Biol Trace Elem Res (2008) 125:13–21 DOI 10.1007/s12011-007-8044-0
MS07116 Sodium Selenosulfate Synthesis and Demonstration of Its In Vitro Cytotoxic Activity Against HepG2, Caco2, and Three Kinds of Leukemia Cells Jinsong Zhang & Hongjuan Lu & Xufang Wang
Received: 8 June 2007 / Revised: 16 August 2007 / Accepted: 23 September 2007 / Published online: 24 October 2007 # Humana Press Inc. 2007
Abstract The biological profile of sodium selenosulfate, Na2SeSO3, is still largely unknown. The present study found that sodium sulfite reacted with elemental selenium at nanoparticle size already at 37°C to yield sodium selenosulfate. Additionally, selenosulfate was obtained by mixing sodium selenite, glutathione, and sodium sulfite at room temperature. In vitro, sodium selenosulfate killed HepG2 or Caco2 cells, in a dosedependent fashion, and 12.5 μM fully suppressed their proliferation. In addition, sodium selenosulfate showed a consistent cytotoxic effect when added to three kinds of leukemia cell lines (HL60, T lymph adenoma, and Daudi). Keywords Selenosulfate . Synthesis . Cytotoxicity . Cancer cells . In vitro
Introduction The ability of selenium compounds to inhibit cell growth and to induce tumor cell apoptosis has been widely demonstrated [1–3] and has potential for therapeutic applications [4–6]. Among inorganic selenium compounds, most studies have been conducted with sodium selenite or selenate. In vivo, these salts are reduced to the pharmacologically active selenium species, whereas selenite is more readily reduced than selenate. We, therefore, felt that it would be advantageous to employ selenium compounds containing the element in a more reduced form. One such compound is sodium selenosulfate, Na2SeSO3, but to our knowledge thus far, it has not been used as selenium source in human or animal nutrition, and its biological profile is still largely unknown. Industrially, sodium selenosulfate, or preferably the potassium salt, is used for the preparation of nanoparticles of selenium-containing compounds, such as cadmium selenide, silver selenide, and copper selenide [7–9]. Potassium selenosulfate is more often used than the corresponding sodium salt because it is easily obtained by refluxing potassium sulfite J. Zhang (*) : H. Lu : X. Wang School of Chemistry and Material Science, University of Science and Technology of China, Hefei 230052 Anhui, People’s Republic of China e-mail:
[email protected]
14
Zhang et al.
with bulk powder of black elemental selenium [10]. The sodium salt is not well accessible in that way due to lower solubility of sodium selenite. In the present study, we describe improved laboratory methods for the preparation of sodium selenosulfate. As to the biological effects of sodium selenosulfate, we previously reported that sodium selenosulfate increased the activity of glutathione peroxidase and thioredoxin reductase in HepG2 cells; however, the bioavailability of selenosulfate in terms of increasing selenoenzymes and selenium accumulation was slightly but significantly less than selenite in selenium-deficient mice at a nutritional dose [11]. In the present study, we describe its cytotoxic effect against HepG2, Caco2, and three kinds of leukemia cells.
Materials and Methods Materials 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), sodium selenite, glutathione (GSH), sodium sulfite, and bovine serum albumin (BSA) were all obtained from Sigma (St. Louis, MO, USA). Other chemicals were of the highest grade available. Preparation of Sodium Selenosulfate from Sodium Sulfite and Elemental Selenium Elemental selenium reacts with sodium sulfite to form sodium selenosulfate according to the equation: Na2SO3 +Se → Na2SeSO3 (Fig. 1). The reaction is typically conducted by refluxing a mixture of 5 mM bulk powder of black elemental selenium and 20 mM sodium sulfite for 3 h at 90°C [12, 13]. Nanoparticles have huge surface area that renders a chemical reaction faster as compared with bulk powder; therefore, it could be extrapolated that sodium selenosulfate may be rapidly obtained if black elemental selenium is substituted by nanoparticles of red elemental selenium (Nano-Se). To testify, Nano-Se with different sizes was prepared according to previously reported methods [14–17]. Briefly, 1 ml of 25 mM sodium selenite was mixed with 4 ml 25 mM GSH containing 200, 20, and 2 mg Fig. 1 Chemical structure of sodium selenosulfate and sodium selenite
Formation and Cytotoxicity of Selenosulfate
15
Fig. 2 Transmission electron microscopy photographs of different sizes of red elemental selenium. a 5– 15 nm, b 20–60 nm, c 80–200 nm, d coarse particles
BSA. Transmission electron microscopy showed that the sizes of Nano-Se were 5–15 nm (Fig. 2a), 20–60 nm (Fig. 2b), and 80–200 nm (Fig. 2c), respectively. Nano-Se in these sizes was well dispersed in the protein solution. When BSA was decreased to 0.2 mg, the selenium atoms aggregated into coarse particles leading to lose solubility (Fig. 2d). Incubating black elemental selenium with sulfite at 37°C for 24 h revealed no evidence of a reaction. In contrast, the reaction of Nano-Se with a particle size of 5–15 nm in a 20 mM solution of sodium sulfite at 37°C was complete in less than 3 min, 20–60 nm Nano-Se took approximately 15 min, 80–200 nm Nano-Se took roughly 40 min, and coarse particles of red elemental selenium took more than 2 h. These results demonstrate that sodium selenosulfate can be easily obtained by substituting black elemental selenium with NanoSe, particularly at smaller sizes. Preparation of Sodium Selenosulfate from Sodium Selenite, GSH, and Sodium Sulfite Sodium selenosulfate can also be prepared by the reaction of sodium selenite with GSH, followed by adding sodium sulfite at the molar ratio of 1:4:4. An intermediate in this reaction is selenodiglutathione (GSSeSG) [18–20] at pH lower than 4. GSSeSG is unstable when the pH value increases to 6–7, decomposing into red elemental selenium and GSH
16
Zhang et al.
disulfide. After adding sodium sulfite, the pH becomes alkaline, and it causes the abrupt formation of red elemental selenium. The elemental selenium reacts with sulfite instantly to selenosulfate. Based on this principle, sodium selenosulfate was prepared by mixing 5 mM sodium selenite with 20 mM GSH, then adding 20 mM sodium sulfite at room temperature. Obviously, this protocol is more convenient for the preparation of sodium selenosulfate. It
related to control (%)
a
1, 30 µM sulfite. 2, 10 µM selenosulfate by refluxing black elemental Se with sulfite. 3, 10 µM selenosulfate by incubating Nano-Se with sulfite. 4, 10 µM selenosulfate by mixing selenite, glutathione and sulfite.
120 100 80 60 40 20 0 1
related to control (%)
b
2
3
4 selenite
120
selenosulfate
100 80 60 40 20 0 6
c related to day 0 (%)
Fig. 3 Cytotoxic effect of selenosulfate and selenite on HepG2 cells. a Cell killing effect of selenosulfate prepared by three different methods (mean±SD, n= 3); the cells were treated with selenium for 72 h; b cell killing effect of selenosulfate and selenite (mean±SD, n=3); the cells were treated with selenium for 72 h; c proliferation inhibitory effect of selenosulfate and selenite at 12.5 μM (mean±SD, n=8)
700
12 Se (µM )
18
control
600
selenite selenosulfate
500 400 300 200 100 0
0
2
4 day
6
Formation and Cytotoxicity of Selenosulfate
a
selenite
120 related to control (%)
Fig. 4 Cytotoxic effect of selenosulfate and selenite on Caco2 cells. a Cell killing effect of selenosulfate and selenite; the cells were treated with selenium for 72 h (mean±SD, n=3); b proliferation inhibitory effect of selenosulfate and selenite at 12.5 μM (mean±SD, n=8)
17
selenosulfate
100 80 60 40 20 0 3
6 Se (µM )
18
b 2500 related to day 0 (%)
control 2000
selenite selenosulfate
1500 1000 500 0 0
2
4
6
day
is worth noting that the fourfold excess sulfite over selenium is necessary [12, 13]. Thus, when the molar ratio of sulfite to elemental selenium was 1, selenosulfate could not be formed. At the ratio of 2, the resulting selenosulfate was not stable as evidenced by the formation of red elemental selenium within 3 h. When the ratio reached 4, the resulting selenosulfate was stable at room temperature for at least 10 h without the appearance of red elemental selenium. Cell Lines and Treatments Human hepatoma HepG2 and colon carcinoma Caco2 cells were cultured in MEM medium supplemented with FBS (10%), penicillin (100 U/ml), and streptomycin (100 mg/ml) in the atmosphere of 5% CO2 at 37°C. Leukemia cells (Daudi, T lymph adenoma, and HL60) were cultured under the same conditions as described herein except substituting MEM medium to RPMI1640 medium. For observation of cell proliferation, HepG2 or Caco2 cells were seeded in 96-well plate with each well containing 1,000 cells. Then sodium selenite and freshly prepared sodium selenosulfate were diluted in the medium at the concentration of 12.5 μM and added to cells for the times indicated. Each treatment contained eight replications. Cell viability was determined using MTT assay. Data are presented as the percent of cells at the beginning of selenium supplementation.
18
Zhang et al.
For the observations of cell killing, cells were seeded in 96-well plate with each well containing 5,000 cells. Sodium selenite and freshly prepared sodium selenosulfate were diluted in corresponding medium at the indicated concentrations and added to cells for 72 h. Each treatment contained three replications. Cell viability was determined using the MTT assay. Data are presented as the percent of cells remaining compared to cells without selenium supplementation.
a
selenite
related to control (%)
140
selenosulfate
120 100 80 60 40 20 0 3
6 Se (µM )
b related to control (%)
12 selenite
140
selenosulfate
120 100 80 60 40 20 0 3
6 Se (µM )
c
12 selenite
100 related to control (%)
Fig. 5 Cell killing effect of selenosulfate and selenite on leukemia cells (mean±SD, n=3). The cells were treated with selenium for 72 h. a Daudi cells, b T lymph adenoma cells, c HL60 cells
selenosulfate
80 60 40 20 0 3
6 Se (µM )
12
Formation and Cytotoxicity of Selenosulfate
19
Fig. 6 DNA fragmentation analysis after Se treatments. HL60 cells were treated with selenium for 6 h. 1 λ-DNA/EcoRI+HindIII markers, 2–4 selenosulfate 15, 10, and 5 μM, respectively, 5–7 selenite 15, 10, and 5 μM, respectively, 8 control
DNA Fragmentation Test About 1×106 HL60 cells in 3 ml RPMI1640 medium were seeded in 35-mm dish. After 3 h of incubation, different concentrations of sodium selenite and freshly prepared sodium selenosulfate were added. The cells were lysed at 6 h after selenium supplementation for DNA extraction. The DNA samples were separated by electrophoresis on a 1.2% agarose gel, then stained with ethidium bromide and scanned. Statistical Analysis The differences between the groups were examined using Student’s t test. A p value of less than 0.05 was considered statistically significant.
Results and Discussion Considering the use of sodium selenosulfate in biological studies, it must be taken into account that it is unstable in acidic solutions, decomposing with formation of red elemental selenium. Solutions of selenosulfate are also sensitive to oxygen. At room temperature in neutral or mildly alkaline solutions, the oxidation occurs slowly, allowing it to be used in in vitro biological studies. For example, the aerobic oxidation of the selenosulfate generated in solution from selenite, GSH, and sulfite at the molar ratio of 1:4:4 occurs only to a minor extent during the first 10 h and requires at least 96 h to completion. Therefore, to prepare and handle selenosulfate, several precautions must be kept in mind: Firstly, a fourfold molar sulfite can prevent the resulting selenosulfate from decomposing into red elemental selenium; secondly, the duration after its preparation for use should not exceed 10 h to avoid its oxidation; and thirdly, addition of hydrochloric acid to a selenosulfate solution will instantly form red elemental selenium and is a useful method to assess whether selenosulfate has been oxidized. Sodium selenosulfate prepared by the mentioned three methods was first tested in HepG2 cells to compare the killing effect at 10 μM. Because there was 30 μM additional sulfite in 10 μM selenosulfate, this concentration of sulfite alone was also included in the tests as an additional control. Compared to the control cells without treatment, 30 μM sulfite alone had no effect on HepG2 cell viability (Fig. 3a), whereas 10 μM selenosulfate
20
Zhang et al.
generated from all three methods could kill cells by approximately 60% (Fig. 3a). These results demonstrate that additional sulfite ions do not contribute to the cytotoxic effect of selenosulfate and selenosulfate prepared from different methods has equal cytotoxic activity. Therefore, further investigations of selenosulfate cytotoxicity were only performed from the mixture of selenite, GSH, and sulfite at the molar ratio 1:4:4. In HepG2 cells, selenosulfate ranging from 3 to 18 μM killed cells in a dose-dependent fashion (Fig. 3b). Selenosulfate was more potent than selenite at 12 and 18 μM (all p< 0.001; Fig. 3b). Furthermore, in a cell proliferation test, compared with the cells without selenium treatment, 12.5 μM selenosulfate suppressed growth and killed cells, whereas 12.5 μM selenite had only a weak effect on suppressing proliferation (Fig. 3c). The difference between the two selenium forms found in HepG2 cells was also observed in Caco2 cells (Figs. 4a,b). In addition, in the three kinds of leukemia cell lines (Daudi, T lymph adenoma, and HL60), 3–12 μM selenosulfate killed the cells in a dose-dependent fashion after 72 h of treatment (Fig. 5). The same concentrations of selenite over 72 h had no significant impact on cell viability except in HL60 cells at 12 μM (p
