Tomofumi Okuno

Identification and metabolism of selenocysteine-glutathione selenenyl sulfide (CySeSG) in small intestine of mice orally exposed to selenocystine

Abstract This investigation was carried out to elucidate the chemical form of selenium-containing metabolite in small intestine of ICR male mice orally administered selenocystine (CySeSeCy). The metabolite in intestinal cytosol of mice treated with CySeSeCy (50 mg/kg) was identified as selenocysteine-glutathione selenenyl sulfide (CySeSG) by high performance liquid chromatography using a gel filtration and reversed phase column. Hydrogen selenide formation was caused as a result of the anaerobic reaction between the CySeSG and liver cytosol containing selenocysteine β-lyase, which specifically acts on selenocysteine (CySeH). Effects of GSH or glutathione reductase on hydrogen selenide formation from CySeSG reacted with the liver cytosol were examined. The CySeSG was nonenzymatically reduced to CySeH by excess GSH in the liver cytosol. It was also recognized that CySeSG was enzymatically reduced to CySeH by glutathione reductase in the presence of NADPH. These results indicate that the chemical form of this metabolite is CySeSG, which has a molecular weight of 473, the CySeSG is then reduced by excess GSH and/or gluta- thione reductase yielding CySeH, which is decomposed by selenocysteine β-lyase to hydrogen selenide. CySeSG may be a stable precursor of hydrogen selenide in animals.

Effects of selenium status and supplementary seleno-chemical sources on mouse T-cell mitogenesis.

Although selenium is thought to be essential for various immune responses, the excess supplementation may have an adverse effect on certain immunological functions. The present study was designed to determine the effective chemical forms of selenium and their optimal levels on T-cell mitogenesis with splenic cells from mice given a selenium-deficient diet for 8 weeks to avoid effects of cellular selenium sources. Although selenium in tissues, except for spleen and thymus, was almost depleted by feeding selenium-deficient diet, the lymphoid organs still contained low levels of selenium. Both activities of cellular glutathione peroxidase (cGPx) and thioredoxin reductase (TR) in liver and splenic cells showed a tendency to decrease by selenium deficiency. However, splenic cells were tolerant against decrease of the selenoenzyme activities, and TR was also more tolerant than cGPx. T-cell proliferation of the selenium-insufficient splenic cells induced by concanavalin A was increased by addition of Na2SeO3, Na2SeO4, Na2Se, seleno-DL-cystine, seleno-L-methionine and selenocystamine. Their promoting action was observed at levels lower than 0.1 micromol/L and was completely suppressed at the highest concentration (1 micromol/L), except for selenocystamine. Na2SeO3 was one of the efficient selenocompounds for the mitogenesis, which was concomitant with the significant induction of cGPx and TR. However, recovery of cGPx activity in the selenium-insufficient cells by supplementary Na2SeO3 was only partial,while TR activity was readily recovered from selenium deficiency. These results therefore indicate that only low levels of selenium is essential for T-cell mitogenesis even in selenium-insufficient splenic cells, and TR, which is readily recovered by Na2SeO3, may be the critical enzyme.

Purification and characterization of mouse hepatic enzyme that converts selenomethionine to methylselenol by its α, γ-elimination

The objective of this study was to purify and characterize a mouse hepatic enzyme that directly generates CH3SeH from seleno-l-methionine (l-SeMet) by the α,γ-elimination reaction. The l-SeMet α,γ-elimination enzyme was ubiquitous in tissues from ICR mice and the activity was relatively high in the large intestine, brain, and muscle, as well as the liver. Aging and sex of the mice did not have any significant influence on the activity in the liver. The enzyme was purified from the mouse liver by ammonium sulfate precipitation and four kinds of column chromatography. These procedures yielded a homogeneous enzyme, which was purified approx 1000-fold relative to mouse liver extract. Overall recovery was approx 8%. The purified enzyme had a molecular mass of approx 160 kDa with four identical subunits. The Km value of the enzyme for the catalysis of l-SeMet was 15.5 m M, and the Vmax was 0.29 units/mg protein. Pyridoxal 5′-phosphate (pyridoxal-P) was required as a cofactor because the holoenzyme could be resolved to the apoenzyme by incubation with hydroxylamine and reconstituted by addition of pyridoxal-P. The enzyme showed the optimum activity at around pH 8.0 and the highest activity at 50°C; it catalyzed the α,γ-elimination reactions of several analogs such as d,l-homocysteine and l-homoserine in addition to l-SeMet. This enzyme also catalyzed the α,β-elimination reaction of Se-methylseleno-l-cysteine. However, l-methionine was inerts. Therefore, the purified enzyme was different from the bacterial l-methionine γ-lyase that metabolizes l-SeMet to CH3SeH, in terms of the substrate specificity. These results were the first identification of a mammalian enzyme that specifically catalyzes the α,γ-elimination reaction of l-SeMet and immediately converts it to CH3SeH, an important metabolite of Se.

Cystathionine γ-lyase contributes to selenomethionine detoxification and cytosolic glutathione peroxidase biosynthesis in Mouse Liver

We earlier found that seleno-l-methionine (L-SeMet) as a food source of selenium (Se) is directly converted to methylselenol (CH3SeH), α-ketobutyrate, and ammonia by the mouse hepatic cystathionine γ-lyase. The purpose of this study was to clarify the biological role of cystathionine γ-lyase in Se detoxification and cytosolic glutathione peroxidase (cGPx) biosynthesis because another metabolic pathway to CH3SeH via seleno-l-cystathionine and seleno-l-cysteine (l-SeCyH) from l-SeMet has been shown by several enzymatic reactions. When mice were treated with either toxic doses of l-SeMet or a Se-deficient diet, the cystathionine γ-lyase activity for l-SeMet was invariable, suggesting that this enzyme was effective in both detoxification and biotransformation of Se. Concerning Se biotransformation into cGPx, production of H2Se as the possible precursor was not observed by the in vitro reaction of the liver cytosol with CH3SeH. When l-SeMet was administered at the nutritional dose to mice fed a Se-deficient diet, levels of both cGPx mRNA and cGPx protein were significantly restored. This recovery was not comparatively suppressed by coadministration of periodate-oxidized adenosine, an inhibitor of S-adenosylhomo-cystenase, where the conversion of l-SeMet to l-SeCyH is inhibited. However, the recovery was strongly suppressed when propargylglycine, an inhibitor of cystathioine γ-lyase that catalyzes the α,γ-elimination reaction of both l-SeMet and seleno-l-cystathionine, was treated. These results suggest that cystathionine γ-lyase is a notable enzyme, in SeMet metabolism and that CH3SeH produced by the enzymatic reaction is utilized for cGPx biosynthesis.

Identification of mouse selenomethionine α,γ-elimination enzyme : Cystathionine γ-lyase catalyzes its reaction to generate methylselenol

The purpose of this study was to identify the seleno-l-methionine (l-SeMet) α,γ-elimination enzyme that catalyzes l-SeMet to generate methylselenol (CH3SeH), a notable intermediate for the metabolism of selenium compounds, in mammalian tissues. The enzyme purified from ICR mouse liver was separated by one-dimensional gel electrophoresis, and the specific band was subjected to in-gel trypsin digestion followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometric analysis. In the peptide mass fingerprinting search, the mass numbers of 14 peptides produced by tryptic digestion of the enzyme were consistent with the theoretical mass numbers calculated from the amino acid sequence of murine cystathionine γ-lyase (E.C. 4.4.1.1). The peptide sequence tags search was also performed to obtain the amino acid sequence data of five tryptic peptides. These peptides were significantly identical to the partial amino acid sequences of cystathionine γ-lyase. This enzyme was clearly shown to catalyze the α, γ-elimination reaction of l-cystathionine by the enzymological research. The Km value for the catalysis of l-cystathionine was 0.81 mM and Vmax was. 0.0013 unit/mg protein. These results suggested that cystathionine γ-lyase catalyzes l-SeMet to generate CH3SeH by its α,γ-elimination reaction.The purpose of this study was to identify the seleno-L-methionine (L-SeMet) alpha,gamma-elimination enzyme that catalyzes L-SeMet to generate methylselenol (CH3SeH), a notable intermediate for the metabolism of selenium compounds, in mammalian tissues. The enzyme purified from ICR mouse liver was separated by one-dimensional gel electrophoresis, and the specific band was subjected to in-gel trypsin digestion followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometric analysis. In the peptide mass fingerprinting search, the mass numbers of 14 peptides produced by tryptic digestion of the enzyme were consistent with the theoretical mass numbers calculated from the amino acid sequence of murine cystathionine gamma-lyase (E.C. 4.4.1.1). The peptide sequence tags search was also performed to obtain the amino acid sequence data of five tryptic peptides. These peptides were significantly identical to the partial amino acid sequences of cystathionine gamma-lyase. This enzyme was clearly shown to catalyze the alpha,gamma-elimination reaction of L-cystathionine by the enzymological research. The Km value for the catalysis of L-cystathionine was 0.81 mM and Vmax was 0.0013 unit/mg protein. These results suggested that cystathionine gamma-lyase catalyzes L-SeMet to generate CH3SeH by its alpha,gamma-elimination reaction.

Ozonated Olive Oil Enhances the Growth of Granulation Tissue in a Mouse Model of Pressure Ulcer

The curative effect of ozonated olive oil was evaluated using mouse models of cut wounds and pressure ulcers (decubitus or bedsores). Although ozonated olive oil did not significantly accelerate or decelerate wound contraction in either model, some histological modifications were observed. Ozonated olive oil induced blood coagulation in the hypodermis and cell infiltration in the dermis 1 day after its application. Moreover, it enhanced the formation of granulation tissue 10 days after application. These results indicate that ozonated olive oil promotes granulation tissue formation and is effective in the healing of wounds and pressure ulcers.

Chemical form of selenium-containing metabolite in small intestine and liver of mice following orally administered selenocystine.

The chemical form of a selenium-containing metabolite in the small intestine following a single oral administration of selenocystine was investigated with ICR male mice. Selenium content in the small intestine of animals treated with 50 mg/kg selenocystine significantly increased 15 min, 1 h and 6 h after treatment. In contrast, selenocystine significantly depressed the intestinal reduced glutathione (GSH) level at 1 h after administration. A significant negative correlation between the selenium level and the level of GSH in the small intestine was observed (r=−0.83, p<0.001). Analysis of the intestinal metabolite of selenocystine showed that selenium-containing metabolites elute in two fractions from a Sephadex G-25 column: the low-molecular fraction (peak I) contained the selenocystine, while the high-molecular fraction (peak II) contained selenocysteine-containing metabolite. An in vitro experiment was performed to gain insight into the mechanism for selenocysteine-containing metabolite production in the intestinal cytosol. When selenocystine or selenocysteine reacted with excess GSH in the presence of intestinal homogenate, the peak II fraction which involved the selenocysteine-containing metabolite was recognized in the Sephadex G-25 chromatogram. From an examination of the distribution of the selenocysteine-containing metabolite, it was recognized that this metabolite exists in plasma and liver cytosol of mice after oral administration of selenocystine. These results suggested that the mice treated with selenocystine produce selenocysteine-containing metabolite by reaction of selenocystine with excess GSH in the small intestine, and the metabolite is then transported to the liver through blood plasma.

Low-dose ethanol aggravates allergic dermatitis in mice

Alcohol injures dendritic cells and suppresses cellular immunity, while some evidence indicates that drinking alcohol aggravates allergic asthma. This study investigated the effect of low doses of ethanol in enhancing allergic reactions in the skin of mice. Liquid food containing alcohol was administered to conventional NC/Nga mice to induce alcoholic hepatic steatosis, and spontaneous dermatitis was evaluated. BALB/c mice were administered approximately 1 g/kg body weight of ethanol by gavage, and contact hypersensitivity (CHS) or active cutaneous anaphylaxis (ACA) was induced. Spleens were collected 24 h after the elicitation of CHS and mRNA expressions of interferon (IFN)-γ, interleukin (IL)-4, IL-6, IL-10, and IL-18 were measured by quantitative RT-PCR. Alcohol-containing diet exaggerated spontaneous dermatitis in conventional NC/Nga mice and contact hypersensitivity in BALB/c mice. Ethanol administered by gavage for 5 days enhanced contact hypersensitivity in BALB/c mice. Ethanol administration with gavage also enhanced ACA of BALB/c mice. Ethanol did not affect mRNA expression of IFN-γ and IL-4, but did enhance IL-6, IL-10, and IL-18 mRNA expression. Histological evaluation revealed an absence of hepatic steatosis in mice administered ethanol by gavage for 5 days. In ethanol-administered mice, inflamed areas presented as lesions or a local extreme accumulation of mononuclear cells in the epidermis. These findings suggest that ethanol enhances the expression of inflammatory cytokines independently from T helper (Th)1/Th2 cytokine phenotypes, causing abnormalities in the epidermis resulting in exacerbated allergic reactivity.

Identification of mouse selenomethionine α, γ-elimination enzyme

The purpose of this study was to identify the seleno-l-methionine (l-SeMet) α,γ-elimination enzyme that catalyzes l-SeMet to generate methylselenol (CH3SeH), a notable intermediate for the metabolism of selenium compounds, in mammalian tissues. The enzyme purified from ICR mouse liver was separated by one-dimensional gel electrophoresis, and the specific band was subjected to in-gel trypsin digestion followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometric analysis. In the peptide mass fingerprinting search, the mass numbers of 14 peptides produced by tryptic digestion of the enzyme were consistent with the theoretical mass numbers calculated from the amino acid sequence of murine cystathionine γ-lyase (E.C. 4.4.1.1). The peptide sequence tags search was also performed to obtain the amino acid sequence data of five tryptic peptides. These peptides were significantly identical to the partial amino acid sequences of cystathionine γ-lyase. This enzyme was clearly shown to catalyze the α, γ-elimination reaction of l-cystathionine by the enzymological research. The Km value for the catalysis of l-cystathionine was 0.81 mM and Vmax was. 0.0013 unit/mg protein. These results suggested that cystathionine γ-lyase catalyzes l-SeMet to generate CH3SeH by its α,γ-elimination reaction.The purpose of this study was to identify the seleno-L-methionine (L-SeMet) alpha,gamma-elimination enzyme that catalyzes L-SeMet to generate methylselenol (CH3SeH), a notable intermediate for the metabolism of selenium compounds, in mammalian tissues. The enzyme purified from ICR mouse liver was separated by one-dimensional gel electrophoresis, and the specific band was subjected to in-gel trypsin digestion followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometric analysis. In the peptide mass fingerprinting search, the mass numbers of 14 peptides produced by tryptic digestion of the enzyme were consistent with the theoretical mass numbers calculated from the amino acid sequence of murine cystathionine gamma-lyase (E.C. 4.4.1.1). The peptide sequence tags search was also performed to obtain the amino acid sequence data of five tryptic peptides. These peptides were significantly identical to the partial amino acid sequences of cystathionine gamma-lyase. This enzyme was clearly shown to catalyze the alpha,gamma-elimination reaction of L-cystathionine by the enzymological research. The Km value for the catalysis of L-cystathionine was 0.81 mM and Vmax was 0.0013 unit/mg protein. These results suggested that cystathionine gamma-lyase catalyzes L-SeMet to generate CH3SeH by its alpha,gamma-elimination reaction.

Oxidized dietary oils enhance immediate- and/or delayed-type allergic reactions in BALB/c mice

BACKGROUND The consumption of cooking oils may exacerbate some allergic diseases. In the present study, the effects of naturally oxidized olive oil on immediate- and/or delayed-type allergic reactions were investigated in BALB/c mice. METHODS Mouse models of 3 types of allergic reactions: contact hypersensitivity (CHS), active cutaneous anaphylaxis (ACA), and DNFB-induced hypersensitivity, were orally administered naturally oxidized olive oil that was obtained by keeping the oil at room temperature for more than 3 years. The effects of ultraviolet ray (UV)-irradiated olive oil and other dietary oils as well as their possible oxidation products on CHS were also investigated. RESULTS Naturally oxidized olive oil had a high peroxide value (POV) and exacerbated CHS, ACA, and DNFB-induced hypersensitivity in a POV-dependent manner. UV-irradiated olive oil, corn oil, sesame oil and triolein had high POVs, but almost the same acid value (AV) and thiobarbituric acid-reactive substance (TBARS) level as fresh oils. Fresh olive oil and the representative oxidation product with a high AV or TBARS level had no effect on CHS, whereas all UV-irradiated oils and naturally oxidized olive oil exacerbated it. CONCLUSIONS Oxidized dietary oils that have high POVs exacerbated immediate- and/or delayed-type allergic reactions regardless of the different oil constituents or oxidation processes.