Correlation between dielectric property by dielectrophoretic levitation and growth activity of cells exposed to electric field

The purpose of this study is to develop a system analyzing cell activity by the dielectrophoresis method. Our previous studies revealed a correlation between the growth activity and dielectric property (Re[K(ω)]) of mouse hybridoma 3-2H3 cells using dielectrophoretic levitation. Furthermore, it was clarified that the differentiation activity of many stem cells could be evaluated by the Re[K(ω)] without differentiation induction. In this paper, 3-2H3 cells exposed to an alternating current (AC) electric field or a direct current (DC) electric field were cultivated, and the influence of damage by the electric field on the growth activity of the cells was examined. To evaluate the activity of the cells by measuring the Re[K(ω)], the correlation between the growth activity and the Re[K(ω)] of the cells exposed to the electric field was examined. The relations between the cell viability, growth activity, and Re[K(ω)] in the cells exposed to the AC electric field were obtained. The growth activity of the cells exposed to the AC electric field could be evaluated by the Re[K(ω)]. Furthermore, it was found that the adverse effects of the electric field on the cell viability and the growth activity were smaller in the AC electric field than the DC electric field.     

Comprehensive Analysis of Human Cells Motion under an Irrotational AC Electric Field in an Electro-Microfluidic Chip

AC electrokinetics is a versatile tool for contact-less manipulation or characterization of cells and has been widely used for separation based on genotype translation to electrical phenotypes. Cells responses to an AC electric field result in a complex combination of electrokinetic phenomena, mainly dielectrophoresis and electrohydrodynamic forces. Human cells behaviors to AC electrokinetics remain unclear over a large frequency spectrum as illustrated by the self-rotation effect observed recently. We here report and analyze human cells behaviors in different conditions of medium conductivity, electric field frequency and magnitude. We also observe the self-rotation of human cells, in the absence of a rotational electric field. Based on an analytical competitive model of electrokinetic forces, we propose an explanation of the cell self-rotation. These experimental results, coupled with our model, lead to the exploitation of the cell behaviors to measure the intrinsic dielectric properties of JURKAT, HEK and PC3 human cell lines.     

Dielectrophoretic study of human erythrocytes

This paper is concerned with the dielectrophoretic study of human erythrocytes under cylindrical field geometry. The influence of physical variables such as the frequency and voltage of the applied electric field, conductivity of the medium in which the cells are suspended, cell concentration and exposure time of the cell to the non-uniform electric field on dielectrophoretic collection rate (DCR) is determined in a systematic manner. It is interesting to note from the DCR spectrum of human erythrocytes that the DCR is minimum at one frequency, maximum at another and there is practically no yield over a certain frequency range. This may be attributed to the variation of complex dielectric constant of the particle and medium over that frequency range. From the DCR spectrum of different groups, it is clear that DCR behaviour is different in the frequency range from 0.3–1.5 MHz, under similar conditions of temperature, conductivity and concentration of erythrocyte suspension and strength of applied AC field. The response of DCR with voltage of the applied field, concentration of cell suspension and square root of elapsed time of the cells confirms the theory of dielectrophoresis.     

Protein synthesis in a maize callus exposed to NaCl and mannitol

A maize (Zea mays, L) callus was exposed to media containing different levels of NaCl (0 to 3%) and mannitol (0 to 18.2%) for a period of 4 weeks, and the changes in growth and protein synthesis were determined. Cells are able to tolerate and grow in NaCl up to 1% (0.17 M) or mannitol up to 9.1% (0.5 M), but the relative overall growth rates are about 1/6 and 1/8 of the control, respectively. Protein synthesis, as assessed by pulselabeling of the cells with ³⁵S-methionine after exposure to the stress reagents at various times of incubation, suggests that the relative rates of amino acid uptake and its incorporation into proteins are inhibited as early as 4 h after exposure, and the extent of inhibition does not increase appreciably until after 1 week. Severe inhibition of uptake and protein synthesis results from prolonged exposures at growth-inhibitory concentrations of NaCl and mannitol. In general, the overall mean inhibition of cellular uptake and protein synthesis in the first 2-week period are approximately 50% and 35% for the NaCl (1%) and mannitol (7.3 %) treatments, respectively. No detectable differences are apparent in the abundant, steady state protein population as revealed by SDS-PAGE and on staining with Coomassie blue or silver, but random losses of individual proteins occur after 2 weeks at 2% and 3% NaCl and at 18.2% mannitol. Of the newly-synthesized proteins, discernible changes are found in 7 and 4 polypeptides in NaCl and mannitol treatments, respectively. Apparently three new proteins (74 kd, 28.5 kd, and 26.2 kd) are induced de novo under both treatments. Other proteins (39.5 kd, 39.0 kd, 30 kd, and 16.5 kd) show an increased or decreased level of synthesis. NaCl levels above 0.5% or mannitol levels above 3.6% do not after the pattern of newly-synthesized proteins. This altered expression of newly-made proteins in the maize callus occurs only after a week of exposure to salinity or osmotic stress and coincides with the cell growth phase.     

Effects of exposure to a 1950 MHz radio frequency field on expression of Hsp70 and Hsp27 in human glioma cells

Human glioma MO54 cells were used to investigate whether radio frequency (RF) field exposure could activate stress response genes. Cells were exposed to continuous wave 1950 MHz or sham conditions for up to 2 h. Specific absorption rates (SARs) were 1, 2, and 10 W/kg. For the cell growth experiment, cell numbers were counted at 0-4 days after exposure. Expression of Hsp27 and Hsp70, as well as the level of phosphorylated Hsp27 (78Ser) protein, was determined by Western blotting. It was found that sham exposed and RF exposed cells demonstrated a similar growth pattern up to 4 days after RF field exposure. RF field exposure at both 2 and 10 W/kg did not affect the growth of MO54 cells. In addition, there were no significant differences in protein expression of Hsp27 and Hsp70 between sham exposed and RF exposed cells at a SAR of 1, 2, or 10 W/kg for 1 and 2 h. However, exposure to RF field at a SAR of 10 W/kg for 1 and 2 h decreased the protein level of phosphorylated Hsp27 (78Ser) significantly. Our results suggest that although exposure to a 1950 MHz RF field has no effect on cell proliferation and expression of Hsp 27 and Hsp70, it may inhibit the phosphorylation of Hsp27 at Serine 78 in MO54 cells.     

Preconditioning of human smooth muscle cells via cyclopentenone prostaglandins protects against toxic effects of oxidized low-density lipoprotein

Human vascular smooth muscle cells (SMC) exhibit upregulation of inducible heat shock protein 70 (Hsp70), upon exposure to oxidized low-density lipoproteins (LDL(ox)). The presence of Hsp70 is thought to protect the cell against the toxic effects of the modified lipoprotein. In order to test this hypothesis, Hsp70 in SMC was upregulated by exposure to Delta(12) prostaglandin J(2) (Delta(12)PGJ(2)) before cells were exposed to LDL(ox). Hsp70 levels were measured after exposure to Delta(12)PGJ(2) and before exposure to LDL(ox). Cell protection was monitored after LDL(ox) exposure by determination of cell toxicity measured by cell lactate dehydrogenase (LDH) release into the medium. Cells treated with Delta(12)PGJ(2) exhibited a 23-fold increase in Hsp70 levels and 56% lower LDH activity release after exposure to LDL(ox) when compared to cells that were not pretreated with Delta(12)PGJ(2). In addition, cells pretreated with prostaglandins that did not induce Hsp70 did not exhibit increased tolerance against the toxic effects of LDL(ox). The results support a protective role for Hsp70 against the toxic effects of LDL(ox) and hint at the potential for the use of small molecules for the prevention of deleterious effects of LDL(ox) through heat shock protein upregulation.     

Experimental investigation on neural cell survival after dielectrophoretic trapping

Negative dielectrophoretic forces can effectively be used to trap cortical rat neurons. The creation of dielectrophoretic forces requires electric fields of high non-uniformity. High electric field strengths, however, can cause excessive membrane potentials by which cells may unrecoverably be changed or it may lead to cell death. In a previous study it was found that cells trapped at 3 Vtt/14 MHz did not change morphologically as compared to cells that were not exposed to the electric field. This study investigates the viability of fetal cortical rat neurons after being trapped by negative dielectrophoretic forces at frequencies up to 1 MHz. A planar quadrupole micro-electrode structure was used for the creation of a non-uniform electric field. The sinusoidal input signal was varied in amplitude (3 and 5 Vtt) and frequency (10 kHz-1 MHz). The results presented in this paper show that the viability of dielectrophoretically trapped postnatal cortical rat cells was greatly frequency dependent. To preserve viability frequencies above 100 kHz (at 3 Vtt) or 1 MHz (5 Vtt) must be used.     

In vitro analysis of the cellular resistance to chemotherapeutic BCNU.

Our assays in vitro show that BCNU inhibits cell proliferation in the C6 cell line experimental glioma and is dose-dependent, starting from 0.5 microgram/ml of the drug with just an hour of exposure. For every tested concentration of BCNU it is shown that, from the fifth day after exposure, cellular resistance appeared. This resistance is justified by the capacity of cell DNA reparation. A study of the clonogenic capacity of the C6 cells exposed to BCNU also shows the appearance of cellular resistance for doses of 0.5 microgram/ml and 1 microgram/ml. Furthermore, the exposure of C6 cell cultures to BCNU at these levels produces a cellular evolution towards more differentiated morphological patterns.     

Nuclear-cytoplasmic relationships in human cells in tissue culture. III. Auto-radiographic study of interrelation of nuclear and cytoplasmic ribonucleic acid

The movement of ribonucleic acid (RNA) from nucleus to cytoplasm has been studied, by autoradiographic techniques, in cells of the human amnion grown in tissue culture. Cells were exposed to cytidine-H(3) for 1 hour after which time only the RNA of the nuclei was labelled. After this 1 hour exposure the cells were placed in a medium containing an excess amount of unlabelled cytidine. Periodically, cells from this medium were fixed. Autoradiographs showed that there was a progressive movement of the label from nucleus to cytoplasm, such that after 24 hours essentially all the label was in the RNA of the cytoplasm. A study of the incorporation of the cytidine-H(3) in deoxyribonucleic acid (DNA), in the same population of cells at the same times, indicated that the presence of excess amounts of unlabelled cytidine almost instantaneously inhibited further utilization of cytidine-H(3). It is concluded that RNA moves from nucleus to cytoplasm as a complex polynucleotide structure.     

50-Hz extremely low frequency electromagnetic fields enhance cell proliferation and DNA damage: possible involvement of a redox mechanism

HL-60 leukemia cells, Rat-1 fibroblasts and WI-38 diploid fibroblasts were exposed for 24-72 h to 0.5-1.0-mT 50-Hz extremely low frequency electromagnetic field (ELF-EMF). This treatment induced a dose-dependent increase in the proliferation rate of all cell types, namely about 30% increase of cell proliferation after 72-h exposure to 1.0 mT. This was accompanied by increased percentage of cells in the S-phase after 12- and 48-h exposure. The ability of ELF-EMF to induce DNA damage was also investigated by measuring DNA strand breaks. A dose-dependent increase in DNA damage was observed in all cell lines, with two peaks occurring at 24 and 72 h. A similar pattern of DNA damage was observed by measuring formation of 8-OHdG adducts. The effects of ELF-EMF on cell proliferation and DNA damage were prevented by pretreatment of cells with an antioxidant like alpha-tocopherol, suggesting that redox reactions were involved. Accordingly, Rat-1 fibroblasts that had been exposed to ELF-EMF for 3 or 24 h exhibited a significant increase in dichlorofluorescein-detectable reactive oxygen species, which was blunted by alpha-tocopherol pretreatment. Cells exposed to ELF-EMF and examined as early as 6 h after treatment initiation also exhibited modifications of NF kappa B-related proteins (p65-p50 and I kappa B alpha), which were suggestive of increased formation of p65-p50 or p65-p65 active forms, a process usually attributed to redox reactions. These results suggest that ELF-EMF influence proliferation and DNA damage in both normal and tumor cells through the action of free radical species. This information may be of value for appraising the pathophysiologic consequences of an exposure to ELF-EMF.     

Synergic effect of retinoic acid and extremely low frequency magnetic field exposure on human neuroblastoma cell line BE(2)C

The aim of the present study was to assess whether exposure to a sinusoidal extremely low frequency magnetic field (ELF‐MF; 50 Hz, 1 mT) can affect proliferation and differentiation in the human neuroblastoma cell line BE(2)C, which is representative of high risk neuroblastomas. Cells were subjected to ELF‐MF exposure in the presence or absence of a neuronal differentiating agent (all‐trans‐retinoic acid, ATRA) for 24–72 h. In each experiment, ELF‐MF‐exposed samples were compared to sham‐exposed samples. Cells exposed to ELF‐MF combined with retinoic treatment showed a decreased cellular proliferation and an increased proportion of G₀/G₁ phase cells compared to cells exposed to either treatment alone. Moreover, ELF‐MF‐ and ATRA‐treated cells showed more differentiated morphological traits (a higher neurite number/cell, an increased neurite length), together with a significant increase of mRNA levels of p21^WAF1/CIP1 and cdk5 genes, both involved in neuronal differentiation. In addition, the expression of cyp19 gene, which is involved both in neuronal differentiation and stress response, was evaluated; cyp19 gene expression was enhanced by ATRA treatment and significantly enhanced further by ELF‐MF exposure combined with ATRA. In conclusion, our data suggest that ELF‐MF exposure can strengthen ATRA effects on neuroblastoma cells. Bioelectromagnetics 31:425–433, 2010. © 2010 Wiley‐Liss, Inc.     

Lysostaphin-Induced, Osmotically Fragile Staphylococcus aureus Cells

Staphylococcus aureus FDA 209P cells when suspended in 24% (w/v) NaCl were rendered osmotically fragile by exposure to lysostaphin for time intervals ranging from 2 to 60 min. Such cells were analyzed chemically and serologically for evidence of residual cell wall material, were plated in hypertonic sucrose medium to determine revertibility to normal, and were subjected to manometric studies to determine metabolic capabilities. Most of the cells (95%) which were exposed to lysostaphin (0.5 or 1.0 unit/ml) for 2 min, although osmotically fragile, retained their cell wall hexosamine and were capable of reverting to osmotically normal cells when plated in hypertonic medium. Cells exposed to lysostaphin for 5 and 10 min also retained much of their cell wall hexosamine, but lost their ability to revert to normal staphylococci. Cells exposed to lysostaphin for 2 to 10 min continued to react with staphylococcus anti-k antiserum. Complete removal of cell wall hexosamine was attained only after exposure to lysostaphin for 20 min or more; these cells failed to react with k antiserum. Lysostaphin-induced L-type colonies were extremely rare in our experiments, even if incubation times and media were optimal for their detection. Lysostaphin-induced staphylococcal protoplasts were as active metabolically in manometric studies as were untreated staphylococci.     

Effects of exposure to a time-varying 1.5 T magnetic field on the neurotransmitter-activated increase in intracellular Ca in relation to actin fiber and mitochondrial functions in bovine adrenal chromaffin cells

Background

It has been reported that exposure to electromagnetic fields influences intracellular signal transduction. We studied the effects of exposure to a time-varying 1.5 T magnetic field on membrane properties, membrane cation transport and intracellular Ca²⁺ mobilization in relation to signals. We also studied the mechanism of the effect of exposure to the magnetic field on intracellular Ca²⁺ release from Ca²⁺ stores in adrenal chromaffin cells.

Methods

We measured the physiological functions of ER, actin protein, and mitochondria with respect to a neurotransmitter-induced increase in Ca²⁺ in chromaffin cells exposed to the time-varying 1.5 T magnetic field for 2 h.

Results

Exposure to the magnetic field significantly reduced the increase in [Ca²⁺]i. The exposure depolarized the mitochondria membrane and lowered oxygen uptake, but did not reduce the intracellular ATP content. Magnetic field-exposure caused a morphological change in intracellular F-actin. F-actin in exposed cells seemed to be less dense than in control cells, but the decrease was smaller than that in cytochalasin D-treated cells. The increase in G-actin (i.e., the decrease in F-actin) due to exposure was recovered by jasplakinolide, but inhibition of Ca²⁺ release by the exposure was unaffected.

Conclusions and general significance

These results suggest that the magnetic field-exposure influenced both the ER and mitochondria, but the inhibition of Ca²⁺ release from ER was not due to mitochondria inhibition. The effect of eddy currents induced in the culture medium may indirectly influence intracellular actin and suppress the transient increase in [Ca²⁺]i.     

Extremely low frequency 7 Hz 100 microT electromagnetic radiation promotes differentiation in the human epithelial cell line HaCaT

Electromagnetic therapy is a treatment method in which an electromagnetic or magnetic stimulus is used to achieve physiological changes in the body. The specific aim of the present work concerns the effectiveness of low frequency electromagnetic fields to modify the biochemical properties of human keratinocytes (HaCaT). Cells exposed to a 7 Hz 100 microT electromagnetic field for one hour (twice daily), indicated modification in shape and morphology. These modifications were also associated with different actin distribution as revealed by phalloidin fluorescence analysis. Indirect immunofluorescence with fluorescent antibodies against involucrin and beta-Catenin, both differentiation and adhesion markers, revealed an increase in involucrin and beta-Catenin expression, supporting the conclusion that exposure to electromagnetic field carries keratinocytes to an upper differentiation level. This study confirms our previous observation and supports the hypothesis that 7 Hz electromagnetic field, may modify cell biochemistry interfering in the differentiation and cellular adhesion of normal keratinocytes.     

Effect of 72 Hz pulsed magnetic field exposure on macromolecular synthesis in CCRF-CEM cells.

Cells from the T-lymphoblastoid cell line, CCRF-CEM, have been exposed in vitro to a quasirectangular, asymmetric electromagnetic field pulsed at 72 Hz at 37 degrees for periods of 30 min to 24 h. RNA synthesis, assessed by incorporation of 3H-uridine, increased (relative to control cells) 2-fold after 30 min in exposed cells and achieved its greatest increase of 3.2-fold relative to controls after 2 h exposure. Increased precursor incorporation was observed at all subsequent exposure times up to 24 h. Synthesis of mRNA was similar, but not identical to that observed with total cellular RNA. Additionally, protein synthesis, determined by incorporation of radioactive precursor into acid-precipitable material, was increased 2.8-fold, compared to controls, after 2 h exposure. Longer exposure times resulted in an exponential decrease in precursor incorporation to 1.1-times control levels after 24 h. Using a dye reduction assay, mitochondrial activity was also found to be increased over a 24 h exposure period. No effect of electromagnetic field exposure was found on cellular synthesis of DNA. These data are generally consistent with other reports documenting effects of electromagnetic field exposure on macromolecular synthesis in vitro.     

Reversible and irreversible oxidant injury to PC12 cells by hydrogen peroxide.

A simple and reproducible model to identify biochemical changes associated with the transition from reversible to irreversible oxidant injury and cell death was established using rat pheochromocytoma PC12 cells. Cells were subjected to a transient oxidative stress induced by exposure to hydrogen peroxide (H2O2). Reversible loss of high-energy phosphates, induced by exposing cells to 0.2 mM H2O2, was preceded by transient increases in cytosolic calcium with no loss of plasma membrane integrity, as indexed by release of cytosolic enzymes. In contrast, permanent loss of high-energy phosphates, induced by treating cells with 0.5 mH H2O2, was associated with sustained rises in cytosolic-free calcium and increased oxidation of pyruvate and palmitate, two mitochondrial substrates. Initial production of pyruvate and lactate was inhibited by exposure to 0.5 mM H2O2 but returned to values comparable to control values at one hour after treatment with H2O2. Compromise of the plasma membrane was a late event, occurring between 1 and 2 hours after exposure to 0.5 mM H2O2. Collectively, these data indicate that irreversible loss of high-energy phosphates and cell death caused by oxidative stress is more closely associated with altered mitochondrial function than with impaired glycolysis.     

Citric Acid Fermentation

Abstract

Dielectrophoresis is the motion of particles caused by electrical polarization effects in inhomogeneous (nonuniform) electric fields. Unlike electrophoresis, the particles do not require a net electrical charge for motion to occur and AC rather than DC fields are employed to exploit the dielectric properties of the particles. Factors controlling the effective dielectric properties of cells and microorganisms include electrical double layers associated with surface charges, the conductivity and permittivity of their membranes and any cell walls, and their morphologies and structural architectures. In recent years, several laboratories have developed separation and manipulation techniques for cells and microorganisms based on dielectrophoresis, using both static and traveling AC fields. In this article, the basic physical factors influencing the dielectrophoretic behavior of particles are outlined, and ways in which these can be employed to achieve selective separation of cells and microorganisms are described.     

Nuclear-Cytoplasmic Relationships in Human Cells in Tissue Culture : III. Autoradiographic Study of Interrelation of Nuclear and Cytoplasmic Ribonucleic Acid

The movement of ribonucleic acid (RNA) from nucleus to cytoplasm has been studied, by autoradiographic techniques, in cells of the human amnion grown in tissue culture. Cells were exposed to cytidine-H³ for 1 hour after which time only the RNA of the nuclei was labelled. After this 1 hour exposure the cells were placed in a medium containing an excess amount of unlabelled cytidine. Periodically, cells from this medium were fixed. Autoradiographs showed that there was a progressive movement of the label from nucleus to cytoplasm, such that after 24 hours essentially all the label was in the RNA of the cytoplasm. A study of the incorporation of the cytidine-H³ in deoxyribonucleic acid (DNA), in the same population of cells at the same times, indicated that the presence of excess amounts of unlabelled cytidine almost instantaneously inhibited further utilization of cytidine-H³. It is concluded that RNA moves from nucleus to cytoplasm as a complex polynucleotide structure.