Selenite or selenomethionine interaction with methylmercury on uptake and toxicity showing a weak selenite protection: studies on cultured K-562 cells

Selenium and methylmercuric chloride (MMC) interactions regarding cellular uptake and selenium protection on MMC toxicity have been studied. Human K-562 cells were pretreated or simultaneously treated with either selenite (5 or 50 μM) or selenomethionine (10 or 50 μM) together with (3.5 or 5 μM) MMC. Cells simultaneously treated with selenite or selenomethionine and 3.5 μM MMC showed a decreased mercury concentration with increased selenium dose especially seen in the selenite combinations. The simultaneous selenite and MMC 3.5 μM combinations showed growth curves with an increasing number of viable cells with increased selenite dose. All combinations with 5 μM MMC were toxic to the cells. Interactions between selenite or selenomethionine and MMC regarding cellular uptake of mercury and selenium were observed and indications of selenite protection against MMC toxicity in human K-562 cells were noticed.     

Selenium protection against mercury-induced apoptosis and growth inhibition in cultured K-562 cells

Selenium and mercuric chloride (MC) interactions regarding effects on cell growth and cell death have been studied. Human K-562 cells were pretreated or simultaneously treated with either selenite (5 or 50 μM) or selenomethionine (10 or 50 μM) and with MC (35 or 50 μM). The 35-μM MC treatments resulted in a clear inhibition of cell growth with no obvious difference between mercury-treated and mercury-selenium-treated cells. Furthermore, the apoptotic frequency was similar at all observations for all selenium treatments with 35 μM MC. In the simultaneously treated selenite and 50-μM MC combinations, a selenite-dependent protection was shown both by increased cell growth and by lower apoptotic frequency at 48 and 96 h of exposure. Both treatments with selenomethionine showed protection observed as an increased cell growth at 48 and 96 h and as decreased apoptotic frequency at 96 h of exposure.     

Differences in the growth inhibition of cultured K-562 cells by selenium, mercury or cadmium in two tissue culture media (RPMI-1640, Ham's F-10)

Effects of some metals on the growth of cultured human erythroleukemia K-562 cells were investigated when grown in two different types of media based upon RPMI-1640 or Ham's F-10. The study on proliferation, using RPMI-1640 supplemented with sodium selenite, selenomethionine, mercuric chloride, methylmercuric chloride and cadmium nitrate showed no inhibition of growth at concentrations of 2.5, 25, 25, 2.5 and 25 M, while at 75, 250, 50, 5 and 50 M toxicity was apparent. Selenite at 5–50 M and selenomethionine at 50–100 M inhibited the growth. In Ham's F-10 supplemented with the same compounds no inhibition was found at concentrations of 5, 10, 25, 1 and 50 M, while at 50, 100, 50, 5 and 75 M toxic effects were noted. Selenite 10 M and selenomethionine 25-50 M inhibited the proliferation. Measurements of trace element levels in pellets of K-562 cells grown in RPMI-1640 or Ham's F-10 unveiled higher cell contents of cadmium and selenium in cells grown in RPMI-1640, being consistent with higher concentrations of these elements in that medium. Manganese and mercury concentrations were higher in cells grown in Ham's F-10 correlating with a higher medium concentration of these elements. The growth responses and cellular uptake differed between the metals and the selenocompounds and although extrapolating the results to humans is difficult the selenium exposures were in approximately the same order of magnitude as in human exposures. The compounds could be ranked according to decreasing toxicity as: methylmercuric chloride > mercuric chloride, cadmium nitrate, sodium selenite > selenomethionine.     

The metabolism of selenite and selenomethionine in mouse fibroblasts grown in tissue culture

Recent reports have provided evidence that selenium is an essential growth factor for cells grown in tissue culture. The aim of the work reported in this paper was to evaluate mouse fibroblasts as a model for the study of selenium metabolism in mammalian cells. The results showed that transformed mouse lung fibroblasts grown in media containing 9.1% bovine serum did not show a growth response to added selenium as selenite over the range of 10–1000 ng/mL. Uptake of selenium by cells was a direct function of the selenium concentration in the medium. The rate of uptake varied with the time of exposure of the cells to the selenium, and to the form of selenium in the medium. Experiments using radioactive selenium showed that⁷⁵Se from selenite was rapidly absorbed into the cell wall, but slowly incorporated into the soluble protein fraction.⁷⁵Se from selenomethionine was more slowly absorbed into the cells, but once inside, it became rapidly incorporated into soluble cytoplasmic proteins. Cell fractionation and gel filtration procedures established that⁷⁵Se from selenite was rapidly incorporated into glutathione peroxidase (GSHpx), whereas⁷⁵Se from selenomethionine was initially incorporated into a wide spectrum of proteins and only after a longer period did the⁷⁵Se peak become associated with GSHpx. These findings suggest fundamental differences exist in the manner in which mammalian cells initially absorb and metabolize different selenium compounds.     

小鼠腹水型肝癌细胞对竹红菌乙素的摄取和滞留

采用 SDS 胶束增溶法较详细地研究了小鼠腹水型肝癌细胞对竹红菌乙素的摄取和滞留过程.从实验结果表明:a.细胞摄取乙素与弗伦德里希吸附等温式符合较好,细胞的饱和吸附量为每个细胞吸附1.23×10⁸乙素分子.b.摄取速度很快,37℃下3min 即可达到饱和.c.被细胞摄取的乙素较难重新释放到溶液,而且未发生明显代谢变化.d.血清可携带乙素在细胞间传递.e.乙素趋向于进入细胞膜双分子层的深处.还结合实验结果对所采用的 SDS 胶束增溶法进行了评述.     

Effects of selenium and serum on selenoprotein W in cultured L8 muscle cells

When rat L8 muscle cells were cultured to examine the effects of serum and selenium concentration on selenoprotein W levels and glutathione peroxidase (GPX) activities, no significant differences (P > 0.05) were found in selenoprotein W levels and GPX activities during differentiation. With three different forms of selenium, selenoprotein W levels and GPX activities were shown to increase in L8 myotubes cultured in media with these selenocompounds. Selenite was utilized more efficiently than selenocysteine for both selenoprotein W and GPX activity, but selenium as selenomethionine was less available. Both the protein content and mRNA levels for selenoprotein W were affected by the selenium content of the media. Northern blot data indicated that the expression of selenoprotein W mRNA increased significantly when L8 myotubes were cultured with selenium (P > 0.05). L8 myotubes cultured in 10% calf serum (CS) versus 2% CS with or without addition of 10 m selenium indicated that the increase of selenoprotein W level in L8 myotubes cultured with higher serum concentration (10% CS) is due to the higher selenium concentration in media rather than serum itself.     

Uptake and release of tetracycline by cultured carrot cells

Summary The nature of tetracycline uptake by carrot cell suspension cultures is described. Tetracycline enters the cells by diffusion and the intracellular level of the antibiotic increases with the amount added. Exposure of carrot cells to high levels of tetracycline for a limited time (24 hr) followed by the removal of the drug and the resuspension of the cells in drug-free medium does not affect cell growth and has no inhibitory effect on protein synthesis (¹⁴C-leucine incorporation).     

Uptake of Selenium and its Antioxidant Activity in Ryegrass When Applied as Selenate and Selenite Forms

Selenium (Se) is an essential micronutrient for animal and human nutrition, but whether it is essential to plants remains controversial. However, there are increasing experimental evidences that indicate a protective role of Se against the oxidative stress in higher plants through Se-dependent glutathione peroxidase (GSH-Px) activity. The effects of the Se chemical forms, selenite and selenate, the rate of their application on shoot Se concentration and their influence on the antioxidative system of ryegrass (Lolium perenne cv. Aries), through the measurement of GSH-Px activity and lipid peroxidation, were evaluated in an Andisol of Southern Chile. Moreover, a soil–plant relationship for Se was determined and a simple method to extract available Se from acid soils is proposed. In a 55-day experiment ryegrass seeds were sown in pots and soil was treated with sodium selenite or sodium selenate (0–10 mg Se kg⁻¹). The results showed that the Se concentration in shoots increased with the application of both selenite and selenate. However, the highest shoot Se concentrations were obtained in selenate-treated plants. For both sources of Se, there was a significant positive correlation between the shoot Se concentration and the GSH-Px activity; and the Se-dependence of this enzymatic activity was related especially with the chemical form of applied Se rather than the Se concentration in plant tissues. Furthermore, the lipid peroxidation, as measured by Thiobarbituric Acid Reactive Substances (TBARS), decreased at low levels of shoot Se concentration, reaching the lowest level at approximately 20 mg Se kg⁻¹ in plants and then increased steadily above this level. In addition, the acid extraction method used to evaluate available Se in soil showed a positive good correlation between soil Se and shoot Se concentrations irrespective of chemical form of Se applied.     

Difference in Selenite Absorption Between High- and Low-Selenium Rice Cultivars and its Mechanism

Two rice cultivars, Xiushui 48 and S. Andrea, with significant difference in selenium (Se) concentrations in brown rice grains, were chosen to study the Se absorption and its mechanism in excised roots. The results showed that the high-selenium cultivar Xiushui 48 absorbed higher amounts of Na₂SeO₃ than low-selenium S. Andrea at different Se levels and treatment periods. It was found that Na₂SO₃ markedly inhibited Na₂SeO₃ absorption by the excised roots of both cultivars. This inhibition might be due to the competition for uptake on the fact that Na₂SO₃ might share a common uptake pathway with Na₂SeO₃. Treatment with ZnCl₂ significantly decreased Na₂SeO₃ absorption of both cultivars possibly by inhibiting the activity of cysteine synthase. It was therefore postulated that the difference in cysteine synthase activity might be one of the reasons which resulted in difference in selenite absorption possibly between the two cultivars. Both HgCl₂ and AgNO₃ treatments can inhibit selenite absorption by rice roots greatly. We propose that selenite enters rice roots through aquaporins as the form of H₂SeO₃.     

Uptake and genotoxic effects of ochratoxin A in cultured porcine urinary bladder epithelial cells

Uptake of radiolabelled ochratoxin A (OTA) into porcine urinary bladder epithelial cells (PUBEC) was measured at neutral (pH 7.5) or acidic (pH 5.0) conditions. Genotoxicity of OTA was evaluated with the Comet assay and cytotoxicity with the neutral red uptake assay. At acidic pH-conditions, the bladder cells were able to take up more OTA than at neutral conditions. Cytotoxic effects were not increased at pH 5.0 compared to pH 7.5, but higher OTA uptake correlated with stronger genotoxic effects in the Comet assay at pH 5.0 compared to pH 7.5. These results demonstrate that uptake of OTA has to be regarded as an important factor for the toxicity of OTA as adverse effects depend on the amount of OTA taken up by the cells. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

Retention of autonomous replicating plasmids in cultured Drosophila cells

Summary Six kinds of autonomously replicating sequences (ARSs) derived from Drosophila or tobacco were inserted into the vector pDSV, constructed with pSV2-gpt and the copia long terminal repeat (LTR). The resulting ARS-containing plasmids, pDSV-ARSs, were transfected into the cultured Drosophila cells of GM1 S1cl1. Most of the plasmids remained for about 2 weeks and some for about 1 month in these cells. The retention time of the plasmid was not directly correlated with autonomously replicating activity of ARSs detected in the yeast. Two plasmids, one carrying ARS of Drosophila nuclear DNA and the other carrying tobacco DNA, showed the longest retention time in transformed cells and replication was confirmed in these cells. Some of these long lived plasmids were recovered, however, as modified forms. Other plasmids had disappeared 1 month after transfection. Two months following transfection, none of plasmids were recovered but they were detected in nuclear DNA as the integrated form. The integration patterns in all the cells transformed by different kinds of ARS-containing plasmids were similar to each other, and to the distribution pattern of copia LTR in the genome. These results suggest that copia LTR sequences contained in the pDSV-ARSs may participate in the integration process of these plasmids into Drosophila DNA.     

Uptake of selenite,selenomethionine and selenate by brush border membrane vesicles isolated from rat small intestine

The uptake of selenite, selenate and selenomethionine (SeMet) was performed with brush border membrane vesicles (BBMV) prepared from rats fed selenium-deficient and supplemented diets. At equilibrium (60 min), the uptake of ⁷⁵Se from [⁷⁵Se]selenite ranged from 16.5 to 18.9 nmol mg^-1 protein. There was a curvilinear relationship in the uptake of selenite over a concentration range of 10–1000 m. About 2 nmol mg^-1 protein was obtained with selenomethionine (SeMet) which occurred between 90 and 180 s. In contrast to selenite, there was a linear relationship in the initial uptake of SeMet over a concentration range of 10–1000 m. The uptake of selenate was approximately 50-fold lower than selenite, reaching 350 pmol mg^-1 protein. Dietary selenium level had no effect on the rate of ⁷⁵Se accumulation by BBMV. Dramatic differences are found in the uptake and binding of selenium by BBMV incubated with different selenocompounds.     

Uptake and transport of selenite and selenate by soybean seedlings of two genotypes

In an attempt to address the role of biological behavior on Se uptake by soybean crop and the genotype effects, experiments with time and concentration sequences of Se uptake by seedlings in Hoagland solution are conducted using selenite and selenate respectively. Two soybean cultivars Tong-ai 405 (TA) and Qidong Green-skin (QG) are used as different genotypes. In presence of selenite, Se uptake by both roots and shoots exhibited a linear increase with the growing time at 5 M and with the solution Se concentrations. However, in presence of selenate, the linear response to growing time is only valid before 24 h of growing. While root Se uptake is much slower under selenate than under selenite in the time sequence experiment, shoot Se levels are similar between the two different Se form treatments. Nevertheless, in the experiment of concentration sequence, either root Se or shoot Se responses linearly to solution Se concentration regardless of the Se forms supplied. A big discrepancy of root Se level with a similarity of shoot Se between the two cultivars is observed in the concentration sequence experiment. This supports a much faster passive uptake of selenite but more or less an active uptake of selenate by soybean seedlings. Comparatively, cultivars TA have a consistently higher Se concentration than QG both in roots and shoots under selenate, while no difference of concentration ratio of shoot to root is recognized between them. The higher Se level in seed grains, therefore, may be accounted for not by Se transport form root to shoot but by greater ability of Se uptake and retention under selenate by the former cultivar. Therefore, not only forms of Se supply but also genotype difference affects the Se bioavailability by different soybean cultivars. This should be taken into account for screening the high Se-efficiency plants or cultivars to improve the Se supply of the food chain.     

Bioaccumulation of rare earth elements in cultured hela cells

HeLa S-3 cells were grown in minimal essential medium supplemented with 10% calf serum and 1 mM L-glutamine without adding any rare earth elements (REEs). Exponentially growing cells were collected, and dried materials were used to analyze their REE content by inductively coupled plasma-mass spectrometry. The results showed that the cells accumulated REEs in individually different manners; namely the accumulation ratio was higher in the lighter REEs than in the heavier REEs. To deduce the implication of the accumulation of REEs in HeLa cells, the accumulation ratios for REEs were compared with those of other biologically important elements. It was seen that the accumulation ratios obtained for REEs (from 31.8 [Ce] to 14.7 [Lu]) were intermediate among those of many bioelements: Fe (124), Mg (54.5), K (38.8), Cr (12.7), Na (11.8), Mn (11.3), Zn (10.7), Ca (8.8), and V (6.7).     

Serum-induced expression of metallothionein isoforms in K-562 cells

The metallothionein (MT) expression was studied in the hematopoietic precursor cell line K-562, after serum deprivation and reconstitution of the cells in medium with 10% (v/v) FCS. Serum deprivation for 72 h markedly downregulated the MT mRNA expression, only the isoforms most abundant in normal K-562 cells were clearly detectable. Within 1-1.5 h after serum supplementation however, a definite induction of MT mRNA was noticed, and all isoforms were induced. Forty-eight hours after serum stimulation, the MT mRNA expression of all isoforms decreased again. Also MT protein levels increased twofold 24 h after serum stimulation. These results suggest that MT has a function in the re-entry of resting cells into the cell cycle, this function however could not be assigned to a specific MT isoform. The induction of MT after serum stimulation was independent of protein synthesis, but dependent on phosphorylation.     

Uptake of the Herbicidal Glyphosate by Escherichia coli K-12

The uptake of the aminoacid biosynthesis inhibitor, used as the broad-spectrum herbicide ingredient, glyphosate (N-[phosphonomethyl]-glycine) was investigated in E. coli as a model to study mechanisms of cell resistance to antimetabolites as drugs and pesticides. Unlike the glyphosate-degrading Arthrobacter sp. strain for which the first successful measurement of glyphosate uptake and its inhibition by orthophosphate was reported [15], E. coli K-12 cannot take up this inhibitor either in the presence of orthophosphate, or after a prolonged starvation for it. However, cells made competent after an overnight cold CaCl₂ exposure followed by dimethyl sulfoxide (DMSO) treatment could take up this compound (K _m for glyphosate uptake, 274 M). Neither amino acids, belonging to a single transport system, nor orthophosphate gave essential inhibition of glyphosate uptake by these cells.     

微囊化K562细胞生长周期及代谢特性的研究

以K562细胞为模型,分别进行微囊化和游离培养,运用流式细胞术考察两种培养体系下细胞周期和生长代谢变化;建立数学模型,模拟了两种培养体系下细胞的生长活性和代谢特性。实验发现：微囊化培养过程中的K562细胞处于DNA合成期（S期）的百分含量显著高于游离培养,并且细胞保持较高的增殖活性。模型计算表明,所建模型动力学参数能够很好地描述微囊化和游离两种培养体系下细胞的代谢情况;对细胞活性的理论计算表明,微囊化的细胞具有较高的增殖和代谢活性,同时细胞能够较长时间保持此活性;模型参数表明,两种培养体系下,葡萄糖对细胞生长的影响无显著差别 (k^Free_L≈k^APA_L),乳酸对游离培养细胞的生长具有明显抑制作用,但对微囊化培养细胞抑制作用较小(kFreeL>≈kAPAL)。