Teruaki Sakurai

Dimethylarsinic acid causes apoptosis in HL-60 cells via interaction with glutathione.

Abstract Inducibility of apoptosis in cultured human HL-60 cells by arsenic compounds, such as arsenite, arsenate, methylarsonic acid (MAA), and dimethylarisinic acid (DMAA), was investigated, together with the role of glutathione (GSH) in the induction. Among the arsenic compounds DMAA was the most potent in terms of the ability to cause the morphological changes (formation of nuclear fragmentation and apoptotic bodies) characteristic of apoptosis. Furthermore, fragmentation of internucleosomal DNA was also induced by DMAA. Depletion of cell GSH by L-buthionine-SR-sulfoximine, a selective inhibitor of γ-glutamylcysteine synthetase, enhanced the cytotoxicity of arsenite, arsenate, and MAA, while such depletion suppressed the cytotoxicity of DMAA. The depletion of GSH also suppressed the morphological changes and the fragmentation of internucleosomal DNA caused by DMAA, both of which are characteristic features of apoptosis. The results suggest that the death of cells caused by DMAA is due to apoptosis and that GSH is involved in the induction of apoptosis by this arsenic compound.

Enhancement of murine alveolar macrophage functions by orally administered β-glucan

The effect of orally administered SSG, a beta-1,3-glucan obtained from the culture filtrate of the fungus Sclerotinia sclerotiorum IFO 9395, on alveolar macrophage (AM) functions of CDF1 mice was examined. SSG administered orally (20, 40, 80 or 160 mg/kg) for 10 consecutive days enhanced the lysosomal enzyme activity of AM. The greatest enhancing effect was observed at 80 mg/kg of SSG. Multiple oral administrations of SSG (10 consecutive days) were needed to induce significant enhancing effects. Phagocytic activity and interleukin-1 (IL-1) production of AM were also augmented by oral administration of SSG, and the kinetics of the activated state differed depending on the kind of activity. However, H2O2 production of AM was not affected by SSG. Orally administered SSG also (40 or 80 mg/kg, 10 consecutive days) increased the number of AM and the greatest increment was observed 14 days after the first administration. On the other hand, the supernatant of Peyers patch (PP) cells from mice administered SSG (80 mg/kg) orally stimulated the lysosomal enzyme activity of AM in vitro, and enhanced colony stimulating activity (CSA) was detected from this supernatant. These results demonstrate that SSG given by the oral route can activate murine AM both qualitatively and quantitatively, and it would mediated, at least in part, by the activation of PP cells in the intestine.

Polymer-mediated extraction of 8-quinolinolato metal chelates for the determination of metal ions in water with graphite furnace atomic absorption spectrometry.

Water-insoluble 8-quinolinolato metal chelates were formed and were stably solubilized in the aqueous solution of a water-soluble polymer, poly (N-isopropylacrylamide)(PNIPAAm), at room temperature. When the solution was heated at 50 degrees C, PNIPAAm precipitated and then formed a gum-like aggregate (polymer phase) having a very small volume. Accompanying the polymer precipitation, hydrophobic 8-quinolinolato chelates with cobalt(II), iron(III), nickel(II), and copper(II) ions were efficiently incorporated into the polymer phase. At 0.5% (w/v) of PNIPAAm and 8.0 mM of 8-quinolinol, the recoveries in the incorporation of four metal chelates were quantitative. The fluorescence spectra of a probe suggests that the hydrated polymer in the aqueous solution provides hydrophobic portions which can incorporate hydrophobic metal chelates. The polymer phase was easily taken out from the solution and was dissolved with a small amount of acetonitrile. The resulting solution could be directly introduced into a graphite furnace of atomic absorption spectrometry. The signal intensities for the absorbance of cobalt after concentrating the chelate were 100-fold greater than those before the concentration.

Different role of serum components and cytokines on alveolar macrophage activation by soluble fungal (1→3)-β-d-glucan

In this study, we investigated the mechanism of alveolar macrophage activation by systemic administration of SSG, a soluble highly branched (1-->3)-beta-D-glucan obtained from a fungus Sclerotinia sclerotiorum IFO 9395. Multiple i.v. administration (10 mg/kg; once daily for 10 consecutive days) of SSG enhanced some functions of alveolar macrophages, such as lysosomal enzyme activity and nitric oxide secretion, on day 1 after the last administration, and it also elevated the concentrations of serum protein, interferon gamma and SSG in bronchoalveolar lavage fluid on the same day. On the in vitro assay system, stimulation by SSG alone (500 microg/ml) slightly augmented the lysosomal enzyme activity of alveolar macrophages, but it had no effect on nitric oxide production of cells. Stimulation by serum (1 or 10% mouse serum) or serum components, such as fibronectin (25 microg/ml) and albumin (500 microg/ml), alone strongly augmented only the lysosomal enzyme activity of alveolar macrophages, but it had no effect on nitric oxide secretion from cells, and no synergism or additive-like effect was observed between serum components and SSG. In contrast, stimulation by crude lymphokine (5%) or recombinant murine interferon gamma (100 U/ml) alone did not induce augmentation of lysosomal enzyme activity and nitric oxide production of alveolar macrophages in vitro, but when cells were incubated together with crude lymphokine or recombinant murine interferon gamma and SSG (500 microg/ml), a significant combined effect was observed on both functions of alveolar macrophages. In addition, pretreatment of crude lymphokine or recombinant murine interferon gamma enhanced the expression of beta-D-glucan specific binding sites on the alveolar macrophage surface in vitro though pretreatment by serum components had no effect. Based on these findings, the enhancement of alveolar macrophage functions by systemic administration of SSG appears to be mediated, at least in part, by both the simple effect of serum components including fibronectin and albumin leaked from pulmonary peripheral blood into the alveoli and the synergistic effect between lymphokines released from activated pulmonary T cells and SSG itself entering the alveoli after SSG injection via the priming effect of lymphokines which enhances the expression of beta-D-glucan specific binding sites on the alveolar macrophage surface.

Cytotoxicological aspects of organic arsenic compounds contained in marine products using the mammalian cell culture technique

Arsenobetaine, arsenocholine, trimethylarsine oxide and tetramethylarsonium iodide, which are contained in marine fishery products, were examined for their potencies on cell growth inhibition, chromosomal aberration and sister chromatid exchange (SCE). Arsenobetaine, the major water-soluble organic arsenic compound in marine animals, exhibited very low cytotoxicity towards mammalian cells. This compound showed no cell growth inhibition at a concentration of 10 mg cm -3 and the cytotoxicity was lower than 1/14 000th of that of sodium arsenite and 1/1600th of that of sodium arsenate towards BALB/c 3T3 cells. The chromosomal aberrations caused by arsenobetaine at a concentration of 10 mg cm -3 consisted mainly of chromatid gaps and chromatid breaks, but in this concentration chromosomal breakage owing to its osmotic pressure is likely to be considerable. No SCE was observed at a concentration of 1 mg cm -3 . Arsenocholine and trimethylarsine oxide also showed no cell growth inhibited at a concentration of 10 mg cm -3 . However, tetramethylarsonium iodide inhibition the growth of BALBIc 3T3 at a concentration of 8 mg cm -3 . These compounds exhibited a low ability to induce chromosomal aberrations at a concentration range of 2-10 mg cm -3 and no SCE was observed at a concentration of 1.0 mg cm -3 . These results suggested that the major and minor organic arsenic compounds contained in marine fishery products are much less cytotoxic inorganic arsenic, methylarsonic acid and dimethylarsinic acid.

Effects of fungal beta-glucan and interferon-gamma on the secretory functions of murine alveolar macrophages.

We investigated the effect of a fungal component, soluble β‐glucan, on secretory functions of murine alveolar macrophages (AMs) in vitro. Stimulation by β‐glucan (500 μg/mL) or interferon‐γ (IFN‐γ; 100 U/mL) alone had a slight effect on AM functions, but when AMs were incubated together with β‐glucan and IFN‐γ, the production and secretion of some immune mediators, such as nitric oxide, interleukin‐1 (IL‐1), IL‐6, and tumor necrosis factor‐α (TNF‐α), were markedly augmented. This combined effect of β‐glucan and IFN‐γ was based on a priming effect of IFN‐γ, because prestimulation with IFN‐γ followed by β‐glucan induced high nitric oxide production of AMs, but reversal of the sequence of treatments had only a slight effect. We also found that preincubation of AMs with IFN‐γ enhanced the binding of fluorescein‐labeled β‐glucan on the AM surface, and this increased binding was abrogated to the control level by the addition of three species of soluble unlabeled (1→3)‐β‐d‐glucans but not by soluble α‐glucan. These data imply that the priming effect of IFN‐γ on the AM response to β‐glucan was dependent, at least in part, on the enhancement of β‐glucan specific binding sites on the AM surface. It was suggested that IFN‐γ is one of the principal factors controlling the pulmonary immune system against both severe fungal infection and inflammation via AM activation at the alveoli.

Accumulation of arsenic in a unicellular alga Chlamydomonas reinhardtii

The unicellular alga Chlamydomonas reinhardtii accumulated and biomethylated arsenic efficiently. A wall-less cell strain (CW-15) of Chlamydomonas reinhardtii proliferated in a low level arsenic-containing medium (0.01-0.1 mmol dm -3 ) more than that in an arsenic-free medium. Although the growth of the algal cells was only slightly more inhibited in a growth medium containing arsenic at a concentration of 1.0 mmol dm -3 than that in an arsenic-free medium, it was completely inhibited at concentrations of 10 and 100 mmol dm -3 . Furthermore, transformed strains were obtained by random introduction of plasmid pJD67, carrying an Arg + gene, into a wall-less cell Arg - mutant CC425 strain. Finally we selected a strain, named AS1, among the transformed CC425 of the arsenic-sensitive group. The accumulation of arsenic by the AS1 strain was about three-fold higher than that by the CW-15 strain and 80-90% of the inorganic arsenic was transformed into a dimethylarsenic compound. It is suggested that the AS1 strain is a suitable model for investigation of the accumulation and biomethylation of arsenic by microalgae in freshwater environments.

Immunotoxicity of organic arsenic compounds in marine animals

In the present study, we demonstrated for the first time the immunotoxic effects of organic arsenic compounds in marine animals, namely arsenocholine [AsCho; trimethyl(2-hydroxyethyl)arsonium cation], arsenobetaine [AsBe; the trimethyl(carboxymethyl)arsonium zwitterion] and the tetramethylarsonium ion (TetMA), to murine principal immune effector cells (macrophages and lymphocytes), comparing them with the effects of inorganic arsenicals in vitro. Inorganic arsenicals (arsenite and arsenate) showed strong cytotoxicity to both macrophages and lymphocytes. The concentration of arsenite that reduced the number of surviving cells to 50% of that in untreated controls (IC 50 ) was 3-5 μmol dm -3 , and the cytotoxicity of arsenate (IC 50 =100 μ-1 m mol dm -3 ) was lower than that of arsenite. Compared with these findings, trimethylarsenic compounds in marine animals, AsCho and AsBe, were less toxic even at a concentration over 10 mmol dm -3 to both macrophages and lymphocytes; however, TetMA had weak, but significant, cytotoxicity to these cells (IC 50 was about 6 mmol dm -3 ).

Effect of soluble fungal (1→3)-β-d-glucan obtained from Sclerotinia sclerotiorum on alveolar macrophage activation

In this study, we examined the effect of systemic administration of SSG, a soluble highly branched (1-->3)-beta-D-glucan obtained from a fungus Sclerotinia sclerotiorum IFO 9395, on pulmonary immune responses in mice. SSG (10 mg/kg) administered intravenously (i.v.) rapidly leaked into the alveolar space and enhanced several functions of alveolar macrophages (AMs), such as phagocytic activity, lysosomal enzyme activity, active oxygen secretion and cytokine production, on day 1 post-administration. However, kinetic changes of influx of SSG into alveoli and AM activation after SSG treatment were different. The enhanced AM functions decreased to control value on day 2 when SSG still existed at the alveolar space. Additionally, a high dose (500 micrograms/ml) of SSG was needed to activate AMs in vitro. These data imply that the stimulation by SSG alone is not effective on AM activation. SSG administered i.v. also augmented interferon gamma (IFN gamma) mRNA expression in the lung tissue, and the kinetic change of the expression was similar to that of AM activation. Additionally, a synergistic effect of SSG and IFN gamma was observed on AM activation in vitro. It may be possible that IFN gamma produced by pulmonary T cells is one of the important factors for AM activation in vivo by SSG injection. Furthermore, SSG administered i.v. enhanced candidacidal activity and cytolytic activity against pulmonary metastatic Lewis lung carcinoma (3LL) cells of AMs, and inhibited significantly the experimental pulmonary metastasis of 3LL cells. These observations are very useful for the clinical application of SSG as a biological response modifier (BRM).

Modulation of cell adhesion and viability of cultured murine bone marrow cells by arsenobetaine, a major organic arsenic compound in marine animals.

In this study, we investigated the biological effects of trimethyl (carboxymethyl) arsonium zwitterion, namely arsenobetaine (AsBe), which is a major organic arsenic compound in marine animals using murine bone marrow (BM) cells and compared them with those of an inorganic arsenical, sodium arsenite, in vitro. Sodium arsenite showed strong cytotoxicity in BM cells, and its IC50 was 6 μM. In contrast, AsBe significantly enhanced the viability of BM cells in a dose‐dependent manner during a 72‐h incubation; about a twofold increase in the viability of cells compared with that of control cells cultured with the medium alone was observed with a μM level of AsBe. In morphological investigations, AsBe enhanced the numbers of large mature adherent cells, especially granulocytes, during a 72‐h BM culture. When BM cells were cultured together with AsBe and a low dose (1 u ml−1) of recombinant murine granulocyte/macrophage colony‐stimulating factor (rMu GM‐CSF), significant additive‐like increasing effects were observed on the numbers of both granulocytes and macrophages originated from BM cells. However, AsBe did not cause proliferation of BM cells at all as determined by colony‐forming assay using a gelatious medium. These findings demonstrate the unique and potent biological effects in mammalian cells of AsBe, a major organic arsenic compound in various marine animals which are ingested daily as seafood in many countries.