The effect of electromagnetic field on reactive oxygen species production in human neutrophils in vitro

The present study was undertaken in order to determine the effect of low frequency electromagnetic field (EMF) on reactive oxygen species (ROS) production in human neutrophils in peripheral blood in vitro. We investigated how differently generated EMF and several levels of magnetic induction affect ROS production. To evaluate the level of ROS production, two fluorescent dyes were used: 2′7′-dichlorofluorscein-diacetate and dihydrorhodamine. Phorbol 12-myristate 13-acetate (PMA), known as strong stimulator of the respiratory burst, was also used. Alternating magnetic field was generated by means of Viofor JPS apparatus. Three different levels of magnetic induction have been analyzed (10, 40 and 60 μT). Fluorescence of dichlorofluorescein and 123 rhodamine was measured by flow cytometry. The experiments demonstrated that only EMF tuned to the calcium ion cyclotron resonance frequency was able to affect ROS production in neutrophils. Statistical analysis showed that this effect depended on magnetic induction value of applied EMF. Incubation in EMF inhibited cell activity slightly in unstimulated neutrophils, whereas the activity of PMA-stimulated neutrophils has increased after incubation in EMF.     

Production of reactive oxygen species by Hemocytes from the marine mussel,Mytilus edulis: Lysosomal localization and effect of xenobiotics

Hemolymph of M. edulis is rich in phagocytic hemocytes. Hemocytes contain numerous lysosomes which, in turn, contain various hydrolytic enzymes. Phagocytic activity of M. edulis hemocytes is thought to be associated with NAD(P)H-oxidase activity of the plasma membrane. The laser dye, dihydrorhodamine 123 (DHR), was used for cytochemical and biochemical detection of the generation of reactive oxygen species (ROS) by isolated M. edulis hemocytes. Hemocytes readily take up DHR from the suspension medium and selectively concentrate it in the lysosomes, wherein DHR is oxidized to fluorescent rhodamine 123. Concomitant uptake of DHR with Superoxide dismutase or the spin-trap, tert-phenylbutyl nitrone, but not catalase markedly reduced fluorescence in the lysosomes implicating Superoxide anion (O₂⁻) but not hydrogen peroxide (H₂O₂) in DHR oxidation. Uptake of the anthraquinone, purpurin, and FeEDTA with DHR greatly amplified fluorescence within the lysosomes. These data are consistent with uptake of xenobiotics by hemocytes and their concentration in lysosomes wherein, ROS are generated in response to their accumulation. The rate of DHR oxidation by hemocytes was not stimulated by zymosan, a known stimulator of the oxidative burst. In vitro studies using the xanthine oxidase/hypoxanthine reaction to generate O₂⁻ and selective inhibitors of ROS production indicated that DHR is oxidized by O₂⁻ and H₂O₂ but not by · OH and that iron can participate in the reaction. Incubating isolated hemocytes promoted low-level, SOD-sensitive, FeEDTA-stimulated production of ethylene from α-keto-γ-methiolbutyric acid, indicating the in situ formation of · OH via production of O₂⁻. The above suggest that enhanced production of ROS in M. edulis hemocytes by xenobiotic accumulation within the lysosomal compartment should be considered in the toxic sequelae of exposure of marine molluscs to chemical pollutants.     

Oxidative pathways in response to polyunsaturated aldehydes in the marine diatom Skeletonema marinoi (Bacillariophyceae)

Polyunsaturated aldehydes (PUA) have recently been shown to induce reactive oxygen species (ROS) and possibly reactive nitrogen species (RNS, e.g., peroxynitrite) in the diatom Skeletonema marinoi (S. marinoi), which produces high amounts of PUA. We now are attempting to acquire better understanding of which reactive molecular species are involved in the oxidative response of S. marinoi to PUA. We used flow cytometry, the dye dihydrorhodamine 123 (DHR) as the main indicator of ROS (but which is also known to partially detect RNS), and different scavengers and inhibitors of both nitric oxide (NO) synthesis and superoxide dismutase activity (SOD). Both the scavengers Tempol (for ROS) and uric acid (UA, for peroxynitrite) induced a lower DHR‐derived green fluorescence in S. marinoi cells exposed to the PUA, suggesting that both reactive species were produced. When PUA‐exposed S. marinoi cells were treated with the NO scavenger 2‐4‐carboxyphenyl‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (cPTIO), an opposite response was observed, with an increase in DHR‐derived green fluorescence. A higher DHR‐derived green fluorescence was also observed in the presence of sodium tungstate (ST), an inhibitor of NO production via nitrate reductase. In addition, two different SOD inhibitors, 2‐methoxyestradiol (2ME) and sodium diethyldithiocarbamate trihydrate (DETC), had an effect, with DETC inducing the strongest inhibition after 20 min. These results indicate the involvement of O₂^? generation and SOD activity in H₂O₂ formation (with downstream ROS generation dependent from H₂O₂) in response to PUA exposure. This is relevant as it refines the biological impact of PUA and identifies the specific molecules involved in the response. It is speculated that in PUA‐exposed S. marinoi cells, beyond a certain threshold of PUA, the intracellular antioxidant system is no longer able to cope with the excess of ROS, thus resulting in the observed accumulation of both O₂^?? and H₂O₂. This might be particularly relevant for population dynamics at sea, during blooms, when cell lysis increases and PUA are released. It can be envisioned that in the final stages of blooms, higher local PUA concentrations accumulate, which in turn induces intracellular ROS generation that ultimately leads to cell death and bloom decay.     

Accumulation of reactive oxygen species in arbuscular mycorrhizal roots

We investigated the accumulation of reactive oxygen species (ROS) in arbuscular mycorrhizal (AM) roots from Medicago truncatula, Zea mays and Nicotiana tabacum using three independent staining techniques. Colonized root cortical cells and the symbiotic fungal partner were observed to be involved in the production of ROS. Extraradical hyphae and spores from Glomus intraradices accumulated small levels of ROS within their cell wall and produced ROS within the cytoplasm in response to stress. Within AM roots, we observed a certain correlation of arbuscular senescence and H₂O₂ accumulation after staining by diaminobenzidine (DAB) and a more general accumulation of ROS close to fungal structures when using dihydrorhodamine 123 (DHR 123) for staining. According to electron microscopical analysis of AM roots from Z. mays after staining by CeCl₃, intracellular accumulation of H₂O₂ was observed in the plant cytoplasm close to intact and collapsing fungal structures, whereas intercellular H₂O₂ was located on the surface of fungal hyphae. These characteristics of ROS accumulation in AM roots suggest similarities to ROS accumulation during the senescence of legume root nodules.     

Impact of Inhomogeneous Static Magnetic Field (31.7–232.0 mT) Exposure on Human Neuroblastoma SH-SY5Y Cells during Cisplatin Administration

Beneficial or adverse effects of Static Magnetic Fields (SMFs) are a large concern for the scientific community. In particular, the effect of SMF exposure during anticancer therapies still needs to be fully elucidated. Here, we evaluate the effects of SMF at induction levels that cisPt-treated cancer patients experience during the imaging process conducted in Low field (200–500 mT), Open field (300–700 mT) and/or inhomogeneous High field (1.5–3 T) Magnetic Resonance Imaging (MRI) machines. Human adrenergic neuroblastoma SH-SY5Y cells treated with 0.1 µM cisPt (i.e. the lowest concentration capable of inducing apoptosis) were exposed to SMF and their response was studied in vitro. Exposure of 0.1 µM cisPt-treated cells to SMF for 2 h decreased cell viability (30%) and caused overexpression of the apoptosis-related cleaved caspase-3 protein (46%). Furthermore, increase in ROS (Reactive Oxygen Species) production (23%) and reduction in the number of mitochondria vs controls were seen. The sole exposure of SMF for up to 24 h had no effect on cell viability but increased ROS production and modified cellular shape. On the other hand, the toxicity of cisPt was significantly prevented during 24 h exposure to SMF as shown by the levels of cell viability, cleaved caspase-3 and ROS production. In conclusion, due to the cytoprotective effect of 31.7–232.0 mT SMF on low-cisPt-concentration-treated SH-SY5Y cells, our data suggest that exposure to various sources of SMF in cancer patients under a cisPt regimen should be strictly controlled.     

Static magnetic field effect on microcirculation,direct versus baroreflex-mediated approach

We compared in conscious rabbits, sedated using pentobarbital intravenous (i.v.) infusion (5 mg kg^{? 1} h^{? 1}), the effect of a static magnetic field (SMF), generated by Nd₂–Fe₁₄–B magnets, on microcirculation during its 40 min local exposure to the microvascular network in cutaneous tissue [20 sham exposure and 20 SMF (0.25 T) exposure runs] or to sinocarotid baroreceptors [14 sham exposure and 14 SMF (0.35 T) exposure runs]. Mean femoral artery blood pressure (BP), heart rate (HR), arterial baroreflex sensitivity (BRS), assessed from HR and BP responses to i.v. bolus of nitroprusside and phenylephrine, and microcirculatory blood flow, using microphotoelectric plethysmography (MPPG), were simultaneously monitored. SMF significantly increased microcirculation on a 17.8% in microvascular and on a 23.3% in baroreceptor exposure series. In baroreceptor exposure series, SMF significantly decreased BP, increased heart rate variability, BRS and sodium nitroprusside (NO-donor) i.v. bolus microcirculatory vasodilatory effect. These suggest augmentation of the arterial baroreflex capacity support NO-dependent vasodilation, by increased sensitivity of vessels to NO, to be a new physiological mechanism of BP buffering and microcirculatory control. A significant positive correlation was also found between increase in BRS and in MPPG (r = 0.66, p < 0.009), indicating baroreflex participation in the regulation of the microcirculation and its enhancement after SMF exposure. Both direct and baroreflex-mediated approaches demonstrate SMF significant vasodilatory effect with potential clinical implication in macro- and microcirculatory disorders.     

Chronic exposure to a 2 mT static magnetic field affects the morphology,the metabolism and the function of in vitro cultured swine granulosa cells

In recent years, the exposure of organisms to static magnetic fields (SMFs) is continuously increasing. Thus, we investigated the effect of chronic exposure to a 2 mT SMF on in vitro cultured swine granulosa cells (GCs). In particular, the culture expansion (cell viability and doubling time), the cell phenotype (cell morphology and orientation, actin and α-tubulin cytoskeleton), the cell metabolism (intracellular Ca²⁺ concentration [Ca²⁺]_i and mitochondrial activity) and the cell function (endocrine activity) were assessed. It has been found that the exposure to the field did not affect the cell viability, but the doubling time was significantly reduced (p < 0.05) in exposed samples after 72 h of culture. At the same time, the cell length and thickness significantly changed (p < 0.05), while the cell orientation was unaffected. Evident modifications were induced on actin and α-tubulin cytoskeleton after 3 days of exposure and, simultaneously, a change in [Ca²⁺]_i and mitochondrial activity started to become evident. Finally, the SMF exposure of GCs longer than 72 h determined a significant alteration of progesterone and estrogen production (p < 0.05). In conclusion, our results demonstrate that the chronic exposure of swine GCs to a 2 mT SMF exerts a negative effect on cell proliferation, morphology, biochemistry and endocrine function in an in vitro model.     

Cellular ATP content was decreased by a homogeneous 8.5 T static magnetic field exposure: role of reactive oxygen species

The literature on the impact of strong static magnetic fields (SMF) on human health is vast and contradictory. The present study focused on the cellular effects of strong homogeneous SMF in human–hamster hybrid (A_L) cells, mitochondria‐deficient (ρ⁰ A_L) cells, and double‐strand break (DSB) repair‐deficient (XRS‐5) cells. Adenosine triphosphate (ATP) content was significantly decreased in A_L cells exposed to 8.5 Tesla (T) but not 1 or 4 T SMF for either 3 or 5 h. In addition, ATP content significantly decreased in the two deficient cell lines exposed to 8.5 T SMF for 3 h. With further incubation of 12 or 24 h without SMF exposure, ATP content could retrieve to the control level in the A_L cells but not ρ⁰ A_L and XRS‐5 cells. Under a fluorescence reader, the levels of reactive oxygen species (ROS) in the three cell lines were significantly increased by exposure to 8.5 T SMF for 3 h. Concurrent treatment with ROS inhibitor, DMSO, dramatically suppressed the ATP content in exposed A_L cells. However, the CD59 mutation frequency and the cell cycle distribution were not significantly affected by exposure to 8.5 T SMF for 3 h. Our results indicated that the cellular ATP content was reduced by 8.5 T SMF for 3 h exposure, which was partially mediated by mitochondria and the DNA DSB repair process. Moreover, ROS were involved in the process of the cellular perturbations from the SMF. Bioelectromagnetics 32:94–101, 2011. © 2010 Wiley‐Liss, Inc.     

The pattern of exposure to static magnetic field of nurses involved in activities related to contrast administration into patients diagnosed in 1.5 T MRI scanners

Static magnetic fields (SMFs) and time-varying electromagnetic fields exposure is necessary to obtain the diagnostic information regarding the structure of patient's tissues, by the use of magnetic resonance imaging (MRI) scanners. A diagnostic procedure may also include the administration of pharmaceuticals called contrast, which are to be applied to a patient during the examination. The nurses involved in administering contrast into a patient during the pause in examination are approaching permanently active magnets of MRI scanners and are exposed to SMF. There were performed measurements of spatial distribution of SMF in the vicinity of MRI magnets and parameters of personal exposure of nurses (i.e. individual exposimetric profiles of variability in time of SMF affecting the nurse who is performing tasks in the vicinity of magnet, characterized by statistical parameters of recorded magnetic flux density affecting the nurse). The SMF exposure in the vicinity of various MRI magnets depends on both magnetic flux density of the main field B ₀ (applicable to a patient) and the construction of the scanner, but the most important factor determining the workers' exposure is the work practice. In the course of a patient's routine examination in scanners of B ₀ = 1.5 T, the nurses are present over ～0.4–2.9 min in SMF exceeding 0.03% of B ₀ (i.e. 0.5 mT), but only sometimes they are present in SMF exceeding 5% of B ₀ (i.e. 75 mT). When patients need more attention because of their health status/condition, the nurses' exposure may be significantly longer – it may even exceed 10 min and 30% of B ₀ (i.e. 500 mT). We have found that the level of exposure of nurses to SMF may vary from < 5% of the main field (a median value: 0.5–1.5%; inter-quartile range: 0.04–8.8%; max value: 1.3–12% of B ₀) when a patient is moved from the magnets bore before contrast administration, up to the main field level (B ₀) when a patient stays in the magnets bore and nurse is crawling into the bore.     

Geomagnetic field modulates artificial static magnetic field effect on arterial baroreflex and on microcirculation

Spreading evidence suggests that geomagnetic field (GMF) modulates artificial magnetic fields biological effect and associated with increased cardiovascular morbidity. To explore the underlying physiological mechanism we studied 350 mT static magnetic field (SMF) effect on arterial baroreflex-mediated skin microcirculatory response in conjunction with actual geomagnetic activity, reflected by K and K _p indices. Fourteen experiments were performed in rabbits sedated by pentobarbital infusion (5 mg/kg/h). Mean femoral artery blood pressure, heart rate, and the ear lobe skin microcirculatory blood flow, measured by microphotoelectric plethysmogram (MPPG), were simultaneously recorded before and after 40 min of NdFeB magnets local exposure to sinocarotid baroreceptors. Arterial baroreflex sensitivity (BRS) was estimated from heart rate/blood pressure response to intravenous bolus injections of nitroprusside and phenylephrine. We found a significant positive correlation between SMF-induced increase in BRS and increment in microvascular blood flow (ΔBRS with ΔMPPG, r=0.7, p<0.009) indicated the participation of the arterial baroreflex in the regulation of the microcirculation and its enhancement after SMF exposure. Geomagnetic disturbance, as opposed to SMF, decreased both microcirculation and BRS, and counteracted SMF-induced increment in microcirculatory blood flow (K-index with ΔMPPG; r _s=−0.55, p<0.041). GMF probably affected central baroreflex pathways, diminishing SMF direct stimulatory effect on sinocarotid baroreceptors and on baroreflex-mediated vasodilatatory response. The results herein may thus point to arterial baroreflex as a possible physiological mechanism for magnetic-field cardiovascular effect. It seems that geomagnetic disturbance modifies artificial magnetic fields biological effect and should be taken into consideration in the assessment of the final effect. An erratum to this article can be found at     

Synaptosomal response to oxidative stress: Effect of vinpocetine

It has been suggested that reactive oxygen species (ROS) play a role in the neuronal damage occurring in ischemic injury and neurodegenerative disorders and that their neutralization by antioxidant drugs may delay or minimize neurodegeneration. In the present study we examine whether vinpocetine can act as an antioxidant and prevent the formation of ROS and lipid peroxidation in rat brain synaptosomes. After ascorbate/Fe²⁺ treatment a significant increase in oxygen consumption (about 5-fold) and thiobarbituric acid reactive substances (TBARS) formation (about 7-fold) occurred as compared to control conditions. Vinpocetine inhibited the ascorbate/Fe²⁺ stimulated consumption of oxygen and TBARS accumulation, an indicator of lipid peroxidation, in a concentration-dependent manner. The ROS formation was also prevented by vinpocetine. Oxidative stress increased significantly the fluorescence of the probes 2′,7′-dichlorodihydrofluorescein (DCFH₂-DA) (about 6-fold) and dihydrorhodamine (DHR) 123 (about 10-fold), which is indicative of intrasynaptosomal ROS generation. Vinpocetine at 100 μM concentration decreased the fluorescence of DCFH₂-DA and DHR 123 by about 50% and 83%, respectively. We conclude that the antioxidant effect of vinpocetine might contribute to the protective role exerted by the drug in reducing neuronal damage in pathological situations.     

Effect of static magnetic field on morphology and growth metabolism of Flavobacterium sp. m1-14

Increasing evidence shows that static magnetic fields (SMFs) can affect microbial growth metabolism, but the specific mechanism is still unclear. In this study, we have investigated the effect of moderate-strength SMFs on growth and vitamin K₂ biosynthesis of Flavobacterium sp. m1-14. First, we designed a series of different moderate-strength magnetic field intensities (0, 50, 100, 150, 190 mT) and exposure times (0, 24, 48, 72, 120 h). With the optimization of static magnetic field intensity and exposure time, biomass and vitamin K₂ production significantly increased compared to control. The maximum vitamin K₂ concentration and biomass were achieved when exposed to 100 mT SMF for 48 h; compared with the control group, they increased by 71.3% and 86.8%, respectively. Interestingly, it was found that both the cell viability and morphology changed significantly after SMF treatment. Second, the adenosine triphosphate (ATP) and glucose-6-phosphate dehydrogenase (G6PDH) metabolism is more vigorous after exposed to 100 mT SMF. This change affects the cell energy metabolism and fermentation behavior, and may partially explain the changes in bacterial biomass and vitamin K₂ production. The results show that moderate-strength SMFs may be a promising method to promote bacterial growth and secondary metabolite synthesis.

     

Exposure to a MRI‐type high‐strength static magnetic field stimulates megakaryocytic/erythroid hematopoiesis in CD34+ cells from human placental and umbilical cord blood

The biological response after exposure to a high‐strength static magnetic field (SMF) has recently been widely discussed from the perspective of possible health benefits as well as potential adverse effects. To clarify this issue, CD34⁺ cells from human placental and umbilical cord blood were exposed under conditions of high‐strength SMF in vitro. The high‐strength SMF exposure system was comprised of a magnetic field generator with a helium‐free superconducting magnet with built‐in CO₂ incubator. Freshly prepared CD34⁺ cells were exposed to a 5 tesla (T) SMF with the strongest magnetic field gradient (41.7 T/m) or a 10 T SMF without magnetic field gradient for 4 or 16 h. In the harvested cells after exposure to 10 T SMF for 16 h, a significant increase of hematopoietic progenitors in the total burst‐forming unit erythroid‐ and megakaryocytic progenitor cells‐derived colony formation was observed, thus producing 1.72‐ and 1.77‐fold higher than the control, respectively. Furthermore, early hematopoiesis‐related and cell cycle‐related genes were found to be significantly up‐regulated by exposure to SMF. These results suggest that the 10 T SMF exposure may change gene expressions and result in the specific enhancement of megakaryocytic/erythroid progenitor (MEP) differentiation from pluripotent hematopoietic stem cells and/or the proliferation of bipotent MEP. Bioelectromagnetics 30:280–285, 2009. © 2009 Wiley‐Liss, Inc.     

Static Magnetic Field Versus Systemic Calcium Channel Blockade Effect on Microcirculation: Possible Mechanisms and Clinical Implementation

The goal was to compare static magnetic field (SMF, generated by Nd₂–Fe₁₄–B magnets) vasodilator capacity with verapamil (VER, a potent, clinically verified Ca²⁺ channel-blocking agent), aimed to assess SMF implementation in conditions with vascular ischemia. Skin microcirculatory blood flow measured by microphotoelectric plethysmogram was recorded in conscious rabbits after 40 min of 0.25 T SMF regional exposure to ear microvascular net (SMF-Vas, n = 20), or 0.35 T to carotid baroreceptors (SMF-Car, n = 14), and compared with that after 30 min VER intravenous infusion (20 µg/kg/min, n = 20). The principal finding is that SMF-Vas, SMF-Car, and VER significantly increased microcirculatory blood flow by 17.9 ± 9.58%, 22.6 ± 11.11%, and 30.5 ± 14.06% (mean ± SEM) respectively, and there was no significant difference between all three treatments (P = 0.986). Microvascular dilation was accompanied by significant decrease of blood pressure in VER and SMF-Car cases. The decrease of arterial baroreflex sensitivity in VER contrasted with its increase in SMF-Car, coupled with improved vessel sensitivity to nitric oxide (NO) dilatory effect. This suggests that SMF can have a strong vasodilator property tailored to address diabetic, mainly NO-deficient, neural, and myogenic microvascular dysfunction, especially employing both SMFs’ vasodilation synergy. Bioelectromagnetics. 2020;41:447–457. © 2020 Bioelectromagnetics Society.     

Implications of using the fluorescent probes, dihydrorhodamine 123 and 2',7'-dichlorodihydrofluorescein diacetate, for the detection of UVA-induced reactive oxygen species

During investigation of UVA-induced oxidative stress in HaCaT keratinocytes with dihydrorhodamine 123 (DHR123) and 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA), exaggerated baseline values were observed within control samples, suggesting a mechanism of probe oxidation and subsequent change in fluorescence intensity (FI) independent of cellular ROS generation. The effects of diluent, UVA pre-treatment and loading protocols upon the FI of the probes have therefore been investigated. The study confirmed the capacity of Dulbecco's Modified Eagle's Medium (DMEM) to confer fluorescence intensity changes in both probes, most notably DCF-DA. In addition, UVA pre-treatment compromises the effectiveness of DHR123 and DCF-DA to detect ROS generated in a cell-free system. In vitro data shows a greater UVA-induced FI increase in HaCaT cells loaded with probe before rather than after UVA treatment. This study has important implications for future research, the understanding of previous studies and associated confounding effects using DHR123 and DCF-DA as ROS sensitive probes.     

Effects of H2O2 on the growth, secretion, and metabolism of hybridoma cells in culture

Summary The effect of low concentrations of hydrogen peroxide (H₂O₂) (5 × 10⁻⁷−9.5 × 10⁻⁷ M) on cell growth and antibody production was investigated with murine hybridoma cells (Mark 3 and anti-hPL) in culture. Cell growth, measured by flow cytometry with morphological parameters, was significantly stimulated by H₂O₂ (8 × 10⁻⁷ M) but H₂O₂ concentration of 7 × 10⁻⁶ M and above increased cell death. H₂O₂ stimulation of antibody production was nonsignificant. The metabolism of cells treated with 8 × 10⁻⁷ or 1 × 10⁻⁵ M H₂O₂ was similar to that of the control in terms of glucose and glutamine consumption, lactate and ammonia production, and amino acid concentrations in the medium. The concentrations of lactate dehydrogenase, a marker of cell death, in test and control cells were similar. However, concentrations of intracellular free radicals measured by flow cytometry with dihydrorhodamine 123 (DHR 123) and dichlorofluorescein diacetate (DCFH-DA) as fluorochromes were different. The reactive oxygen species content of cells in 8 × 10⁻⁷ M H₂O₂ was similar to that of the controls, but there was a sudden, marked production of superoxide anions (detected with DHR 123) and H₂O₂ or peroxides (detected with DCFH-DA) by cells incubated with 1 × 10⁻⁵ M H₂O₂ which increased with increasing H₂O₂ until cell death.     

Morphofunctional study of 12‐O‐tetradecanoyl‐13‐phorbol acetate (TPA)‐induced differentiation of U937 cells under exposure to a 6 mT static magnetic field

This study deals with the morphofunctional influence of 72 h exposure to a 6 mT static magnetic field (SMF) during differentiation induced by 50 ng/ml 12‐O‐tetradecanoyl‐13‐phorbol acetate (TPA) in human leukaemia U937 cells. The cell morphology of U937 cells was investigated by optic and electron microscopy. Specific antibodies and/or molecules were used to label CD11c, CD14, phosphatidylserine, F‐actin and to investigate the distribution and activity of lysosomes, mitochondria and SER. [Ca²⁺]_i was evaluated with a spectrophotometer. The degree of differentiation in SMF‐exposed cells was lower than that of non‐exposed cells, the difference being exposure time‐dependent. SMF‐exposed cells showed cell shape and F‐actin modification, inhibition of cell attachment, appearance of membrane roughness and large blebs and impaired expression of specific macrophagic markers on the cell surface. The intracellular localization of SER and lysosomes was only partially affected by exposure. A significant localization of mitochondria with an intact membrane potential at the cell periphery in non‐exposed, TPA‐stimulated cells was observed; conversely, in the presence of SMF, mitochondria were mainly localised near the nucleus. In no case did SMF exposure affect cell viability. The sharp intracellular increase of [Ca²⁺]_i could be one of the causes of the above‐described changes. Bioelectromagnetics 30:352–364, 2009. © 2009 Wiley‐Liss, Inc.     

Lateral gradients significantly enhance static magnetic field‐induced inhibition of pain responses in mice—a double blind experimental study

Recent research demonstrated that exposure of mice to both inhomogeneous (3–477 mT) and homogeneous (145 mT) static magnetic fields (SMF) generated an analgesic effect toward visceral pain elicited by the intraperitoneal injection of 0.6% acetic acid. In the present work, we investigated behavioral responses such as writhing, entry avoidance, and site preference with the help of a specially designed cage that partially protruded into either the homogeneous (ho) or inhomogeneous (inh) SMF. Aversive effects, cognitive recognition of analgesia, and social behavior governed mice in their free locomotion between SMF and sham sides. The inhibition of pain response (I) for the 0–5, 6–20, and 21–30 min periods following the challenge was calculated by the formula I = 100 (1 ? x/y) in %, where x and y represent the number of writhings in the SMF and sham sides, respectively. In accordance with previous measurements, an analgesic effect was induced in exposed mice (I_ho = 64%, P < 0.0002 and I_inh = 62%, P < 0.002). No significant difference was found in the site preference (SMF_{ho, inh} vs. sham) indicating that SMF is neither aversive nor favorable. Comparison of writhings observed in the sham versus SMF side of the cage revealed that SMF exposure resulted in significantly fewer writhings than sham (I_ho = 64%, P < 0.004 and I_inh = 81%, P < 0.03). Deeper statistical analysis clarified that the lateral SMF gradient between SMF and sham sides could be responsible for most of the analgesic effect (I_ho = 91%, P < 0.02 and I_inh = 54%, P < 0.02). Bioelectromagnetics 34:385–396, 2013. © 2012 Wiley Periodicals, Inc.     

Involvement of nitrate reductase-dependent nitric oxide production in magnetopriming-induced salt tolerance in soybean

In the present study, experiments were performed to investigate the role of nitric oxide (NO) in magnetopriming-induced seed germination and early growth characteristics of soybean (Glycine max) seedlings under salt stress. The NO donor (sodium nitroprusside, SNP), NO scavenger (2-[4-carboxyphenyl]-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, CPTIO), inhibitors of nitrate reductase (sodium tungstate, ST) or NO synthase (N-nitro-L-Arg-methyl ester, LNAME) and NADPH oxidase inhibitor (diphenylene iodonium, DPI) have been used to measure the role of NO in the alleviation of salinity stress by static magnetic field (SMF of 200 mT, 1 h). Salt stress (50 mM NaCl) significantly reduced germination and early growth of seedlings emerged from non-primed seeds. Pre-treatment of seeds with SMF positively stimulated the germination and consequently promoted the seedling growth. ST, LNAME, CPTIO and DPI significantly decreased the growth of seedling, activities of α-amylase, protease and nitrate reductase (NR), hydrogen peroxide (H₂O₂), superoxide (O₂^•−) and NO content in roots of seedlings emerged from non-primed and SMF-primed seeds. However, the extent of reduction was higher with ST in seedlings of SMF-primed seeds under both conditions, whereas SNP promoted all the studied parameters. Moreover, the generation of NO was also confirmed microscopically using a membrane permanent fluorochrome (4-5-diaminofluorescein diacetate [DAF-2 DA]). Further, analysis showed that SMF enhanced the NR activity and triggered the NO production and NR was maximally decreased by ST as compared to LNAME, CPTIO and DPI. Thus, in addition to ROS, NO might be one of the important signaling molecules in magnetopriming-induced salt tolerance in soybean and NR may be responsible for SMF-triggered NO generation in roots of soybean.     

The development and in vitro characterisation of an intracellular nanosensor responsive to reactive oxygen species

Advances in sensor technologies have enhanced our understanding of the roles played by reactive oxygen species (ROS) in a number of physiological and pathological processes. However, high inter-reactivity and short life spans has made real-time monitoring of ROS in cellular systems challenging. Fluorescent dyes capable of intracellular ROS measurements have been reported. However, these dyes are known to be intrinsically cytotoxic and thus can potentially significantly alter cellular metabolism and adversely influence in vitro data. Reported here is the development and in vitro application of a novel ROS responsive nanosensor, based on PEBBLE (Probes Encapsulated By Biologically Localised Embedding) technology. The ROS sensitive fluorescent probe dihydrorhodamine 123 (DHR 123) was employed as the sensing element of the PEBBLE through entrapment within a porous, bio-inert polyacrylamide nanostructure enabling passive monitoring of free radical flux within the intracellular environment. Successful delivery of the nanosensors into NR8383 rat alveolar macrophage cells via phagocytosis was achieved. Stimulation of PEBBLE loaded NR8383 cells with phorbol-12-myristate-13-acetate (PMA) enabled real time monitoring of ROS generation within the cell without affecting cellular viability. These data suggest that PEBBLE nanosensors could offer significant advantages over existing technologies used in monitoring the intracellular environment.