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

Abstract

1. 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. 2. Sodium arsenite showed strong cytotoxicity in BM cells, and its IC(50) was 6 microM. 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 microM level of AsBe. 3. 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 gelatinous medium. 4. 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.

Figure 1

Primary structure of AsBe and the f.a.b.-m.s. of synthesized AsBe. The f.a.b.-m.s. was performed using a JMS DX-300 mass spectrometer (JEOL Co., Tokyo) equipped with f.a.b. ion source and xenon atoms at 6 KeV. The f.a.b.-m.s. of AsBe showed m/z 135 [M-CO₂+H]⁺, m/z 179 [M+H]⁺, m/z 201 [M+Na]⁺, m/z 357 [2M+H]⁺ and m/z 379 [2M+Na]⁺.

Figure 2

Effect of arsenicals on the viability of thymocytes. Thymocytes isolated from CDF₁ mice were incubated with various doses of arsenite, AsBe or medium alone in the presence or absence of Con A (2.5 μg ml⁻¹) for 72 h at 37°C, and the viability of the cells was determined by AB assay. Results are expressed as arithmetic mean±s.d. of two thymocyte cultures each performed in duplicate dishes (n=4). ^*P<0.05 comparison with control thymocytes incubated with medium alone. ^**P<0.001.

Figure 3

Effect of arsenicals on the viability of BM cells. BM cells isolated from CDF₁ mice were incubated with various doses of arsenite, AsBe, trimethylarsine oxide, glycinebetaine or medium alone for 72 h at 37°C, and the viability of cells was determined by AB assay. Results are expressed as arithmetic mean±s.d. of two BM cultures each performed in duplicate dishes (n=4). ^*P<0.001 comparison with control BM cells incubated with medium alone. ^**P<0.01. ^***P<0.05.

Figure 4

Effect of AsBe on the adherent ability of BM cells. BM cells isolated from CDF₁ mice were incubated with 10 mM AsBe or medium alone on 96-well tissue culture plates for 24 or 72 h at 37°C. After the incubation, the wells were washed twice with warmed PBS to remove any nonadherent cells, and the remaining adherent cells on the culture plates were observed. This experiment has been repeated three times, yielding the same results, and one representative experiment is given. The magnification of the microphotographs is ×200.

Figure 5

Diff-Quik staining of adherent cells generated from BM cells by the incubation with AsBe. BM cells isolated from CDF₁ mice were incubated with 10 mM AsBe or medium alone on 96-well tissue culture plates for 72 h at 37°C. After the incubation, the wells were washed twice with warmed PBS to remove any nonadherent cells, and the remaining adherent cells on the culture plates were stained using a Diff-Quik stain kit. This experiment has been repeated three times, yielding the same results, and one representative experiment is given. The magnification of the microphotographs is ×200.