Cell-density dependent effects of low-dose ionizing radiation on E. coli cells

The changes in genome conformational state (GCS) induced by low-dose ionizing radiation in E. coli cells were measured by the method of anomalous viscosity time dependence (AVTD) in cellular lysates. Effects of X-rays at doses 0.1 cGy--1 Gy depended on post-irradiation time. Significant relaxation of DNA loops followed by a decrease in AVTD. The time of maximum relaxation was between 5-80 min depending on the dose of irradiation. U-shaped dose response was observed with increase of AVTD in the range of 0.1-4 Gy and decrease in AVTD at higher doses. No such increase in AVTD was seen upon irradiation of cells at the beginning of cell lysis while the AVTD decrease was the same. Significant differences in the effects of X-rays and gamma-rays at the same doses were observed suggesting a strong dependence of low-dose effects on LET. Effects of 0.01 cGy gamma-rays were studied at different cell densities during irradiation. We show that the radiation-induced changes in GCS lasted longer at higher cell density as compared to lower cell density. Only small amount of cells were hit at this dose and the data suggest cell-to-cell communication in response to low-dose ionizing radiation. This prolonged effect was also observed when cells were irradiated at high cell density and diluted to low cell density immediately after irradiation. These data suggest that cell-to-cell communication occur during irradiation or within 3 min post-irradiation. The cell-density dependent response to low-dose ionizing radiation was compared with previously reported data on exposure of E. coli cells to electromagnetic fields of extremely low frequency and extremely high frequency (millimeter waves). The body of our data show that cells can communicate in response to electromagnetic fields and ionizing radiation, presumably by reemission of secondary photons in infrared-submillimeter frequency range.     

The effects of the microwaves on E. coli cells depend on oxygen concentration and static magnetic field

The effects of non-thermal microwaves (MW), 10(-4) and 10(-10) W/cm(2), on conformation of nucleoids in E. coli cells were analyzed by the method of anomalous viscosity time dependence (AVTD). MW exposure was performed at different values of static magnetic field and concentration of oxygen, 8-90 microT, and 2.3-7.8 mg/l, respectively. It was shown, that slight changes in both static magnetic field and oxygen concentration result in significant changes of MW effects up to their disappearance. It was established, that changes in static magnetic field affected significantly the time kinetics of the MW effects. The obtained data provide further evidence for strong dependence of the effects of non-thermal microwaves on physical parameters of exposure and physiological factors. These dependences should be taken into account in replication studies. The obtained results encourage further investigation of possible modulation of non-thermal MW effects by additional electromagnetic fields.     

Toxicity and SOS-response to ELF magnetic fields and nalidixic acid in E. coli cells

Extremely low-frequency magnetic fields (ELF-MF) have previously been shown to affect conformation of chromatin and cell proliferation. Possible genotoxic and carcinogenic effects of ELF-MF have also been discussed and tested. In this study, we analysed the effect of ELF-MF on chromatin conformation in E. coli GE499 cells by the anomalous viscosity time-dependence (AVTD) technique. Possible genotoxic effects of the specific combination of static and ELF-MF, which has been proven to affect chromatin conformation, were investigated by a clonogenic assay, by assessing cell-growth kinetics, and by analysis of the SOS-response by means of inducible recA-lacZ fusion-gene products and the β-galactosidase assay. The genotoxic agent nalidixic acid (NAL) was used as a positive control and in combination with ELF-MF. Nalidixic acid at 3-30μg/ml decreased the AVTD peaks and induced a cytotoxic effect. In contrast to NAL, ELF-MF fields increased AVTD, stimulated cell growth, and increased cloning efficiency. These effects depended on the frequency within the range of 7-11Hz. While NAL induced an SOS-response, exposure to ELF-MF did not induce the recA-lacZ fusion-gene product. Exposure to ELF-MF did not modify the genotoxic effects of NAL either. All together, the data show that ELF-MF, under specific conditions of exposure, acted as a non-toxic but cell-growth stimulating agent.     

Frequency-dependent effects of ELF magnetic field on chromatin conformation in Escherichia coli cells and human lymphocytes.

The effects of magnetic fields of extremely low frequency (ELF, 21 microT r.m.s.) on cells of different Escherichia coli K12 strains and human lymphocytes were studied by the method of anomalous viscosity time dependence (AVTD). Within the frequency range of 6-24 Hz, two resonance-type frequency windows with maximal effects at 9 Hz and 16 Hz were observed in response of GE499 strain. Only one frequency window with maximum effect at 8.5 Hz was found for GE500 cells. These data along with previously obtained for two other E. coli strains, AB1157 and EMG2, indicate that frequency windows are dependent on genotype of cells exposed to ELF. Resonance-type effects of ELF were also observed in human lymphocytes in frequency windows around 8 and 58 Hz. These ELF effects differed significantly between studied donors, but were well reproducible in independent experiments with lymphocytes from the same donors. The frequency windows in response of E. coli strains and human lymphocytes to ELF significantly overlapped suggesting that the same targets may be involved in this response. We compared the frequency windows with predictions based on the ion cyclotron resonance (ICR) model and the magnetic parametric resonance model. These models predicted effects of ELF magnetic fields at the 'cyclotron' frequencies of some ions of biological relevance. According to the ICR model, ELF effects should be also observed at harmonics of cyclotron frequencies and, contrary, parametric resonance model predicted effects at subharmonics. While we observed coincidence of each experimental resonance frequency with predictions of one of these two models, all experimentally defined effective frequency windows were in good agreement with relatively narrow frequency ranges of both harmonics and subharmonics for natural isotopes of Na, K, Ca, Mg, and Zn ions. The experimental data support idea that both harmonics and subharmonics of several biologically important ions are involved in frequency-dependent ELF effects in cells of different types.     

Toxicity and SOS response to ELF magnetic field and nalidixic acid in E. coli cells

Extremely low frequency (ELF) magnetic fields have previously been shown to affect conformation of chromatin and cell proliferation. Possible genotoxic and carcinogenic effects of ELF have also been discussed and tested. In this study, we analyzed the effect of ELF on chromatin conformation in E. coli GE499 cells by the anomalous viscosity time dependence (AVTD) technique. Possible genotoxic ELF effects at the specific combination of static and ELF magnetic fields, that has been proven to have effects on chromatin conformation, were investigated by clonogenic assay, cell growth kinetics, and analysis of SOS-response using inducible recA-lacZ fusion and the β-galactosidase assay. Genotoxic agent nalidixic acid (NAL) was used as positive control and in combination with ELF. Nalidixic acid at 3-30μg/ml decreased the AVTD peaks and induced cytotoxic effect. In contrast to NAL, ELF increased AVTD, stimulated cell growth, and increased cloning efficiency. These effects depended on frequency within the frequency range of 7-11Hz. While NAL induced SOS response, ELF exposure did not induce the recA-lacZ fusion. Exposure to ELF did not modify the genotoxic effects of NAL either. All together, the data show that ELF, under specific conditions of exposure, acted as nontoxic but cell growth stimulating agent.     

Cell-to-cell communication in response of E. coli cells at different phases of growth to low-intensity microwaves

Effects of millimeter waves (MMW) at the frequency of 51.755 GHz were studied in logarithmic and stationary E. coli cells at various cell densities. The changes in the genome conformational state (GCS) were analyzed by the method of anomalous viscosity time dependence (AVTD). Before lysis, the cells were adjusted to the cell density of 4x10(7) cells/ml and all AVTD measurements were run at this cell density. Stationary cells responded to MMW by increase in AVTD, while the same MMW exposure decreased AVTD in logarithmic cells. MMW effects depended on cell density during exposure and were stronger for stationary cells. The observed dependence on cell density suggested a cell-to-cell communication between cells during exposure to microwaves. Decrease in power density (PD) resulted in more striking differences between responses at different cell densities. The data provided evidence that intercellular communication in response to MMW depended on cell status and PD of microwaves. The MMW effects were studied in more detail at low intensity of 10(-17) W/cm(2) in the range of cell densities 4x10(7) to 8x10(8) cells/ml. The obtained sigmoid-like dependence of MMW effect on cell density saturated at approximately 5x10(8) cells/ml. The dependence of MMW effect on cell density was very similar in this study and in previous studies with weak extremely low frequency (ELF) electromagnetic fields (EMF). The data suggested that cell-to-cell communication might be involved in response of cells to weak EMF of various frequency ranges.     

Cell density dependent response of E. Coli cells to weak ELF magnetic fields

The effects of weak magnetic fields of extremely low frequency (ELF) on E. coli K12 AB1157 cells were studied by the method of anomalous viscosity time dependencies (AVTD). E. coli cells at different densities within a range of 5 × 10⁵–10⁹ cell/ml were exposed to ELF (sinusoidal, 30 μT peak, 15 min) at a frequency of 9 Hz. A transient effect with maximum 40–120 min after exposure was observed. Kinetics of the per-cell-normalised ELF effects fitted well to a Gaussian distribution for all densities during exposure. A maximum value of these kinetics and a time for this maximum were strongly dependent on the cell density during exposure. These data suggest a cell-to-cell interaction during response to ELF. Both dependencies had three regions close to a plateau within the ranges of 3 × 10⁵ − 2 × 10⁷ cell/ml, 4 × 10⁷ − 2 × 10⁸ cell/ml and 4 × 10⁸–10⁹ cell/ml and two rather sharp transitions between these plateaus. The effect reached a maximum value at a density of 4 × 10⁸ cell/ml. Practically no effect was observed at the lowest density of 3 × 10⁵ cell/ml. The data suggested that the ELF effect was mainly caused by a secondary rather than a primary reaction. The filtrates from exposed cells neither induced significant AVTD changes in unexposed cells nor increased the ELF effect when were added to cells before exposure. The data did not provide evidence for significant contribution of stable chemical messengers, but some unstable compounds such as radicals could be involved in the mechanism of cell-to-cell interaction during response to ELF. The results obtained were also in accordance with a model based on an re-emission of secondary photons during resonance fluorescence. Bioelectromagnetics 19:300–309, 1998. © 1998 Wiley-Liss, Inc.     

The peculiarities of the microwave in the frequency range of 51-52 GHz spectrum effects on E. coli cells

The effects of microwaves on conformation of nucleoids in E. coli cells were studied by the method of anomalous viscosity time dependence (AVTD) at various frequencies in the range of 51-52 GHz and the power flux density of 100 microW/cm(2) . Linearly polarized microwaves resulted in significant effects within specific frequency windows of resonance type. The distances between frequency windows were in the range of 55-180 MHz. Only one of two possible circular polarizations, left-handed or right-handed, was shown to be effective at each frequency window. The sign of effective circular polarization alternated between frequency windows. We show that the effects of microwaves on E. coli cells as measured by the AVTD technique are not caused by adhesion of cells. The half-width of the 51.575 GHz resonance was measured to be 120+/-20 MHz. This value is very close to the half-width of the 51.755 GHz resonance as it has previously been determined at the same power flux density. The obtained data suggest similar targets for effects of microwaves at these two resonance frequencies and provide evidence for non-thermal nature of observed microwave effects.     

Rotation of biological systems in a magnetic field: slitting of spectra for some magnetobiological effects

The interference mechanism for biological reception of weak magnetic fields was studied with consideration for the own molecular rotations of ion-protein complexes. An additional rotation of a biological system is shown to decrease the biological effect of "magnetic vacuum" and split spectral peaks from the effects of static magnetic field.     

ELF magnetic field affects proliferation of SPD8/V79 Chinese hamster cells but does not interact with intrachromosomal recombination

Extremely low-frequency (ELF) magnetic fields have previously been shown to affect conformation of chromatin, cell proliferation, and calcium metabolism. Possible mutagenic and carcinogenic effects of ELF have also been discussed and tested. In this study, intrachromosomal recombination in the hprt gene after exposure to ELF magnetic field was investigated using the SPD8 recombination assay. SPD8 cells, derived from V79 Chinese hamster cells were exposed to ELF at a specific combination of static and ELF magnetic fields, that has been proven to have effects on chromatin conformation in several cell types. The genotoxic agent camptothecin (CPT) was used either as a positive control or simultaneously with ELF. We also analysed the effect of ELF and CPT on chromatin conformation with the anomalous viscosity time dependence (AVTD) technique, cell growth kinetics, and cell survival with clonogenic assay. DNA fragmentation was analysed by pulsed field gel electrophoresis (PFGE). ELF did not induce recombination alone, neither did ELF modify the recombinogenic effect of CPT. Although, there was no effect on cell survival in response to ELF exposure, inhibition of cell growth was observed. On the other hand, ELF exposure partly counteracted the growth inhibition seen with CPT. The data suggest that ELF exposure may stimulate or inhibit cell growth depending on the state of the cells. Although, ELF did not induce recombination, a weak but statistically significant DNA fragmentation comparable with CPT-induced fragmentation was observed with PFGE 48h after exposure to ELF.     

Effect of low-intensity magnetic fields on the development of satellite muscle cells of a newborn rat in the primary culture

The influence of Earth magnetic field shielded down to 0.3 microT and static magnetic field (60-160 microT) on the proliferation and differentiation of satellite muscle cells in the primary culture has been investigated. A stimulatory effect of static magnetic fields on the rate of the formation of massive multinucleated myotubes and an increase in the intracellular calcium concentration ([Ca2+]i) have been detected for magnetic fields of the microtesla range. On the other hand, it was shown that the reduction of earth magnetic fields to 0.3 microT leads to the inhibition of proliferation and differentiation of skeletal muscle cells in the primary culture. Since the formation of contractile myotubes during in vitro experiments is similar to the regeneration of skeletal muscle fibers under muscle damage in vivo, it may be concluded that weak magnetic fields have a strong effect on intracellular processes by influencing all phases of muscle fiber formation. It is necessary to take this fact into consideration when forecasting probable complications of skeletal muscle regeneration during long-term exposure of man to low-intensity magnetic fields and also for the potential use of low static magnetic fields as a tool to recover the affected myogenesis.     

Effect of static magnetic fields on the budding of yeast cells

The effect of static magnetic fields on the budding of single yeast cells was investigated using a magnetic circuit that was capable of generating a strong magnetic field (2.93 T) and gradient (6100 T² m^?1). Saccharomyces cerevisiae yeast cells were grown in an aqueous YPD agar in a silica capillary under either a homogeneous or inhomogeneous static magnetic field. Although the size of budding yeast cells was only slightly affected by the magnetic fields after 4 h, the budding angle was clearly affected by the direction of the homogeneous and inhomogeneous magnetic fields. In the homogeneous magnetic field, the budding direction of daughter yeast cells was mainly oriented in the direction of magnetic field B. However, when subjected to the inhomogeneous magnetic field, the daughter yeast cells tended to bud along the axis of capillary flow in regions where the magnetic gradient, estimated by B(dB/dx), were high. Based on the present experimental results, the possible mechanism for the magnetic effect on the budding direction of daughter yeast cells is theoretically discussed. Bioelectromagnetics 31:622–629, 2010. © 2010 Wiley‐Liss, Inc.     

A simple and sensitive pulsed field gel electrophoresis protocol to study 50 kb apoptotic DNA fragmentation in human lymphocytes

DNA fragmentation of 50 kb is observed in apoptotic human lymphocytes as measured with pulsed field gel electrophoresis (PFGE). Standard PFGE assay involves embedding of cells into agarose blocks followed by lysis in the presence of proteinase K. In this study, we modified the PFGE protocol by omitting the proteinase K. In this study, we modified the PFGE assay by omitting the proteinase K and changing lysis solution according to the method of anomalous viscosity time dependence (AVTD). The conditions of PFGE were adjusted aiming to compress apoptotic fragments, increasing sensitivity and the number of samples that could be loaded on the same gel. Lymphocytes were irradiated with gamma-rays and apoptotic fragmentation of DNA was determined by PFGE using standard lysis with proteinase K and lysis protocol from AVTD method. Both protocols of lysis resulted in the same pattern of DNA fragments. The yield of radiation-induced apoptotic fragmentation was higher with the AVTD protocol of lysis. The novel PFGE protocol is simple and relatively non-expensive, requires only 7 h running time and gives a possibility to analyse simultaneously up to 69 samples in the same gel. The sensitivity of our protocol provides reproducible detection of 50 kb fragmentation after irradiation of human lymphocytes with 5 cGy of gamma-rays. In 2 of 6 donors tested, this DNA fragmentation was detected at dose on 2 cGy. The novel protocol can be used for quantification of 50 kb apoptotic fragments induced by different agents including low dose ionising radiations, chemicals and electromagnetic fields.     

Frequency-dependent effects of ELF magnetic field on chromatin conformation in Escherichia coli cells and human lymphocytes

The effects of magnetic fields of extremely low frequency (ELF, 21 μT r.m.s.) on cells of different Escherichia coli K12 strains and human lymphocytes were studied by the method of anomalous viscosity time dependence (AVTD). Within the frequency range of 6–24 Hz, two resonance-type frequency windows with maximal effects at 9 Hz and 16 Hz were observed in response of GE499 strain. Only one frequency window with maximum effect at 8.5 Hz was found for GE500 cells. These data along with previously obtained for two other E. coli strains, AB1157 and EMG2, indicate that frequency windows are dependent on genotype of cells exposed to ELF. Resonance-type effects of ELF were also observed in human lymphocytes in frequency windows around 8 and 58 Hz. These ELF effects differed significantly between studied donors, but were well reproducible in independent experiments with lymphocytes from the same donors. The frequency windows in response of E. coli strains and human lymphocytes to ELF significantly overlapped suggesting that the same targets may be involved in this response. We compared the frequency windows with predictions based on the ion cyclotron resonance (ICR) model and the magnetic parametric resonance model. These models predicted effects of ELF magnetic fields at the ‘cyclotron’ frequencies of some ions of biological relevance. According to the ICR model, ELF effects should be also observed at harmonics of cyclotron frequencies and, contrary, parametric resonance model predicted effects at subharmonics. While we observed coincidence of each experimental resonance frequency with predictions of one of these two models, all experimentally defined effective frequency windows were in good agreement with relatively narrow frequency ranges of both harmonics and subharmonics for natural isotopes of Na, K, Ca, Mg, and Zn ions. The experimental data support idea that both harmonics and subharmonics of several biologically important ions are involved in frequency-dependent ELF effects in cells of different types.     

Determination of the structure of Escherichia coli glyoxalase I suggests a structural basis for differential metal activation

The metalloenzyme glyoxalase I (GlxI) converts the nonenzymatically produced hemimercaptal of cytotoxic methylglyoxal and glutathione to nontoxic S-D-lactoylglutathione. Human GlxI, for which the structure is known, is active in the presence of Zn(2+). Unexpectedly, the Escherichia coli enzyme is inactive in the presence of Zn(2+) and is maximally active with Ni(2+). To understand this difference in metal activation and also to obtain a representative of the bacterial enzymes, the structure of E. coli Ni(2+)-GlxI has been determined. Structures have also been determined for the apo enzyme as well as complexes with Co(2+), Cd(2+), and Zn(2+). It is found that each of the protein-metal complexes that is catalytically active has octahedral geometry. This includes the complexes of the E. coli enzyme with Ni(2+), Co(2+), and Cd(2+), as well as the structures reported for the human Zn(2+) enzyme. Conversely, the complex of the E. coli enzyme with Zn(2+) has trigonal bipyramidal coordination and is inactive. This mode of coordination includes four protein ligands plus a single water molecule. In contrast, the coordination in the active forms of the enzyme includes two water molecules bound to the metal ion, suggesting that this may be a key feature of the catalytic mechanism. A comparison of the human and E. coli enzymes suggests that there are differences between the active sites that might be exploited for therapeutic use.     

Mononuclear metal complexes with piroxicam: synthesis, structure and biological activity

Piroxicam (=Hpir) is a non-steroidal anti-inflammatory and an anti-arthritic drug. VO(2+), Mn(2+), Fe(3+), MoO(2)(2+) and UO(2)(2+) complexes with deprotonated piroxicam have been prepared and characterized with the use of infrared, UV-Vis, nuclear magnetic resonance and electron paramagnetic resonance spectroscopies. The experimental data suggest that piroxicam acts as a deprotonated bidentate ligand in all complexes and is coordinated to the metal ion through the pyridine nitrogen and the amide oxygen. Molecular mechanics calculations in the gas state have been performed in order to propose a model for the Fe(3+), VO(2+) and MoO(2)(2+) complexes. Potential anticancer cytostatic and cytotoxic effects of piroxicam complexes with VO(2+), Mn(2+) and MoO(2)(2+) on human promyelocytic leukemia HL-60 cells have been investigated. Among all complexes, only VO(pir)(2)(H(2)O) clearly induces apoptosis after 24-h incubation, whereas piroxicam induces apoptosis after 57-h incubation.     

Increased antibiotic resistance of E. coli exposed to static magnetic fields

The present study demonstrates that exposure of bacteria to medium strength static magnetic fields can significantly alter antibiotic sensitivity. Cultures of Escherichia coli were exposed to fields produced by permanent magnets. Samples of bacterial cultures continuously growing in the presence and in the absence of static magnetic fields were left untreated or were treated with an antibiotic and measured at 45 min intervals for cell growth and survival. It was found that exposure of E. coli to the static fields significantly increased antibiotic resistance. Bioelectromagnetics 22:129-137, 2001. Published 2001 Wiley-Liss, Inc.     

Amplitude and frequency dissociation spectra of ion-protein complexes rotating in magnetic fields

A mechanism is presented that predicts new biological effects of static and sinusoidal weak magnetic fields. The model is based on an earlier proposed interference mechanism of quantum states of ions within protein cavities. The quantum dynamics of an ion is studied for the case of ion-protein complexes that rotate in magnetic fields. Both the individual molecular rotation and rotation together with a biological sample are taken into account. A formula is derived for the magnetic field-dependent part of the dissociation probability of an ion-protein in these conditions. The formula explains the unusual amplitude dependence of the known biological effect in PC-12 cells exposed to AC-DC magnetic field. The dependence had the functional motif J(2)(1)(2H(AC)/H(DC)), where J(1) is the first order Bessel function of the first kind. A good fit was obtained assuming individual rotation of the Li-protein complex in MF. The macroscopic rotation of a biological system, even at low speed 1.5-2 Hz, is predicted to reduce the biological effects of a "magnetic vacuum" and to shift the spectral peaks in the field and frequency dependencies of some magnetobiological effects.     

Relationship between radiation induced adaptive response in human fibroblasts and changes in chromatin conformation

Chromatin conformation changes in the normal human fibroblasts VH-10 were studied by the method of anomalous viscosity time dependence (AVTD). Gamma-irradiation of cells in a dose range of 0.1–3 Gy caused an increase in maximal viscosity of cell lysates. Conversely, irradiation of cells with low doses of 0.5 or 2 cGy resulted in a decrease in the AVTD peaks with a maximum effect approximately 40 min after irradiation. The same exposure conditions were used to study a possible adaptive effect of low doses, measured by changes in cell survival. A primary dose of 2 cGy caused significant modification of cell response to a challenge dose. Approximately 20% protection to challenge doses of 0.5 Gy (p < 0.003), 2 Gy (p < 0.02) and 2.5 Gy (p < 0.002) was observed. However, the direction of this effect (adaptation or synergism) was found to be dependent on a challenge dose. The combined effect of 2 cGy and 1 Gy was significantly synergistic, while no modification was observed for 1.5 Gy and 3 Gy. A partial correlation was found between the AVTD changes and cell survival when the combined effect of a primary dose of 2 cGy and challenge dose was examined. The dose of 2 cGy alone increased survival by 16% (p < 0.0003). These results suggest that the low-dose induced effects on survival may be related to chromatin reorganization.