Toshikazu Kaise

The acute toxicity of arsenobetaine

The acute toxicity of arsenobetaine was studied in male mice. No deaths were observed with oral administration of 10 g/kg of arsenobetaine. Therefore the LD50 value was higher than 10 g/kg. This compound was found in urine in the non-metabolized form. No particular toxic symptoms were observed following administration. These results suggest that arsenobetaine has low toxicity and is not metabolized in mice.

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.

The formation of trimethylarsine oxide from arsenobetaine by biodegradation with marine microorganisms

Abstract Bottom sediments collected from coastal waters were aerobically incubated in inorganic medium containing arsenobetaine as a carbon source. An arsenic compound converted from arsenobetaine during incubation was purified from the medium by ion exchange chromatography. The purified compound was identified to be trimethylarsine oxide by TLC, HPLC, FAB-MS and NMR.

Biomethylation of Arsenic in an Arsenic‐rich Freshwater Environment

Arsenic circulation in an arsenic-rich freshwater ecosystem was elucidated to detect arsenic species in the river water and in biological samples living in the freshwater environment. Water-soluble arsenic compounds in biological samples were extracted with 70% methanol. Samples containing arsenic compounds in the extracts were treated with 2 mol dm3 of sodium hydroxide and reduced with sodium borohydride. The detection of arsenic species was accomplished using a hydride generation/cold trap/cryofocus/gas chromatography-mass spectrometry (HG/CT/CF/GC-MS) system. The major arsenic species in the river water, freshwater algae and fish are inorganic arsenic, dimethylarsenic and trimethylarsenic compounds, respectively. Trimethylarsenic compounds are also detected in aquatic macro-invertebrates. The freshwater unicellular alga Chlorella vulgaris, in a growth medium containing arsenate, accumulated arsenic and converted it to a dimethylarsenic compound. The water flea Daphnia magna, which was fed on arsenic-containing algae, converted it to a trimethylarsenic species.

Rapid determination of arsenic species in freshwater organisms from the arsenic-rich Hayakawa River in Japan using HPLC-ICP-MS

Speciation analyses of water-soluble arsenicals from freshwater and biological samples collected from the Hayakawa River (Kanagawa, Japan), which contains a high concentration of arsenic, were performed using high performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC-ICP-MS). River water contained only arsenate, which is a pentavalent inorganic arsenical. The water bug Stenopsyche marmorata contained inorganic arsenicals accounting for 77% of the water-soluble arsenicals, followed by oxo-arsenosugar-glycerol, which is a type of dimethylarsinoylriboside (arsenosugar). The freshwater green macroalga Cladophora glomerata contained oxo-arsenosugar-glycerol and oxo-arsenosugar-phosphate as 64% of the water-soluble arsenicals. Production of the same types of arsenosugars was confirmed in the freshwater green microalga Chlamydomonas reinhardtii CC125 experimentally exposed to arsenate. The muscle tissues of all freshwater fish and crustaceans analyzed contained arsenobetaine, oxo-arsenosugar-glycerol, and/or oxo-arsenosugar-phosphate in various concentrations. Seven freshwater fish (Cobitis biwae, Leuciscus hakonensis, Phoxinus lagowski steindachneri, Plecoglossus altivelis, Rhinogobius sp. CB, Rhinogobius sp. CO, Sicyopterus japonicus) and the crustacean Macrobracbium nipponenese contained arsenobetaine in their muscle tissues as the predominant form, contributing up to 80% of the water-soluble arsenicals, while the freshwater fish Anguilla japonica muscle tissues primarily contained dimethylarsinic acid as 77% of the water-soluble arsenicals, followed by arsenobetaine. The freshwater fish Zacco platypus muscle tissues predominantly contained oxo-arsenosugar-phosphate, accounting for 51% of the water-soluble arsenicals, followed by dimethylarsinic acid and arsenobetaine. These biological samples possessed non-extractable arsenical(s) accounting for more than 50% of the total arsenic concentration.

Speciation of arsenic trioxide metabolites in blood cells and plasma of a patient with acute promyelocytic leukemia

Arsenic trioxide (As2O3) has been widely accepted as the second-best choice for the treatment of relapsed and refractory acute promyelocytic leukemia (APL) patients. However, a few studies have been conducted on a detailed speciation of As2O3 metabolites in blood samples of patients. To clarify the speciation of arsenic, the blood samples were collected at various time points from a patient with APL after remission induction therapy and during consolidation therapy. The total amounts of arsenic in blood cells and plasma, and the plasma concentrations of inorganic arsenic and methylated metabolites were determined by inductively coupled plasma mass spectrometry (ICP-MS) and high-performance liquid chromatography/ICP-MS, respectively. The total amounts of arsenic in the blood cells were 4–10 times higher than those in plasma. Among all arsenic metabolites, the pentavalent arsenate (AsV) in plasma was more readily eliminated. During the drug-withdrawal period, the initial plasma concentrations of trivalent arsenic (AsIII) declined more rapidly than those of methylarsonic acid and dimethlyarsinic acid, which are known as the major methylated metabolites of AsIII. On the other hand, during the consecutive administration in the consolidation therapy period, the plasma concentrations of total arsenic and arsenic metabolites increased with time. In conclusion, these results may support the idea that methylated metabolites of As2O3 contribute to the efficacy of arsenic in APL patients. These results also suggest that detailed studies on the pharmacokinetics as well as the pharmacodynamics of As2O3 in the blood cells from APL patients should be carried out to provide an effective treatment protocol.

Rapid Biotransformation of Arsenate into Oxo-Arsenosugars by a Freshwater Unicellular Green Alga, Chlamydomonas reinhardtii

We examined the short-term metabolic processes of arsenate for 24 h in a freshwater unicellular green alga, Chlamydomonas reinhardtii wild-type strain CC-125. The arsenic species in the algal extracts were identified by high-performance liquid chromatography/inductively coupled plasma mass spectrometry after water extraction using a sonicator. Speciation analyses of arsenic showed that the levels of arsenite, arsenate, and methylarsonic acid in the cells rapidly increased for 30 min to 1 h, and those of dimethylarsinic acid and oxo-arsenosugar-glycerol also tended to increase continuously for 24 h, while that of oxo-arsenosugar-phosphate was quite low and fluctuated throughout the experiment. These results indicate that this alga can rapidly biotransform arsenate into oxo-arsenosugar-glycerol for at least 10 min and then oxo-arsenosugar-phosphate through both reduction of incorporated arsenate to arsenite and methylation of arsenite and/or arsenate retained in the cells to dimethylarsinic acid via methylarsonic acid as an possible intermediate.

Effects of Naturally Occurring Polymethyoxyflavonoids on Cell Growth, P-Glycoprotein Function, Cell Cycle, and Apoptosis of Daunorubicin-Resistant T Lymphoblastoid Leukemia Cells

ABSTRACT Effects of polymethoxyflavonoids tangeretin and nobiletin and the related polyphenolic compounds baicalein, wogonin, quercetin, and epigallocatechin gallate on the cell growth, P-glycoprotein function, apoptosis, and cell cycle of human T lymphoblastoid leukemia MOLT-4 and its daunorubicin-resistant cells were investigated. The IC50 values of these compounds on the cell growth were 7.1–32.2 μmol/L, and the inhibitory effects were observed to be almost equal to the parent MOLT-4 and the daunorubicin-resistant cells. Tangeretin and nobiletin showed the strongest effects with the IC50 values of 7.1–14.0 μmol/L. These polymethoxyflavonoids inhibited the P-glycoprotein function and significantly influenced the cell cycle (p<.05), whereas they did not induce apoptosis.

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.

Contribution of aquaporin 9 and multidrug resistance-associated protein 2 to differential sensitivity to arsenite between primary cultured chorion and amnion cells prepared from human fetal membranes

Arsenic trioxide (arsenite, As(III)) has shown a remarkable clinical efficacy, whereas its side effects are still a serious concern. Therefore, it is critical to understand the effects of As(III) on human-derived normal cells for revealing the mechanisms underlying these side effects. We examined the effects of As(III) on primary cultured chorion (C) and amnion (A) cells prepared from human fetal membranes. A significant dose-dependent As(III)-mediated cytotoxicity was observed in the C-cells accompanied with an increase of lactate dehydrogenase (LDH) release. Higher concentrations of As(III) were required for the A-cells to show cytotoxicity and LDH release, suggesting that the C-cells were more sensitive to As(III) than the A-cells. The expression levels of aquaporin 9 (AQP9) were approximately 2 times higher in the C-cells than those in the A-cells. Both intracellular arsenic accumulation and its cytotoxicity in the C-cells were significantly abrogated by sorbitol, a competitive AQP9 inhibitor, in a dose-dependent manner. The protein expression levels of multidrug resistance-associated protein (MRP) 2 were downregulated by As(III) in the C-cells, but not in the A-cells. No significant differences in the expression levels of MRP1 were observed between C- and A-cells. The protein expression of P-glycoprotein (P-gp) was hardly detected in both cells, although a detectable amount of its mRNA was observed. Cyclosporine A, a broad-spectrum inhibitor for ABC transporters, and MK571, a MRP inhibitor, but not PGP-4008, a P-gp specific inhibitor, potently sensitized both cells to As(III)-mediated cytotoxicity. These results suggest that AQP9 and MRP2 are involved in controlling arsenic accumulation in these normal cells, which then contribute to differential sensitivity to As(III) cytotoxicity between these cells.