Time dependent modifications of Hep G2 cells during exposure to static magnetic fields

Morphological modifications, i.e., cell shape, cell surface sugar residues, cytoskeleton, and apoptosis of Hep G2 cells during 24 h exposure to 6 mT static magnetic field (static MF) were studied by means of light and electron microscopy and cytochemistry. Progressive modifications of cell shape and surface were observed during the entire period of exposure to static MF. Control cells were polyhedric with short microvilli covering the cell surface, while those exposed to static MF, were elongated with many irregular microvilli randomly distributed on the cell surface. At the end of the exposure period, the cells had a less flat shape due to partial detachment from the culture dishes. However, throughout the period of exposure under investigation, the morphology of the organelles remained unmodified and cell proliferation was only partially affected. In parallel with cell shape changes, the microfilaments and microtubules, as well as the quantity and distribution of surface ConA-FITC and Ricinus communnis-FITC labeling sites, were modified in a time dependent manner. Apoptosis, which was almost negligible at the beginning of experiment, increased to about 20% after 24 h of continuous exposure. The induction of apoptosis was likely due to the increment of [Ca2+]i during exposure. In conclusion, the data reported in the present work indicates that 6 mT static MF exposure exerts time dependent biological effects on Hep G2 cells.     

Investigation on the effect of static magnetic field up to 30 mT on viability percent,proliferation rate and IC50 of HeLa and fibroblast cells

Abstract

We have investigated the effects of static magnetic field (SMF) on the viability of the human cervical cancer (HeLa) cell line and fibroblast cells. The cells were cultured in DMEM medium and treated several times (24, 48,72 and 96?h) and at several intensities (5, 10, 20 and 30?mT) of magnetic field (MF). The cytotoxicity and cell viability percent in treated cells were performed using MTT assay by evaluating mitochondrial dehydrogenase activity. The MF ability on inducing cell death or inhibiting biochemical function was reported as cell death percent. The results showed that the increase of MF intensity and the time that cells were exposed to this treatment increased sharply cell death percent and proliferation rate in HeLa cell compare to fibroblast cells. Our data suggest that SMF biological effects on cell death were different in our selected targets. Cell type and time of exposure have been therefore found to be significant factors. These findings could be used to improve new effective method using SMF in conjunction with the common therapeutic approaches.     

Biological effects of 6 mT static magnetic fields: a comparative study in different cell types

The present work was a comparative study of the bio-effects induced by exposure to 6 mT static magnetic field (MF) on several primary cultures and cell lines. Particular attention was dedicated to apoptosis. Cell viability, proliferation, intracellular Ca(2+) concentration and morphology were also examined. Primary cultures of human lymphocytes, mice thymocytes and cultures of 3DO, U937, HeLa, HepG2 and FRTL-5 cells were grown in the presence of 6 mT static MF and different apoptosis-inducing agents (cycloheximide, H(2)O(2), puromycin, heat shock, etoposide). Biological effects of static MF exposure were found in all the different cells examined. They were cell type-dependent but apoptotic inducer-independent. A common effect of the exposure to static MF was the promotion of apoptosis and mitosis, but not of necrosis or modifications of the cell shape. Increase of the intracellular levels of Ca(2+) ions were also observed. When pro-apoptotic drugs were combined with static MF, the majority of cell types rescued from apoptosis. To the contrary, apoptosis of 3DO cells was significantly increased under simultaneous exposure to static MF and incubation with pro-apoptotic drugs. From these data we conclude that 6 mT static MF exposure interfered with apoptosis in a cell type- and exposure time-dependent manner, while the effects of static MF exposure on the apoptotic program were independent of the drugs used.     

Effects of magnetic field on the antioxidant defense system of recirculation-cultured Chlorella vulgaris

Little is known about the influence of magnetic fields (MF) on growth of microalgae such as Chlorella vulgaris, which has been consumed as health food for various nutritional and pharmacological effects. This preliminary study investigated whether static MF can modulate the antioxidant system in C. vulgaris by exposing the cells to static MF generated by dual yoke electromagnets with magnetic flux density of 10-50 mT for 12 h. After exposure to 10-35 mT for 12 h, the activity of superoxide dismutases and peroxidase increased significantly compared to control cells. However, a remarkable increase of catalase activity occurred at 45 and 50 mT. The lipid peroxidation of algae cells determined by production of thiobarbituric acid-reactive substances was much increased when exposed to 35, 45, and 50 mT of MF. The scavenging ability of 2,2-diphenyl-1-picrylhydrazyl radical was decreased markedly while there was no variation of total carotenoids content in C. vulgaris cells. Assay of specific growth rate in 72 h cultivation after MF exposure was also conducted. In groups after exposure to 10-35 mT of MF, specific growth rate was significantly increased. These results suggest that 10-35 mT of static MF exposure could promote the growth of C. vulgaris and regulate its antioxidant defense system to protect cells efficiently, which could possibly enhance the growth of C. vulgaris in industrialized cultivation by MF.     

Interaction of Actinobacillus actinomycetemcomitans outer membrane vesicles with HL60 cells does not require leukotoxin

Outer membrane derived vesicles (MVs) secreted by Actinobacillus actinomycetemcomitans JP2 contain a membranolytic leukotoxin and are toxic to human HL60 cells. To determine how MVs interact with human target cells, HL60 cells were incubated with vesicles, reacted with anti-vesicle antibodies and a FITC-labelled reporter, and visualized by confocal scanning laser microscopy. Target cells rapidly became reactive with anti-vesicle antibodies upon exposure to vesicles. Confocal microscopy showed that labelling occurred primarily in the cytoplasmic membrane and that very little internal fluorescence was observed. The cytoplasmic membrane of HL60 cells was also strongly labelled after exposure to MVs that contained the fluorescent phospholipid, SP-DiOC18. In contrast, incubation of cells with free SP-DiOC18 resulted primarily in the labelling of internal structures of HL60 cells. These results suggest that A. actinomycetemcomitans MVs associate with, or are incorporated into the cytoplasmic membrane of HL60 cells. The leukotoxin is a membranolytic cytotoxin and cells exposed to MVs were lysed by vesicle-associated toxin in a time and dose-dependent manner. However, cells became reactive with anti-vesicle antibodies when MVs were added in the presence of inhibitors of leukotoxin-mediated lysis or when sublytic doses of MVs were analysed. In addition, MVs produced by an isogenic leukotoxin-deficient strain of A. actinomycetemcomitans JP2 were non-toxic but rapidly interacted with HL60 cells. These results suggest that A. actinomycetemcomitans MVs can deliver leukotoxin to HL60 cells but that the association of vesicles with the cytoplasmic membrane occurs independently of the leukotoxin polypeptide.     

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.     

Establishment, identification and characterization of a new sheep sinus tumor cell line

A cell line designated SRT was established from a sheep sinus tumor. Following primary culture, the cells were serially passaged 40 times. SRT cells maintained an epithelioid fibroblast-like appearance and had a population doubling time of approximately 18 hr. Karyotype analysis of 14th passage cells showed the modal 2n chromosome number to be between 46 to 60, due to a large variation in acrocentric chromosome number. The electrophoretic mobilities of enzymes extracted from SRT cells were identical with those from normal sheep sinus cells. It propagated a number of ovine, bovine and canine viruses. Some virus-like particles (80-120 nm) were observed under the electron microscope. The tumor origin, good growth and wide range of virus susceptibility make SRT a highly suitable cell line for in vitro cancer research and for comparative virology studies.     

Evaluation of isolation methods and culture conditions for rat bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (bMSCs) are multipotent and preferred for cell therapy. However, the content of bMSCs is very low. To propagate a large number of primary bMSCs rapidly has become a prerequisite for bMSC study and application. Different methods of isolating and culturing bMSC were used and compared among groups: bMSCs of group A are isolated using direct adherence method and cultured by conventional medium changing; of group B are isolated using direct adherence method and cultured by low volume medium changing; of group C are isolated using density gradient centrifugation and cultured by conventional medium changing; of group D are isolated using density gradient centrifugation and cultured by low volume medium changing. The average population doubling time (PDT), average generation time and the cumulative cell doubling level were calculated for every group. bMSCs cultured with complete medium containing 10, 11 and 15 % FBS were allocated into group a, b and c separatedly. Cell numbers were counted everyday under a microscope, the population doubling level curve was plotted and PDT was calculated. The growth curve of bMSC in group a, b and c was made. Both density gradient centrifugation and direct adherence methods obtained relatively pure bMSCs. A larger quantity of primary bMSCs were obtained by direct adherence. bMSC proliferation was faster when cultured via the low volume medium changing method at a serum concentration of 11 % than the other methods. Isolating bMSC by direct adherence and culturing by low volume medium changing at a serum concentration of 11 % is preferential for bMSC propagation.     

Phosphatidyl choline and the growth in serum-free medium of vascular endothelial and smooth muscle cells,and corneal endothelial cells

Liposomes made by sonication of egg yolk phosphatidyl choline support the proliferation of low-density bovine vascular and corneal endothelial cells, and vascular smooth muscle cells maintained on basement laminacoated dishes and exposed to a defined medium supplemented with transferrin. The optimal growth-promoting effect of phosphatidyl choline was observed at concentrations of 25 μg/ml for low-density cultures of vascular smooth muscle cells, and 100 μg/ml for vascular and corneal endothelial cells. The growth rate and final cell density of vascular endothelial cells exposed to a synthetic medium supplemented with transferrin and either high-density lipoproteins or phosphatidyl choline has been compared. Although cultures exposed to phosphatidyl choline reached a final cell density similar to that of cultures exposed to high-density lipoproteins, they had a longer average doubling time (17 h vs. 12 h) during their logarithmic growth phase and a shorter lifespan (17 generations vs. 30 generations). Similar observations were made in the case of vascular smooth muscle cells or bovine corneal endothelial cells maintained in medium supplemented with transferrin, fibroblast growth factor (FGF) or epidermal growth factor (EGF), and insulin and exposed to either high-density lipoproteins or phosphatidyl choline. Since phosphatidyl choline can, for the most part, replace highdensity lipoproteins in supporting the proliferation of various cell types, it is likely that the growth stimulating signal conveyed by high-density lipoproteins is associated with its polar lipid fraction, which is composed mostly of phosphatidyl cholines.     

Surface Phosphatidylserine Is Responsible for the Internalization on Microvesicles Derived from Hypoxia-Induced Human Bone Marrow Mesenchymal Stem Cells into Human Endothelial Cells

Background

Previous data have proven that microvesicles derived from hypoxia-induced mesenchymal stem cells (MSC-MVs) can be internalized into endothelial cells, enhancing their proliferation and vessel structure formation and promoting in vivo angiogenesis. However, there is a paucity of information about how the MSC-MVs are up-taken by endothelial cells.

Methods

MVs were prepared from the supernatants of human bone marrow MSCs that had been exposed to a hypoxic and/or serum-deprivation condition. The incorporation of hypoxia-induced MSC-MVs into human umbilical cord endothelial cells (HUVECs) was observed by flow cytometry and confocal microscopy in the presence or absence of recombinant human Annexin-V (Anx-V) and antibodies against human CD29 and CD44. Further, small interfering RNA (siRNA) targeted at Anx-V and PSR was delivered into HUVECs, or HUVECs were treated with a monoclonal antibody against phosphatidylserine receptor (PSR) and the cellular internalization of MVs was re-assessed.

Results

The addition of exogenous Anx-V could inhibit the uptake of MVs isolated from hypoxia-induced stem cells by HUVECs in a dose- and time-dependent manner, while the anti-CD29 and CD44 antibodies had no effect on the internalization process. The suppression was neither observed in Anx-V siRNA-transfected HUVECs, however, addition of anti-PSR antibody and PSR siRNA-transfected HUVECs greatly blocked the incorporation of MVs isolated from hypoxia-induced stem cells into HUVECs.

Conclusion

PS on the MVs isolated from hypoxia-induced stem cells is the critical molecule in the uptake by HUVECs.     

Magnetic field inhibits isolated lymphocytes' proliferative response to mitogen stimulation

We aimed to find out how the exposure of isolated lymphocytes to a pulsed magnetic field (MF) affected their in vitro proliferative response to mitogenic stimulation. Cells were exposed to MF of various intensities (0.3, 0.6, and 1.2 T) at a constant frequency of 30 Hz, for a period of 60, 180, and 330 s. Then, the proliferative response of splenocytes was induced by optimal concentrations of concanavalin A (Con A; mitogenic toward T cells), bacterial lipopolysaccharide (LPS; mitogenic toward B cells), or pokeweed mitogen (PWM; mitogenic toward both populations). We found that the exposure of lymphocytes to the MF profoundly inhibited their proliferative response to mitogens. The suppressive action of the MF on B and T cell proliferation was intensified when a cooperative response of those two lymphocyte populations was simultaneously induced by PWM. The inhibitory effect of MF depended on the exposure time and MF intensity. Prolonged exposure and/or a stronger intensity of the MF weakened its inhibitory influence on the response of lymphocyte to mitogenic stimulation. The data show that an exposure to MF may influence the activity of lymphocytes in their response to mitogenic stimuli.     

No evidence of cellular alterations by MilliTesla-level static and 50 Hz magnetic fields on S. cerevisiae

In an attempt to determine whether magnetic field (MF) exposures might induce cellular alterations, S. cerevisiae yeast cells were exposed to static or sinusoidal 50?Hz homogeneous MF (0.35?mT, 1.4?mT, and 2.45?mT) for 1?h and 72?h. Unsynchronized cells grown exponentially while exposed to MF, containing cells in all stages of the mitotic cell cycle. MF was generated by a pair of Helmholtz coils (40?cm in diameter, coaxial, separated by 20?cm). Survival, cell cycle distribution, colony forming ability, and mutation frequency were assayed. No differences in the above-mentioned parameters were observed in MF exposed samples in relation to unexposed controls, suggesting that homogeneous MF at these intensities do not produce appreciable cellular alterations in this organism under typical in vitro growth conditions.     

No Evidence of Cellular Alterations by MilliTesla-Level Static and 50 Hz Magnetic Fields on S. cerevisiae

In an attempt to determine whether magnetic field (MF) exposures might induce cellular alterations, S. cerevisiae yeast cells were exposed to static or sinusoidal 50?Hz homogeneous MF (0.35?mT, 1.4?mT, and 2.45?mT) for 1?h and 72?h. Unsynchronized cells grown exponentially while exposed to MF, containing cells in all stages of the mitotic cell cycle. MF was generated by a pair of Helmholtz coils (40?cm in diameter, coaxial, separated by 20?cm). Survival, cell cycle distribution, colony forming ability, and mutation frequency were assayed. No differences in the above-mentioned parameters were observed in MF exposed samples in relation to unexposed controls, suggesting that homogeneous MF at these intensities do not produce appreciable cellular alterations in this organism under typical in vitro growth conditions.     

Reproducibility and desensitization of the power frequency magnetic field effect on movement of the common cutworm Spodoptera litura

All creatures on Earth, including human beings, can be influenced by the power frequency electromagnetic field (EMF), even though the consequence and degree of the effect may vary due to regional context, species, etc. Most of the outstanding scientific achievements about the EMF effect on life have come from behavioral studies. In such studies, in contrast to the geomagnetic field or static magnetic field (MF), the oscillating MF has attracted far less attention so far. Following a previous report, to attain deep basic knowledge about the effect of an extremely low frequency (ELF) MF on animal behavior, we characterized the 60‐Hz MF‐responsive movement activity of common cutworm larvae using sophisticated experimental schemes. The MF‐exposed third instar larvae showed significantly reduced locomotive activity compared to the matching sham‐exposed larvae. Moreover, repeated MF exposure to the same larvae up to three times also showed similar behavioral responsiveness even though the extent of movement decrease was attenuated by the repetition time. These results suggest that sinusoidal power frequency MF could disrupt the normal locomotory activity of insect larvae, and the insects may show adaptive desensitization to the same MF.     

Microvesicles released from microglia stimulate synaptic activity via enhanced sphingolipid metabolism

Microvesicles (MVs) released into the brain microenvironment are emerging as a novel way of cell-to-cell communication. We have recently shown that microglia, the immune cells of the brain, shed MVs upon activation but their possible role in microglia-to-neuron communication has never been explored. To investigate whether MVs affect neurotransmission, we analysed spontaneous release of glutamate in neurons exposed to MVs and found a dose-dependent increase in miniature excitatory postsynaptic current (mEPSC) frequency without changes in mEPSC amplitude. Paired-pulse recording analysis of evoked neurotransmission showed that MVs mainly act at the presynaptic site, by increasing release probability. In line with the enhancement of excitatory transmission in vitro, injection of MVs into the rat visual cortex caused an acute increase in the amplitude of field potentials evoked by visual stimuli. Stimulation of synaptic activity occurred via enhanced sphingolipid metabolism. Indeed, MVs promoted ceramide and sphingosine production in neurons, while the increase of excitatory transmission induced by MVs was prevented by pharmacological or genetic inhibition of sphingosine synthesis. These data identify microglia-derived MVs as a new mechanism by which microglia influence synaptic activity and highlight the involvement of neuronal sphingosine in this microglia-to-neuron signalling pathway.     

Involvement of midkine expression in the inhibitory effects of low-frequency magnetic fields on cancer cells

Effects of magnetic fields (MFs) on cancer cells may depend on cell type and exposure conditions. Gene expression levels are different among cancer cells. However, the effect of MFs on cancer cells with different gene expressions is still unclear. In this study, the cancer cell lines BGC-823, MKN-45, MKN-28, A549, SPC-A1, and LOVO were exposed to a low-frequency MF. Specific parameters of MFs were determined. Furthermore, the potential of the MF to influence cancer cell growth with midkine (MK) expression was evaluated. Cell proliferation and cell cycle were detected using the CCK-8 assay and flow cytometry. Cell ultrastructure was observed by transmission electron microscopy. BGC-823 cells with over-expression of MK (BGC-MK cells) and stanniocalcin-1 were generated by plasmid construction and transfection. Results showed that exposure to a 0.4-T, 7.5 Hz MF inhibited the proliferation of BGC-823, MKN-28, A549, and LOVO cells, but not MKN-45 and SPC-A1 cells. Moreover, the inhibitory effect of the MF on BGC-MK cells was lower (12.3%) than that of BGC-823 cells (20.3%). Analysis of the cell cycle showed that exposure to the MF led to a significant increase in the S phase in BGC-823 cells, but not in BGC-MK cells. In addition, organelle morphology was modified in BGC-823 cells exposed to the MF. These results suggest that exposure to a 0.4-T, 7.5 Hz MF could inhibit tumor cell proliferation and disturb the cell cycle. The alteration of MK expression in cancer cells may be related to the inhibitory effect of the MF on these cells.     

The effect of weak 50 Hz magnetic fields on the number of free oxygen radicals in rat lymphocytes in vitro

The aim of the work was verification of the hypothesis that weak power frequency (50 Hz) magnetic fields (MF) affected the number of free oxygen radicals in living biological cells and that these changes could be qualitatively explained by the radical pair mechanism. The experiments were performed on rat lymphocytes. One-hour exposure to 50 Hz MF at 20, 40, or 200 microT flux densities was performed inside a pair of Helmholtz coils with axis along or crosswise to the Earth's static MF. Iron ions (FeCl2) were used as a stimulator of the oxidation processes. Oxygen radicals were measured by fluorimetry using a DCF-DA fluorescent probe. Only in the lymphocytes exposed at 40 microT MF directed along the Earth's static MF there was a decrease of fluorescence in relation to non-exposed samples. Our observation seems to confirm the hypothesis that low level power frequency MF affects oxidative processes which occur in living biological cells and that this effect can be explained by the radical pair mechanism.     

Photosensitizers and antioxidants: a way to new drugs?

Photodynamic therapy (PDT) is a relatively new modality of treatment of diseases involving uncontrolled cell proliferation. It is based on the production of reactive species upon illumination of a photosensitizer in the presence of oxygen. Antioxidants are primarily reducing agents prone to scavenge reactive species in one way or another. Their presence in photodynamic reactions usually reduces the efficacy of PDT. Some antioxidants like ascorbic acid, alpha-tocopherol or butyl-4-hydroxyanisole, however, when added to cells at adequate concentrations may enhance the photodamaging activity of PDT. The presence of transition metals and precise timing of antioxidant administration may also be important factors in increasing the efficacy of PDT. Antioxidant carrier sensitizers have been designed, synthesised and tested for their antibacterial PDT activity. The promising results raise the question whether the introduction of antioxidant moieties into sensitizer molecules would lead to the synthesis of highly effective new drugs.     

Effects of a 60 Hz magnetic field on photosynthetic CO2 uptake and early growth of radish seedlings

Photosynthetic CO2 uptake rate and early growth parameters of radish Raphanus sativus L. seedlings exposed to an extremely low frequency magnetic field (ELF MF) were investigated. Radish seedlings were exposed to a 60 Hz, 50 microT(rms) (root mean square) sinusoidal magnetic field (MF) and a parallel 48 microT static MF for 6 or 15 d immediately after germination. Control seedlings were exposed to the ambient MF but not the ELF MF. The CO2 uptake rate of ELF MF exposed seedlings on day 5 and later was lower than that of the control seedlings. The dry weight and the cotyledon area of ELF MF exposed seedlings on day 6 and the fresh weight, the dry weight and the leaf area of ELF MF exposed seedlings on day 15 were significantly lower than those of the control seedlings, respectively. In another experiment, radish seedlings were grown without ELF MF exposure for 14 d immediately after germination, and then exposed to the ELF MF for about 2 h, and the photosynthetic CO2 uptake rate was measured during the short-term ELF MF exposure. The CO2 uptake rate of the same seedlings was subsequently measured in the ambient MF (control) without the ELF MF. There was no difference in the CO2 uptake rate of seedlings exposed to the ELF MF or the ambient MF. These results indicate that continuous exposure to 60 Hz, 50 microT(rms) sinusoidal MF with a parallel 48 microT static MF affects the early growth of radish seedlings, but the effect is not so severe that modification of photosynthetic CO2 uptake can observed during short-term MF exposure.