Essential micronutrient selenium is excreted into the urine and͞or 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 1-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.
A speciation technique for arsenic has been developed using an anion-exchange high-performance liquid chromatography/inductively coupled argon plasma mass spectrometer (HPLC/ICP MS). Under optimized conditions, eight arsenic species [arsenocholine, arsenobetaine, dimethylarsinic acid (DMA(V)), dimethylarsinous acid (DMA(III)), monomethylarsonic acid (MMA(V)), monomethylarsonous acid (MMA(III)), arsenite (As(III)), and arsenate (As(V))] can be separated with isocratic elution within 10 min. The detection limit of arsenic compounds was 0.14-0.33 microg/L. To validate the method, Standard Reference Material in freeze-dried urine, SRM-2670, containing both normal and elevated levels of arsenic was analyzed. The method was applied to determine arsenic species in urine samples from three arsenic-affected districts of West Bengal, India. Both DMA(III) and MMA(III) were detected directly (i.e., without any prechemical treatment) for the first time in the urine of some humans exposed to inorganic arsenic through their drinking water. Of 428 subjects, MMA(III) was found in 48% and DMA(III) in 72%. Our results indicate the following. (1) Since MMA(III) and DMA(III) are more toxic than inorganic arsenic, it is essential to re-evaluate the hypothesis that methylation is the detoxification pathway for inorganic arsenic. (2) Since MMA(V) reductase with glutathione (GSH) is responsible for conversion of MMA(V) to MMA(III) in vivo, is DMA(V) reductase with GSH responsible for conversion of DMA(V) to DMA(III) in vivo? (3) Since DMA(III) forms iron-dependent reactive oxygen species (ROS) which causes DNA damage in vivo, DMA(III) may be responsible for arsenic carcinogenesis in human.
Multiple copies of a cis-acting DNA element, metal-responsive element (MRE) are required for heavy metalinduced transcriptional activation of mammalian metallothionein genes. To approach the regulatory mechanism mediated by these multiple elements, we studied the properties of seven MREs located upstream of the human metallothionein-IIA (hMT-IIA) gene in detail. Transfection assays of reporter gene constructs each containing one of these MREs as the promoter element revealed that only four MREs can mediate zinc response. With respect to the distribution of active MREs over the promoter region, the hMT-IIA gene is largely different from the mouse metallothionein-I gene, suggesting that MRE arrangement is not an important factor for metal regulation. Experiments using various model promoters showed that multiple MRE copies act highly synergistically, supporting the biological significance of the multiplicity. Only the four active MREs efficiently bound the purified transcription factor human MTF-1, and MRE mutants defective in binding this protein lost the ability to support zinc-induced reporter gene expression, strongly suggesting that the direct interaction between human MTF-1 and a set of the selected MREs plays the major role in heavy metal regulation. In protein/DNA binding reactions in vitro, the purified human MTF-1 was activated by zinc but not by other metallothionein-inducing heavy metals, supporting the idea that zinc is the direct modulator of human MTF-1.Keywords: metallothionein; zinc; transcription factor; protein±nucleic acid interaction.Transcription of mammalian metallothionein genes is activated by heavy metals such as cadmium and zinc [1±3]. A cis-acting DNA element, metal-responsive element (MRE), mediates this activation [4±6]. MRE consists of the highly conserved core consensus sequence, TGCRCNC, and semi-conserved GC-rich sequence [7,8]. Several putative regulator proteins that interact with MRE have been reported [9]. Radtke et al. [10] cloned the cDNA encoding one of these factors, mouse MTF-1 (MREbinding transcription factor-1). Using the techniques of antisense RNA expression [11] and gene disruption [12], it has been shown that MTF-1 is essential for the transcriptional activation of metallothionein genes by several heavy metals. In our previous work, a human MRE-binding factor, ZRF (zinc regulatory factor) was purified from HeLa cell nuclei [13]. This protein was expected to be the human counterpart of mouse MTF-1, from amino-acid sequence analysis and the studies of DNAbinding properties [13]. In fact, the cDNA cloned using a probe based on the partial amino acid sequences of ZRF (F. Otsuka, unpublished results and DDBJ D31907) encodes a protein almost identical with the human homologue of mouse MTF-1 [14]. To avoid confusion, the term human MTF-1 is used for the human MTF-1/ZRF protein throughout this paper.Although MTF-1 is likely to play an important role in metal regulation, the molecular mechanisms for metal signal transduction and metallothionein gene activation are yet uncle...
Selenium (Se) is an ultramicro essential nutrient and both inorganic (selenite and selenate) and organic (selenocysteine and selenomethionine) forms of Se can be used as nutritional sources. Metabolic pathways for Se in the body were studied for selenite and selenate, with the use of enriched 82Se, by speciation with separation by gel filtration HPLC and detection by element-specific mass spectrometry with ionization with inductively coupled argon plasma (HPLC-ICP MS). The concentrations of 82Se in organs and body fluids and the distributions of their constituents depending on the dose and time after the intravenous administration of 82Se-selenite and -selenate to rats were determined. Selenite was taken up by red blood cells within several minutes, reduced to selenide by glutathione, and then transported to the plasma, bound selectively to albumin and transferred to the liver. Contrary to selenite, intact selenate was either taken up directly by the liver or excreted into the urine. The 82Se of selenite origin and that of selenate origin were detected in the forms of the two Se peak materials in the liver, A and B. The former one was methylated to the latter in vivo and in vitro. The latter one was identical with the major urinary metabolite and it was identified as Se-methyl-N-acetyl-selenohexosamine (selenosugar). The chemical species-specific metabolic pathway for Se was explained by the metabolic regulation through selenide as the assumed common intermediate for the inorganic and organic Se sources and as the checkpoint metabolite between utilization for the selenoprotein synthesis and methylation for the excretion of Se.
Two unidentified arsenic metabolites were detected in the liver of rats on a gel filtration column by HPLC inductively coupled argon plasma mass spectrometry after an injection of dimethylarsinic (DMA(V)), dimethylarsinous (DMA(III)), monomethylarsonic (MMA(V)), or monomethylarsonous (MMA(III)) acid. The same arsenicals were also produced in vitro by incubation of DMA(III) in the liver supernatant but not by DMA(V). The two arsenic metabolites eluted at the same retention times as those of the two arsenicals prepared by reaction of DMA(V) with either thiosulfate plus disulfite or hydrogen sulfide or sodium sulfide plus sulfuric acid. The faster and slower eluting products on a gel filtration column were assigned as dimethyldithioarsinic acid (dimethylarsinodithioic acid) (DMTA(V)) and dimethylthioarsinous acid (DMTA(III)) from mass spectrometric data at m/z = 170 and 138 by electrospray ionization mass spectrometry with negative and positive ion modes, respectively. They were prepared selectively by reacting DMA(V) with hydrogen sulfide or sodium sulfide plus sulfuric acid under different reaction conditions. DMA(III) but not DMA(V) was transformed to DMTA(III) and DMTA(V) in the presence of sodium sulfide in vitro, suggesting that DMA(V) is reduced to DMA(III) with hydrogen sulfide, thiolated to DMTA(III), and then further thiolated oxidatively to DMTA(V). Metabolically, it is assumed that DMA(III) is transformed to DMTA(III) in the presence of sulfide ions, and then, DMTA(III) is oxidatively thiolated to DMTA(V). As the chemical species produced by reduction with the Reay and Asher method are DMTA(III) and DMTA(V), and different from DMA(III), the studies carried out with DMA(III) with the Reay and Asher method have to be reexamined.
A method for the accurate determination of selenoamino acids in human serum by HPLC-ICPMS was developed using the species-specific isotope dilution analysis principle. A serum sample was enzymatically digested with a mixture of lipase and protease after derivatization of the selenocysteine residues with iodoacetamide. The selenoamino acid fraction was isolated by size exclusion LC followed by the separation of selenomethionine and the carboxymethylated selenocysteine by capillary HPLC. The isotope-specific determination of 77Se and 80Se was achieved on-line by ICP collision cell MS allowing the removal of polyatomic interferences. Quantification was carried out by isotope dilution using a 77Se-labeled selenomethionine spike and the determination of the 77Se/80Se ratio in the cHPLC selenomethionine peak. The accurately determined selenomethionine was used as an internal standard for the selenocysteine determination from the same chromatogram. The modification of the previously developed cHPLC-ICPMS interface allowed the reduction of the absolute detection limits twice (down to the 75-fg level), which resulted in the lowest ever reported procedural detection limits (below 0.5 ng g(-1) for a 450-mg serum sample). The precision was less than 5% RSD. The method was validated by the mass balance of selenium (amino acid incorporated vs total).
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.