible role of Rad17 in the nucleolus upon UV irradiation.

The toxicity of nano-sized particles of mercury (NP-Hg), which are thought to be generated during the detoxification of methyl mercury (MeHg), may differ from that of MeHg, elemental Hg (Hg 0 ), and inorganic Hg (I-Hg). From a human health perspective, it is important to evaluate the presence of NP-Hg in seafoods. We investigated the in vivo formation of NP-Hg in fish and shellfish, which are the main sources of Hg exposure in humans. NP-Hg was measured in 90 fish samples with single-particle inductively coupled plasma mass spectrometry (spICP-MS) after enzyme degradation with pancreatin and lipase. In addition to NP-Hg, total Hg (T-Hg), MeHg, and selenium (Se) concentrations were evaluated. Transient Hg signals were detected as nanoparticles from almost all samples by using spICP-MS. Higher particle number concentrations (C PN ) were observed in the tuna-swordfish group than in the shellfish group (17.7 × 10 7 vs. 1.2 × 10 6 particles/g, respectively). Although the C PN and maximum particle mass increased significantly with increasing T-Hg concentration, the increase in C PN was greater than those in maximum particle mass. Assuming that the NP-Hg detected was HgSe (tiemannite) and spherical based on previous reports, the maximum particle diameter was estimated to be 89 nm. The mean dietary exposures to NP-Hg, T-Hg, and MeHg were estimated to be 0.067, 5.75, and 5.32 μg/person per day, respectively. Generation of NP-Hg was inferred to be widespread in marine animals, with a preferential increase in the number of particles rather than an increase in particle size. The mean dietary exposure to NP-Hg in Japanese people was estimated to be 1.2 ng/kg body weight (BW) per day. Compared to PTWI of 4 μg/kg BW per week (0.57 μg/kg BW per day) derived by JECFA (2011), the health risk from redissolved I-Hg from NP-Hg is small., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Copper (Cu) participates in the biological redox reaction in the body, and its deficiency is fatal to the body. At the same time, Cu is extremely toxic when it exists in excess. Thus, the body has to tightly and spatiotemporally regulate the concentration of Cu within a physiological range by several groups of Cu-regulating proteins. However, entire mechanisms underlying the maintenance of Cu homeostasis in body and cells have not fully understood. It is necessary to analyze Cu itself in a body and in a cell to reveal the Cu homeostasis. In this review, recent advances in the analytical techniques to understand the Cu metabolism such as speciation, imaging and single-cell analysis of Cu were highlighted., Competing Interests: No potential conflicts of interest were disclosed., (Copyright © 2022 JCBN.)

Iron is not only essential but also a toxic trace element. Under iron repletion, ferritin maintains cellular iron homeostasis by storing iron to avoid iron toxicity. Under iron depletion, the ferritin-specific autophagy adaptor NCOA4 delivers ferritin to lysosomes via macroautophagy to enable cells to use stored iron. Here, we show that NCOA4 also plays crucial roles in the regulation of ferritin fate under iron repletion. NCOA4 forms insoluble condensates via multivalent interactions generated by the binding of iron to its intrinsically disordered region. This sequesters NCOA4 away from ferritin and allows ferritin accumulation in the early phase of iron repletion. Under prolonged iron repletion, NCOA4 condensates can deliver ferritin to lysosomes via a TAX1BP1-dependent non-canonical autophagy pathway, thereby preventing relative iron deficiency due to excessive iron storage and reduced iron uptake. Together, these observations suggest that the NCOA4-ferritin axis modulates intracellular iron homeostasis in accordance with cellular iron availability., (© 2022 The Authors.)

Selenium is a chalcogen element that is essential in animals, but is highly toxic when ingested above the nutritional requirement. Selenite is used as a supplement in patients receiving total parenteral nutrition. However, the therapeutic and toxic doses of selenite are separated by a narrow range. This ambivalent character of selenite implies the presence of cellular mechanisms that precisely control selenite homeostasis. Here, we investigated mechanisms that determine cellular susceptibility to selenite exposure. The resistance to selenite exposure was significantly different among cell lines. We determined the expression levels of TPMT (thiopurine S -methyltransferase) and SLC4A1 (solute carrier family 4 member 1), which encode selenium methyltransferase and selenite transporter, respectively. We also examined the effect of inhibition of Band 3 protein activity, which is encoded by SLC4A1 , on the cellular sensitivity to selenite. The data suggest that the expression level of SLC4A1 is the determinant of cellular sensitivity to selenite., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yasumitsu Ogra reports financial support was provided by Japanese Ministry of Education, Culture, Sports, Science and Technology. Yasunori Fukumoto reports financial support was provided by Japanese Ministry of Education, Culture, Sports, Science and Technology. Yu-ki Tanaka reports financial support was provided by Japanese Ministry of Education, Culture, Sports, Science and Technology., (© 2022 The Authors.)

Methidathion [3-(dimethoxyphosphinothioylsulfanylmethyl)-5-methoxy-1,3,4-thiadiazol-2-one; hereinafter DMTP], one of the most widely used organophosphorus pesticides, has been detected in some clinical cases of accidental exposure and suicide in Japan. It has been reported that DMTP concentration is decreased in blood. In this study, it is difficult to recover DMTP in the free form because DMTP is bound to human serum albumin (HSA). We detected DMTP adducts in HSA by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q/TOF-MS). The mass spectra showed that DMTP was preferably bound to the lysine (K), tyrosine (Y), and cysteinylproline (CP) residues of HSA. The concentrations of K-adduct, DMTP-Y-adduct and DMTP-CP-adduct were increased in vitro in a dose-dependent fashion when DMTP concentration was lower than the lethal dose. Furthermore, the DMTP-Y-adduct and DMTP-CP-adduct were also detected in post-mortem blood of an autopsied subject who died by intentional DMTP ingestion. The results suggested that the DMTP-Y-adduct and DMTP-CP-adduct could be used as a biomarker of DMTP poisoning, and the decrease concentration of DMTP in blood after death could be determined on the basis of the concentration of the DMTP-CP-adduct in blood.

Mercury (Hg) is one of the most toxic environmental pollutants, and is biocondensed via the food chain. Selenium (Se) is an essential element that possesses an antagonistic property towards Hg in vivo. The antagonistic property is explained by the assumption that Hg and Se directly interact to form HgSe nanoparticles (HgSe NPs) in organs. It is presumed that the toxic effects of HgSe NPs are lower than that of ionic Hg; however, no precise evaluation has been conducted so far. In the present study, we evaluated the distribution of HgSe NPs ingested in Se-deficient rats. The recovery of serum selenoproteins from a deficient level was not observed in rats orally administered HgSe NPs. In addition, the excretion of Hg and Se via urine was not observed. Interestingly, the biosynthesis of selenoproteins and urinary selenometabolites would have required the production of selenide through the degradation of HgSe NPs. Therefore, it seems that selenide and Hg are not released from HgSe NPs in vivo. The administration of HgSe NPs did not increase Hg and Se concentrations in organs, and almost all HgSe NPs were recovered in feces, indicating no or low bioaccessibility of HgSe NPs even in Se-deficient rats. These results suggest that HgSe NPs are biologically inert and do not become a secondary environmental pollutant of Hg., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The ATR pathway is one of the major DNA damage checkpoints, and Rad17 is a DNA-binding protein that is phosphorylated upon DNA damage by ATR kinase. Rad17 recruits the 9-1-1 complex that mediates the checkpoint activation, and proteasomal degradation of Rad17 is important for recovery from the ATR pathway. Here, we identified several Rad17 mutants deficient in nuclear localization and resistant to proteasomal degradation. The nuclear localization signal was identified in the central basic domain of Rad17. Rad17 Δ230-270 and R240A/L243A mutants that were previously postulated to lack the destruction box, a sequence that is recognized by the ubiquitin ligase/anaphase-promoting complex that mediates degradation of Rad17, also showed cytoplasmic localization. Our data indicate that the nuclear translocation of Rad17 is functionally linked to the proteasomal degradation. The ATP-binding activity of Rad17, but not hydrolysis, is essential for the nuclear translocation, and the ATPase domain orchestrates the nuclear translocation, the proteasomal degradation, as well as the interaction with the 9-1-1 complex. The Rad17 mutant that lacked a nuclear localization signal was proficient in the interaction with the 9-1-1 complex, suggesting cytosolic association of Rad17 and the 9-1-1 complex. Finally, we identified two tandem canonical and noncanonical destruction boxes in the N-terminus of Rad17 as the bona fide destruction box, supporting the role of anaphase-promoting complex in the degradation of Rad17. We propose a model in which Rad17 is activated in the cytoplasm for translocation into the nucleus and continuously degraded in the nucleus even in the absence of exogenous DNA damage., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Glucocorticoid-induced osteoporosis (GIOP) is a major cause of secondary osteoporosis, and the pathogenic mechanisms of GIOP remain to be elucidated. Here, we show a rapid dexamethasone-induced osteoporosis animal model using zebrafish scales. Intraperitoneal injection of dexamethasone over a 5-day period suppressed the regeneration of scales. Furthermore, the circularity of the newly formed regenerated scales was also slightly reduced compared to that of the control group on day 5. The changes in bone-related enzymes, such as cathepsin K, tartrate-resistant acid phosphatase (TRAP) for bone resorption, and alkaline phosphatase (ALP) for bone formation, provide insight into the progression of bone diseases; therefore, we further developed a method to measure the activities of cathepsin K, TRAP, and ALP using zebrafish scales. We found that a lysis buffer with detergent at neutral pH under sonication efficiently helped extract these three enzymes with high activity levels. Interestingly, treatment with a dexamethasone injection produced considerably higher levels of cathepsin K activity and a lower Ca/P ratio than those in the control group, suggesting that dexamethasone increased osteoclast activity, with no significant changes in the activities of TRAP and ALP. Our GIOP model and enzyme assay method could help to design better treatments for GIOP.

Malathion, diethyl 2-[(dimethoxyphosphinothioyl)thio]butanedioate, is one of most widely used organophosphoryl pesticide, and it has been detected in several clinical cases of accidental exposure and suicide. It is reported that the observed malathion concentration in blood of persons who suffer from malathion poisoning is smaller than the expected concentration. Because malathion is bound to human serum albumin (HSA), recovery of malathion in the free form is insufficient. We detected malathion adducts in HSA by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q/TOF-MS). The mass spectra showed that malathion was preferably bound to the lysine (K) and cysteinylproline (CP) residues of HSA. The K- and CP-adducts of malathion were increased in vitro with a dose-dependent fashion when its concentration was smaller than the lethal dose. Further, the K-adduct was also detected in post-mortem blood of an autopsied subject suffering from intentional malathion ingestion. These results suggest that the K-adduct seems to be available to use a biomarker of malathion poisoning, and the determination of the K-adduct could make possible to estimate the amount of malathion ingestion.

The PML/RARα fusion protein is the oncogenic driver in acute promyelocytic leukemia (APL). Although most APL cases are cured by PML/RARα-targeting therapy, relapse and resistance can occur due to drug-resistant mutations. Here we report that thermal stress destabilizes the PML/RARα protein, including clinically identified drug-resistant mutants. AML1/ETO and TEL/AML1 oncofusions show similar heat shock susceptibility. Mechanistically, mild hyperthermia stimulates aggregation of PML/RARα in complex with nuclear receptor corepressors leading to ubiquitin-mediated degradation via the SIAH2 E3 ligase. Hyperthermia and arsenic therapy destabilize PML/RARα via distinct mechanisms and are synergistic in primary patient samples and in vivo , including three refractory APL cases. Collectively, our results suggest that by taking advantage of a biophysical vulnerability of PML/RARα, thermal therapy may improve prognosis in drug-resistant or otherwise refractory APL. These findings serve as a paradigm for therapeutic targeting of fusion oncoprotein-associated cancers by hyperthermia., Significance: Hyperthermia destabilizes oncofusion proteins including PML/RARα and acts synergistically with standard arsenic therapy in relapsed and refractory APL. The results open up the possibility that heat shock sensitivity may be an easily targetable vulnerability of oncofusion-driven cancers. See related commentary by Wu et al., p. 300 ., (©2021 American Association for Cancer Research.)

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.

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.

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., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

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., (© 2020 Suzuki et al.)

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.

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

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., Competing Interests: The authors declare no competing financial interest.

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.

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).

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.

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.

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.

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 O₂ 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 O₂ mass shift mode gave us more accurate abundance ratios of Se than ICP-MS.

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.

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.

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.

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 0.5mg As/kg, followed by speciation analysis of selected organs and body fluids at 10 min and 12h after the injection. MMMTA(V) was excreted into urine in its intact form, and approximately 35% of the dose was recovered in urine at 12h 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 12h 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 12h 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.)

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.

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.

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, 82Se, in rats depleted of endogenous natural abundance isotopes with another Se isotope, 78Se, 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.

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.

Liquid chromatography (LC) hyphenated with both elemental and molecular mass spectrometry has been used for Se speciation in Se-enriched garlic. Different species were separated by ion-pair liquid chromatography-inductively coupled plasma mass spectrometry (LC-ICP-MS) after hot-water extraction. They were identified by on-line reversed-phase liquid chromatography-electrospray ionization tandem mass spectrometry (RPLC-ESI-MS-MS). Se-methionine and Se-methylselenocysteine were determined by monitoring their product ions. Another compound, gamma-glutamyl-Se-methylselenocysteine, shown to be the most abundant form of Se in the garlic, was determined without any additional sample pre-treatment after extraction and without the need for a synthesized standard. Product ions for this dipeptide were detected by LC-ESI-MS-MS for three isotopes of Se-78 Se, 80Se: and 82Se. The method was extended to the species extracted during in-vitro gastrointestinal digestion. Because both Se-methylselenocysteine and gamma-glutamyl-Se-methylselenocysteine have anticarcinogenic properties, their extractability and stability during human digestion are very important. Garlic was also treated with saliva, to enable detection and analysis of species extracted during mastication. Detailed information on the extractability of selenium species by both simulated gastric and intestinal fluid are given, and variation of the distribution of Se among the different species with time is discussed. Although the main species in garlic is the dipeptide gamma-glutamyl-Se-methylselenocysteine, Se-methylselenocysteine is the main compound present in the extracts after treatment with gastrointestinal fluids. Two more, so far unknown compounds were observed in the chromatogram. The extracted species and their transformations were analysed by combining LC-ICP-MS and LC-ESI-MS-MS. In both the simulated gastric and intestinal digests, Se-methionine, Se-methylselenocysteine, and gamma-glutamyl-Se-methylselenocysteine could be determined by LC-ESI-MS-MS by measuring their typical product ions.