Static magnetic fields affect calcium fluxes and inhibit stress-induced apoptosis in human glioblastoma cells

BACKGROUND: Epidemiologic data revealed increased brain tumor incidence in workers exposed to magnetic fields (MFs), raising concerns about the possible link between MF exposure and cancer. However, MFs seem to be neither mutagenic nor tumorigenic. The mechanism of their tumorigenic effect has not been elucidated. METHODS: To evaluate the interference of MFs with physical (heat shock, HS) and chemical (etoposide, VP16) induced apoptoses, respectively, we exposed a human glioblastoma primary culture to 6 mT static MF. We investigated cytosolic Ca(2+) ([Ca(2+)](i)) fluxes and extent of apoptosis as key endpoints. The effect of MFs on HS- and VP16-induced apoptoses in primary glioblastoma cultures from four patients was also tested. RESULTS: Static MFs increased the [Ca(2+)](i) from a basal value of 124 +/- 4 nM to 233 +/- 43 nM (P < 0.05). MF exposure dramatically reduced the extent of HS- and VP16-induced apoptoses in all four glioblastoma primary cultures analyzed by 56% (range, 28-87%) and 44% (range, 38-48%), respectively. However, MF alone did not exert any apoptogenic activity. Differences were observed across the four cultures with regard to apoptotic induction by HS and VP16 and to MF apoptotic reduction, with an individual variability with regard to apoptotic sensitivity. CONCLUSION: The ability of static MFs to reduce the extent of damage-induced apoptosis in glioblastoma cells might allow the survival of damaged and possibly mutated cells.     

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.     

Cell shape and plasma membrane alterations after static magnetic fields exposure

The biological effects of static magnetic fields (MFs) with intensity of 6 mT were investigated in lymphocytes and U937 cells in the presence or absence of apoptosis-inducing drugs by transmission (TEM) and scanning (SEM) electron microscopy. Lectin cytochemistry of ConA-FITC conjugates was used to analyze plasma membrane structural modifications. Static MFs modified cell shape, plasma membrane and increased the level of intracellular [Ca++] which plays an antiapoptotic role in both cell types. Modifications induced by the exposure to static MFs were irrespective of the presence or absence of apoptotic drugs or the cell type. Abundant lamellar-shaped microvilli were observed upon 24 hrs of continuous exposure to static MFs in contrast to the normally rough surface of U937 cells having numerous short microvilli. Conversely, lymphocytes lost their round shape and became irregularly elongated; lamellar shaped microvilli were found when cells were simultaneously exposed to static MFs and apoptosis-inducing drugs. In our experiments, static MFs reduced the smoothness of the cell surface and partially impeded changes in distribution of cell surface glycans, both features being typical of apoptotic cells. Cell shape and plasma membrane structure modifications upon static MFs exposure were time-dependent. Lamellar microvilli were clearly observed before the distortion of cell shape, which was found at long times of exposure. MFs exposure promoted the rearrangement of F-actin filaments which, in turn, could be responsible for the cell surface modifications. Here we report data that support biological effects of static MFs on U937 cells and human lymphocytes. However, the involvement of these modifications in the onset of diseases needs to be further elucidated.     

Sustained enhancement of Ca(2+) influx by glibenclamide induces apoptosis in RINm5F cells

Cytosolic Ca(2+) elevations are known to be involved in triggering apoptosis in many tissues, but the effect of sustained enhancement of Ca(2+) influx on apoptosis in beta cells remains unknown. We have found that the viability of RINm5F cells is decreased dose-dependently by continuous exposure to glibenclamide at concentrations from 10(-7) to 10(-4) M, and that this effect is partially ameliorated by pretreatment with cycloheximide. Electrophoresis of the cells exposed to glibenclamide revealed ladder-like fragmentation characteristic of apoptosis, and which also is suppressed by cycloheximide pretreatment. By using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining, we detected increased DNA fragmentation in the nuclei of the cells exposed to glibenclamide, and staining with Hoechst 33342 and propidium iodide showed a dose-dependent increase in the number of cells with the chromatin condensation and fragmentation in their nuclei that is characteristic of apoptosis. The effects of glibenclamide on cell viability and apoptotic cell death were partially inhibited by treatment with Ca(2+) channel blocker, and by reducing the extracellular Ca(2+) concentration during glibenclamide exposure, suggesting that they may be derived from increased Ca(2+) influx. Furthermore, only the percentage of apoptotic cells, and not that of necrotic cells, increased with the increasing intracellular Ca(2+) concentration during glibenclamide exposure. In conclusion, we have demonstrated that the sustained enhancement of Ca(2+) influx caused by glibenclamide exposure can induce apoptotic cell death in a pure beta cell line.     

Role of methionine 35 in the intracellular Ca2+ homeostasis dysregulation and Ca2+-dependent apoptosis induced by amyloid beta-peptide in human neuroblastoma IMR32 cells

Amyloid beta-peptide (Abeta) plays a fundamental role in the pathogenesis of Alzheimer's disease. We recently reported that the redox state of the methionine residue in position 35 of amyloid beta-peptide (Abeta) 1-42 (Met35) strongly affects the peptide's ability to trigger apoptosis and is thus a major determinant of its neurotoxicity. Dysregulation of intracellular Ca(2+) homeostasis resulting in the activation of pro-apoptotic pathways has been proposed as a mechanism underlying Abeta toxicity. Therefore, we investigated correlations between the Met35 redox state, Abeta toxicity, and altered intracellular Ca(2+) signaling in human neuroblastoma IMR32 cells. Cells incubated for 6-24 h with 10 microM Abeta1-42 exhibited significantly increased KCl-induced Ca(2+) transient amplitudes and resting free Ca(2+) concentrations. Nifedipine-sensitive Ca(2+) current densities and Ca(v)1 channel expression were markedly enhanced by Abeta1-42. None of these effects were observed when cells were exposed to Abeta containing oxidized Met35 (Abeta1-42(Met35-Ox)). Cell pre-treatment with the intracellular Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (1 microM) or the Ca(v)1 channel blocker nifedipine (5 microM) significantly attenuated Abeta1-42-induced apoptosis but had no effect on Abeta1-42(Met35-Ox) toxicity. Collectively, these data suggest that reduced Met35 plays a critical role in Abeta1-42 toxicity by rendering the peptide capable of disrupting intracellular Ca(2+) homeostasis and thereby provoking apoptotic cell death.     

Characterization of calcium release-activated apoptosis of LNCaP prostate cancer cells

Apoptosis inhibition rather than enhanced cellular proliferation occurs in prostate cancer (CaP), the most commonly diagnosed malignancy in American men. Therefore, it is important to characterize residual apoptotic pathways in CaP cells. When intracellular Ca(2+) stores are released and plasma membrane "store-operated" Ca(2+) entry channels subsequently open, cytosolic [Ca(2+)] increases and is thought to induce apoptosis. However, cells incapable of releasing Ca(2+) stores are resistant to apoptotic stimuli, indicating that Ca(2+) store release is also important. We investigated whether release of intracellular Ca(2+) stores is sufficient to induce apoptosis of the CaP cell line LNCaP. We developed a method to release stored Ca(2+) without elevating cytosolic [Ca(2+)]; this stimulus induced LNCaP cell apoptosis. We compared the apoptotic pathways activated by intracellular Ca(2+) store release with the dual insults of store release and cytosolic [Ca(2+)] elevation. Earlier processing of caspases-3 and -7 occurred when intracellular store release was the sole Ca(2+) perturbation. Apoptosis was attenuated in both conditions in stable transfected cells expressing antiapoptotic proteins Bclx(L) and catalytically inactive caspase-9, and in both scenarios inactive caspase-9 became complexed with caspase-7. Thus, intracellular Ca(2+) store release initiates an apoptotic pathway similar to that elicited by the dual stimuli of cytosolic [Ca(2+)] elevation and intracellular store release.     

Effect of extremely low frequency magnetic fields on calpain activation

The effects of low intensity, low frequency magnetic fields (MFs) on catalytic activity of the calcium dependent protease calpain was determined following the enzyme activation both in "in vitro" and "in vivo" conditions. We have observed that a 0.3 mT MF induces a significant increase in the requirement of the protease for this metal ion. This change is detectable at low [Ca(2+)] and disappears when the level of Ca(2+) is raised to saturating amounts. The observed effects are not due to transient MF(-) induced conformational changes occurring in calpain, but to direct effects of the MF on Ca(2+) ions, which become less available for the binding sites present in calpain. Altogether, these results indicate that exposure to low intensity, low frequency MFs alters the intracellular Ca(2+) "availability," thereby modifying the related cell response.     

Calcium is a key signaling molecule in beta-lapachone-mediated cell death

beta-Lapachone (beta-Lap) triggers apoptosis in a number of human breast and prostate cancer cell lines through a unique apoptotic pathway that is dependent upon NQO1, a two-electron reductase. Downstream signaling pathway(s) that initiate apoptosis following treatment with beta-Lap have not been elucidated. Since calpain activation was suspected in beta-Lap-mediated apoptosis, we examined alterations in Ca(2+) homeostasis using NQO1-expressing MCF-7 cells. beta-Lap-exposed MCF-7 cells exhibited an early increase in intracellular cytosolic Ca(2+), from endoplasmic reticulum Ca(2+) stores, comparable to thapsigargin exposures. 1,2-Bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, an intracellular Ca(2+) chelator, blocked early increases in Ca(2+) levels and inhibited beta-Lap-mediated mitochondrial membrane depolarization, intracellular ATP depletion, specific and unique substrate proteolysis, and apoptosis. The extracellular Ca(2+) chelator, EGTA, inhibited later apoptotic end points (observed >8 h, e.g. substrate proteolysis and DNA fragmentation), suggesting that later execution events were triggered by Ca(2+) influxes from the extracellular milieu. Collectively, these data suggest a critical, but not sole, role for Ca(2+) in the NQO1-dependent cell death pathway initiated by beta-Lap. Use of beta-Lap to trigger an apparently novel, calpain-like-mediated apoptotic cell death could be useful for breast and prostate cancer therapy.     

Static and ELF magnetic fields induce tumor growth inhibition and apoptosis

The ability of static and extremely low frequency (ELF) Magnetic Fields (MF) to interfere with neoplastic cell function has been evaluated. In vitro experiments were carried out to study the role of MF characteristics (intensity, frequency, and modulation) on two transformed cell lines (WiDr human colon adenocarcinoma and MCF-7 human breast adenocarcinoma) and one nontransformed cell line (MRC-5 embryonal lung fibroblast). Increase in cell death morphologically consistent with apoptosis was reported exclusively in the two transformed cell lines. Cell-death induction was observed with MF of more than 1 mT. It was independent of the MF frequency and increased when modulated MF (static with a superimposition of ELF at 50 Hz) were used. Based on the in vitro results, four different MF exposure characteristics were selected and used to treat nude mice xenografted with WiDr cells. The treatment of nude mice bearing WiDr tumors subcutaneously. with daily exposure for 70 min to MF for 4 weeks caused significant tumor growth inhibition (up to 50%) by the end of the treatment when modulated MF were used for at least 60% of the whole treatment period and the time-averaged total MF intensity was higher than 3.59 mT. No toxic morphological changes induced by exposure were observed in renewing, slowly proliferating, or static normal cells. A discussion on the possible biophysical mechanism at the base of the observed biological results is also offered.     

Exposure of cells to static magnetic field accelerates loss of integrity of plasma membrane during apoptosis

BACKGROUND: Much attention is being paid to the biologic effects of magnetic fields (MFs). Although MFs enhance tumorigenesis, they are neither mutagenic nor tumorigenic. The mechanism of their tumorigenic effect has not been elucidated. METHODS: To investigate the effect of MFs on apoptosis in HL-60 cells, we exposed the cells to static MFs of 6 mT generated by a magnetic disk of known intensity. Apoptosis was triggered by the DNA topoisomerase I inhibitor, camptothecin (CPT). Activation of caspases in situ using the fluorochrome-labeled inhibitor (FLICA) method and determination of plasma membrane integrity by excluding propidium iodide (PI) were measured by both laser scanning cytometry (LSC) and flow cytometry (FC). LSC and FC identified cells at three sequential stages of their demise: early apoptosis (cells with activated caspases and PI negative); late apoptosis (cells with activated caspases but unable to exclude PI); secondary necrosis (cells with apoptotic morphology no longer stained with FLICA, not excluding PI). RESULTS: MF alone did not induce any apoptogenic or necrogenic effect. CPT exposure led to the sequential appearance of apoptotic cells. In the presence of CPT and MF, the overall proportion of cells undergoing apoptosis was not significantly changed. However, we consistently observed a significant increase in the frequency of late apoptotic/necrotic cells when compared with samples treated with CPT alone (P < 0.001), as well as a decrease in the percentage of early apoptotic cells (P = 0.013). The data obtained by FC and LSC were consistent with each other, showing a similar phenomenon. CONCLUSION: Whereas MF alone or with CPT did not affect overall cell viability, it accelerated the rate of cell transition from apoptosis to secondary necrosis after induction of apoptosis by the DNA-damaging agent, CPT. Modulation of the kinetics of the transition from apoptosis to secondary necrosis by MF in vivo may play a role in inflammation and tumorigenesis.     

Synchronous progression of calcium transient-dependent beating and sarcomere destruction in apoptotic adult cardiomyocytes

During early apoptosis, adult cardiomyocytes show unusual beating, suggesting possible participation of abnormal Ca(2+) transients in initiation of apoptotic processes in this cell type. Simultaneously with the beating, these cells show dynamic structural alteration resulting from cytoskeletal disintegration that is quite rapid. Because of the specialized structure and extensive cytoskeleton of cardiomyocytes, we hypothesized that its degradation in so short a time would require a particularly efficient mechanism. To better understand this mechanism, we used serial video microscopy to observe beta-adrenergic stimulation-induced apoptosis in isolated adult rat cardiomyocytes while simultaneously recording intracellular Ca(2+) concentration and cell length. Trains of Ca(2+) transients and corresponding rhythmic contractions and relaxations (beating) were observed in apoptotic cells. Frequencies of Ca(2+) transients and beating gradually increased with time and were accompanied by cellular shrinkage. As the cells shrank, amplitudes of Ca(2+) transients declined and diastolic intracellular Ca(2+) concentration increased until the transients were lost. Beating and progression of apoptosis were significantly inhibited by antagonists against the L-type Ca(2+) channel (nifedipine), ryanodine receptor (ryanodine), inositol 1,4,5-trisphosphate receptor (heparin), sarco(endo)plasmic Ca(2+)-ATPase (thapsigargin), and Na(+)/Ca(2+) exchanger (KB-R7943). Electron-microscopic examination of beating cardiomyocytes revealed progressive breakdown of Z disks. Immunohistochemical analysis and Western blot confirmed that disappearance of Z disk constituent proteins (alpha-actinin, desmin, and tropomyosin) preceded degradation of other cytoskeletal proteins. It thus appears that, in adult cardiomyocyte apoptosis, Ca(2+) transients mediate apoptotic beating and efficient sarcomere destruction initiated by Z disk breakdown.     

The coxsackievirus 2B protein suppresses apoptotic host cell responses by manipulating intracellular Ca2+ homeostasis

Enteroviruses, small cytolytic RNA viruses, confer an antiapoptotic state to infected cells in order to suppress infection-limiting apoptotic host cell responses. This antiapoptotic state also lends protection against cell death induced by metabolic inhibitors like actinomycin D and cycloheximide. The identity of the viral antiapoptotic protein and the underlying mechanism are unknown. Here, we provide evidence that the coxsackievirus 2B protein modulates apoptosis by manipulating intracellular Ca(2+) homeostasis. Using fluorescent Ca(2+) indicators and organelle-targeted aequorins, we demonstrate that ectopic expression of 2B in HeLa cells decreases the Ca(2+) content of both the endoplasmic reticulum and the Golgi, resulting in down-regulation of Ca(2+) signaling between these stores and the mitochondria, and increases the influx of extracellular Ca(2+). In our studies of the physiological importance of the 2B-induced alterations in Ca(2+) signaling, we found that the expression of 2B suppressed caspase activation and apoptotic cell death induced by various stimuli, including actinomycin D and cycloheximide. Mutants of 2B that were defective in reducing the Ca(2+) content of the stores failed to suppress apoptosis. These data implicate a functional role of the perturbation of intracellular Ca(2+) compartmentalization in the enteroviral strategy to suppress intrinsic apoptotic host cell responses. The putative down-regulation of an endoplasmic reticulum-dependent apoptotic pathway is discussed.     

Effects of 60 Hz 14 microT magnetic field on the apoptosis of testicular germ cell in mice

We recently reported that continuous exposure, for 8 weeks, of extremely low frequency (ELF) magnetic field (MF) of 0.1 or 0.5 mT might induce testicular germ cell apoptosis in BALB/c mice. In that report, the ELF MF exposure did not significantly affect the body weight or testicular weight, but significantly increased the incidence of testicular germ cell death. In the present study, we aimed to further characterize the effect of a 16-week continuous exposure to ELF MF of 14 or 200 microT on testicular germ cell apoptosis in mice. There were no significant effects of MF on body weight and testosterone levels in mice. In TUNEL staining (In situ terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling), germ cells showed a significantly higher apoptotic rate in exposed mice than in sham controls (P < 0.001). TUNEL-positive cells were mainly spermatogonia. In an electron microscopic study, degenerating spermatogonia showed condensation of nuclear chromatin similar to apoptosis. These results indicate that apoptosis may be induced in spermatogenic cells in mice by continuous exposure to 60 Hz MF of 14 microT.     

DNA damage in rat lymphocytes treated in vitro with iron cations and exposed to 7 mT magnetic fields (static or 50 Hz)

The present study was undertaken to verify a hypothesis that exposure of the cells to static or 50 Hz magnetic fields (MF) and simultaneous treatment with a known oxidant, ferrous chloride, may affect the oxidative deterioration of DNA molecules.The comet assay was chosen for the assessment of DNA damage. The experiments were performed on isolated rat lymphocytes incubated for 3h in Helmholtz coils at 7 mT static or 50 Hz MF. During MF exposure, part of the cell samples were incubated with 0.01 microM H(2)O(2) and another one with 10 microg/ml FeCl(2,) the rest serving as controls.Lymphocyte exposure to MF at 7 mT did not increase the number of cells with DNA damage in the comet assay. Incubation of lymphocytes with 10 microg/ml FeCl(2) did not produce a detectable damage of DNA either. However, when the FeCl(2)-incubated lymphocytes were simultaneously exposed to 7 mT MF, the number of damaged cells was significantly increased and reached about 20% for static MF and 15% for power frequency MF. In the control samples about 97% of the cells did not have any DNA damage.It is not possible at present to offer a reasonable explanation for the findings of this investigation - the high increase in the number of lymphocytes showing symptoms of DNA damage in the comet assay, following simultaneous exposure to the combination of two non-cytotoxic factors -10 microg/ml FeCl(2) and 7 mT MF. In view of the obtained results we can only hypothesise that under the influence of simultaneous exposure to FeCl(2) and static or 50 Hz MF, the number of reactive oxygen species generated by iron cations may increase substantially. Further studies will be necessary to confirm this hypothesis and define the biological significance of the observed effect.     

Role of Ca2+ signaling in initiation of stretch-induced apoptosis in neonatal heart cells

Abnormal mechanical load, as seen in hypertension, is found to induce heart cell apoptosis, yet the signaling link between cell stretch and apoptotic pathways is not known. Using an in vitro stretch model mimicking diastolic pressure stress, here we show that Ca(2+) signaling participates essentially in the early stage of stretch-induced apoptosis. In neonatal rat cardiomyocytes, the moderate 20% stretch resulted in tonic elevation of intracellular free Ca(2+) ([Ca(2+)](i)). Buffering [Ca(2+)](i) by EGTA-AM, suppressing ryanodine-sensitive Ca(2+) release, and blocking L-type Ca(2+) channels all prevented the stretch-induced apoptosis as assessed by phosphatidylserine exposure and nuclear fragmentation. Notably, Ca(2+) suppression also prevented known stretch-activated apoptotic events, including caspase-3/-9 activation, mitochondrial membrane potential corruption, and reactive oxygen species production, suggesting that Ca(2+) signaling is the upstream of these events. Since [Ca(2+)](i) did not change without activating mechanosensitive Ca(2+) entry, we conclude that stretch-induced Ca(2+) entry, via the Ca(2+)-induced Ca(2+) release mechanism, plays an important role in initiating apoptotic signaling during mechanical stress.     

Evidence supporting a role for calcium in apoptosis induction by the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO)

The synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) is a novel anticancer agent that induces apoptosis in tumor cells. The cytotoxic stress underpinning CDDO-induced apoptosis has not been established. This study compared and contrasted the effects of CDDO on COLO 16 human skin cancer cells and their respiration-deficient (rho(0)) clones to elucidate the stress signal responsible for initiating apoptosis. CDDO promoted apoptosis in COLO 16 cells in a dose- and time-dependent manner. The rho(0) clones appeared to be more sensitive to CDDO-induced apoptosis implying that the disruption of mitochondrial respiration was not directly associated with triggering cell death. After a 4-h exposure to CDDO, mitochondrial inner transmembrane potential-sensitive dyes revealed mitochondrial hyperpolarization in the COLO 16 cells and mitochondrial depolarization in the rho(0) clones. Electron microscopy illustrated that this exposure also promoted mitochondrial condensation, endoplasmic reticulum dilation, and chromatin condensation in the COLO 16 cells. Endoplasmic reticulum dilation and chromatin condensation were also observed in the rho(0) clones, but the mitochondria in these cells were markedly swollen implying that the disruption of intracellular Ca(2+) homeostasis was associated with cell death. A Ca(2+)-sensitive dye confirmed that CDDO increased cytoplasmic free Ca(2+) in the COLO 16 cells, their rho(0) clones, as well as in malignant breast and lung epithelial cells. A cell-permeant Ca(2+) chelator reduced the CDDO-induced increase in cytoplasmic free Ca(2+), and inhibited caspase activation, the development of apoptotic morphology, and DNA fragmentation in the COLO 16 cells, implying that Ca(2+) played a pivotal role in signaling the initiation of apoptosis.     

Effects of a time varying strong magnetic field on release of cytosolic free Ca2+ from intracellular stores in cultured bovine adrenal chromaffin cells

This study was made to explain the mechanisms for the effects of exposure to a time varying 1.51 T magnetic field on the intracellular Ca(2+) signaling pathway. The exposure inhibited an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in bovine chromaffin cells induced by addition of bradykinin (BK) to a Ca(2+) free medium. The exposure did not change BK induced production of inositol 1,4,5-trisphosphate (IP(3)). [Ca(2+)](i) was markedly increased in IP(3) loaded cells, and this increase was inhibited by the magnetic field exposure. A similar increase in [Ca(2+)](i) by other drugs, which stimulated Ca(2+) release from intracellular Ca(2+) stores, was again inhibited by the same exposure. However, transmembrane Ca(2+) fluxes caused in the presence of thapsigargin were not inhibited by the magnetic field exposure in a Ca(2+) containing medium. Inhibition of the BK induced increase in [Ca(2+)](i) by the exposure for 30 min was mostly recovered 1 h after exposure ended. Our results reveal that the magnetic field exposure inhibits Ca(2+) release from intracellular Ca(2+) stores, but that BK bindings to BK receptors of the cell membrane and intracellular inositol IP(3) production are not influenced.     

Effects of 50 Hz extremely low frequency magnetic field on the morphology and function of boar spermatozoa capacitated in vitro

The aim of this study was to evaluate the effect of an acute exposure to a sinusoidal MF-ELF (50 Hz, 1mT) on the ability of boar mature spermatozoa to acquire the fertilizing competence in vitro. The spermatozoa exposed during the 4h of incubation to the MF-ELF were evaluated for morphological (surface morphology and acrosome integrity) and functional parameters (cell viability, motility, induction of acrosomal reaction, AR, and the ability to in vitro fertilize oocytes). In parallel, the intracellular Ca(2+) levels as well as the major mechanisms of Ca(2+) clearance were assessed: (45)Ca intakes and intracellular Ca(2+) sequestration by analyzing intracellular Ca(2+) elevation induced by thapsigargin or studying mitochondrial function with Mito-Tracker. The MF-ELF exposure did not affect sperm viability and morphology during the first h of incubation when sperm Ca(2+) homeostasis were already compromised. First of all, MF-ELF treated spermatozoa showed resting intracellular Ca(2+) levels significantly lower than those recorded in controls. This result was dependent on a lower extracellular Ca(2+) intake and from the inhibitory role exerted on both intracellular Ca(2+) storages. As a consequence, after 1h of incubation MF-ELF exposed cells displayed a reduced motility, a modest reactivity when coincubated with solubilized zonae pellucidae and a reduction in oocyte penetrating ability. After 2 or 4h of incubation, in addition, signs of morphological damage appeared on plasma membrane and at acrosomal level. In conclusion, MF-ELF influence negatively spermatozoa first by impairing cell Ca(2+) homeostasis then by dramatically affecting sperm morphology and function.