Exposure to 50 Hz Extremely-Low-Frequency Magnetic Fields Induces No DNA Damage in Cells by Gamma H2AX Technology.

The current results for extremely-low-frequency magnetic fields (ELF-MF) on DNA damage are still debated. A sensitive indicator and systematic research are needed to assess the effects of ELF-MF. In this study, we used γH2AX as an early and sensitive molecular marker to evaluate the DNA damage effects of ELF-MF in vitro. Human amnion epithelial cells (FLs), human skin fibroblast cells (HSFs), and human umbilical vein endothelial cells (HUVECs) were exposed to 50 Hz ELF-MF at 0.4, 1, and 2 mT for 15 min, 1 h, and 24 h, respectively. After exposure, cells were subjected to γH2AX immunofluorescence and western blot. The results showed no significant difference in the average number of foci per cell, the percentage of γH2AX foci-positive cells, or the expression of γH2AX between the sham and 50 Hz ELF-MF exposure groups (P > 0.05). In conclusion, 50 Hz ELF-MF did not induce DNA damage in FLs, HSFs, or HUVECs, which was independent of the intensity or duration of the exposure.

Expression of integrins to control migration direction of electrotaxis.

Proper control of cell migration is critically important in many biologic processes, such as wound healing, immune surveillance, and development. Much progress has been made in the initiation of cell migration; however, little is known about termination and sometimes directional reversal. During active cell migration, as in wound healing, development, and immune surveillance, the integrin expression profile undergoes drastic changes. Here, we uncovered the extensive regulatory and even opposing roles of integrins in directional cell migration in electric fields (EFs), a potentially important endogenous guidance mechanism. We established cell lines that stably express specific integrins and determined their responses to applied EFs with a high throughput screen. Expression of specific integrins drove cells to migrate to the cathode or to the anode or to lose migration direction. Cells expressing αMß2, ß1, α2, αIIbß3, and α5 migrated to the cathode, whereas cells expressing ß3, α6, and α9 migrated to the anode. Cells expressing α4, αV, and α6ß4 lost directional electrotaxis. Manipulation of α9 molecules, one of the molecular directional switches, suggested that the intracellular domain is critical for the directional reversal. These data revealed an unreported role for integrins in controlling stop, go, and reversal activity of directional migration of mammalian cells in EFs, which might ensure that cells reach their final destination with well-controlled speed and direction.-Zhu, K., Takada, Y., Nakajima, K., Sun, Y., Jiang, J., Zhang, Y., Zeng, Q., Takada, Y., Zhao, M. Expression of integrins to control migration direction of electrotaxis.

Corrigendum: Diabetic cornea wounds produce significantly weaker electric signals that may contribute to impaired healing.

This corrects the article DOI: 10.1038/srep26525.

A voxel-based analysis of neurobiological mechanisms in placebo analgesia in rats.

Placebo analgesia is the beneficial effect that follows despite a pharmacologically inert treatment. Modern neuroimaging studies in humans have delineated the hierarchical brain regions involved in placebo analgesia. However, because of the lack of proper approaches to perform molecular and cellular manipulations, the detailed molecular processes behind it have not been clarified. To address this issue, we developed an animal model of placebo analgesia in rats and analyzed the placebo analgesia related brain activity using small-animal neuroimaging method. We show here that gabapentin-based Pavlovian conditioning successfully induced placebo analgesia in neuropathic pain model rats and hierarchical brain regions are involved in placebo analgesia in rats, including the prelimbic cortex (PrL) of the medial prefrontal cortex (mPFC), nucleus accumbens (NAc), ventrolateral periaqueductal gray matter (vlPAG), etc. The functional couplings in placebo responders between the mPFC and vlPAG was interrupted by naloxone, an antagonist of µ opioid receptor. Moreover, both local chemical lesion and microinfusion of naloxone in the mPFC suppressed the placebo analgesia. These results suggest that the intrinsic µ opioid system in the mPFC causally contribute to placebo analgesia in rats, and the small-animal neuroimaging approach could provide important insights toward understanding the placebo effect in great detail.

Src activation decouples cell division orientation from cell geometry in mammalian cells.

Orientation of cell division plane plays a crucial role in morphogenesis and regeneration. Misoriented cell division underlies many important diseases, such as cancer. Studies with Drosophila and C. elegance models show that Src, a proto-oncogene tyrosine-protein kinase, is a critical regulator of this aspect of mitosis. However, the role for Src in controlling cell division orientation in mammalian cells is not well understood. Using genetic and pharmacological approaches and two extracellular signals to orient cell division, we demonstrated a critical role for Src. Either knockout or pharmacological inhibition of Src would retain the fidelity of cell division orientation with the long-axis orientation of mother cells. Conversely, re-expression of Src would decouple cell division orientation from the pre-division orientation of the long axis of mother cells. Cell division orientation in human breast and gastric cancer tissues showed that the Src activation level correlated with the degree of mitotic spindle misorientation relative to the apical surface. Examination of proteins associated with cortical actin revealed that Src activation regulated the accumulation and local density of adhesion proteins on the sites of cell-matrix attachment. By analyzing division patterns in the cells with or without Src activation and through use of a mathematical model, we further support our findings and provide evidence for a previously unknown role for Src in regulating cell division orientation in relation to the pre-division geometry of mother cells, which may contribute to the misoriented cell division.

Effects of Single and Repeated Exposure to a 50-Hz 2-mT Electromagnetic Field on Primary Cultured Hippocampal Neurons.

The prevalence of domestic and industrial electrical appliances has raised concerns about the health risk of extremely low-frequency magnetic fields (ELF-MFs). At present, the effects of ELF-MFs on the central nervous system are still highly controversial, and few studies have investigated its effects on cultured neurons. Here, we evaluated the biological effects of different patterns of ELF-MF exposure on primary cultured hippocampal neurons in terms of viability, apoptosis, genomic instability, and oxidative stress. The results showed that repeated exposure to 50-Hz 2-mT ELF-MF for 8 h per day after different times in culture decreased the viability and increased the production of intracellular reactive oxidative species in hippocampal neurons. The mechanism was potentially related to the up-regulation of Nox2 expression. Moreover, none of the repeated exposure patterns had significant effects on DNA damage, apoptosis, or autophagy, which suggested that ELF-MF exposure has no severe biological consequences in cultured hippocampal neurons.

Diabetic cornea wounds produce significantly weaker electric signals that may contribute to impaired healing.

Wounds naturally produce electric signals which serve as powerful cues that stimulate and guide cell migration during wound healing. In diabetic patients, impaired wound healing is one of the most challenging complications in diabetes management. A fundamental gap in knowledge is whether diabetic wounds have abnormal electric signaling. Here we used a vibrating probe to demonstrate that diabetic corneas produced significantly weaker wound electric signals than the normal cornea. This was confirmed in three independent animal models of diabetes: db/db, streptozotocin-induced and mice fed a high-fat diet. Spatial measurements illustrated that diabetic cornea wound currents at the wound edge but not wound center were significantly weaker than normal. Time lapse measurements revealed that the electric currents at diabetic corneas lost the normal rising and plateau phases. The abnormal electric signals correlated significantly with impaired wound healing. Immunostaining suggested lower expression of chloride channel 2 and cystic fibrosis transmembrane regulator in diabetic corneal epithelium. Acute high glucose exposure significantly (albeit moderately) reduced electrotaxis of human corneal epithelial cells in vitro, but did not affect the electric currents at cornea wounds. These data suggest that weaker wound electric signals and impaired electrotaxis may contribute to the impaired wound healing in diabetes.

An Experimental Model for Simultaneous Study of Migration of Cell Fragments, Single Cells, and Cell Sheets.

Recent studies have demonstrated distinctive motility and responses to extracellular cues of cells in isolation, cells collectively in groups, and cell fragments. Here we provide a protocol for generating cell sheets, isolated cells, and cell fragments of keratocytes from zebrafish scales. The protocol starts with a comprehensive fish preparation, followed by critical steps for scale processing and subsequent cell sheet generation, single cell isolation, and cell fragment induction, which can be accomplished in just 3 days including a 36-48 h incubation time. Compared to other approaches that usually produce single cells only or together with either fragments or cell groups, this facile and reliable methodology allows generation of all three motile forms simultaneously. With the powerful genetics in zebrafish our model system offers a useful tool for comparison of the mechanisms by which cell sheets, single cells, and cell fragments respond to extracellular stimuli.

Exposure to 50Hz-sinusoidal electromagnetic field induces DNA damage-independent autophagy.

As electromagnetic field (EMF) is commonly encountered within our daily lives, the biological effects of EMF are of great concern. Autophagy is a key process for maintaining cellular homeostasis, and it can also reveal cellular responses to environmental stimuli. In this study, we aim to investigate the biological effects of a 50Hz-sinusoidal electromagnetic field on autophagy and we identified its mechanism of action in Chinese Hamster Lung (CHL) cells. CHL cells were exposed to a 50Hz sinusoidal EMF at 0.4mT for 30min or 24h. In this study, we found that a 0.4mT EMF resulted in: (i) an increase in LC3-II expression and increased autophagosome formation; (ii) no significant difference in the incidence of γH2AX foci between the sham and exposure groups; (iii) reorganized actin filaments and increased pseudopodial extensions without promoting cell migration; and (iv) enhanced cell apoptosis when autophagy was blocked by Bafilomycin A1. These results implied that DNA damage was not directly involved in the autophagy induced by a 0.4mT 50Hz EMF. In addition, an EMF induced autophagy balanced the cellular homeostasis to protect the cells from severe adverse biological consequences.

Extremely Low Frequency Magnetic Fields Do Not Induce DNA Damage in Human Lens Epithelial Cells In Vitro.

Non-ionizing radiations, e.g., radiofrequency electromagnetic fields, could induce DNA damage and oxidative stress in human lens epithelial cells (LECs) which can be early events in cataractogenesis. Extremely low frequency magnetic fields (ELF MF) as another common form of man-made electromagnetic fields has been considered as suspected human carcinogen by International Agency for Research on Cancer (IARC) and become a focus that people play more and more attentions to. This study aimed to determine whether ELF MF can induce DNA damage in cultured human LECs at a relatively low intensity. Human LECs were exposed or sham-exposed to a 50 Hz ELF MF which produced by a well-designed exposure system at the intensity of 0.4 mT. DNA damage in human LECs was examined by the phosphorylated form of histone variant H2AX (γH2AX) foci formation assay and further explored with western blot, flow cytometry, and alkaline comet assay. Immunofluorescence analysis showed that 0.4 mT ELF MF did not significantly increase γH2AX foci formation in human LECs after 2, 6, 12, 24, or 48 hr exposure. No significant differences had been detected in γH2AX expression level between the ELF MF- and sham-exposure groups, while no obvious chromosomal DNA fragmentation was detected by alkaline comet assay after ELF MF exposure. The results indicate an absence of genotoxicity in ELF MF-exposed human epithelial cells and do not support the hypothesis that environmental ELF MF might be causally led to genomic instability via chromosomal damage response processes. Neither short nor long term continuous exposure to 50 Hz ELF MF at 0.4 mT could induce DNA damage in human lens epithelial cells in vitro.

cAMP and cGMP Play an Essential Role in Galvanotaxis of Cell Fragments.

Cell fragments devoid of the nucleus and major organelles are found in physiology and pathology, for example platelets derived from megakaryocytes, and cell fragments from white blood cells and glioma cells. Platelets exhibit active chemotaxis. Fragments from white blood cells display chemotaxis, phagocytosis, and bactericidal functions. Signaling mechanisms underlying migration of cell fragments are poorly understood. Here we used fish keratocyte fragments and demonstrated striking differences in signal transduction in migration of cell fragments and parental cells in a weak electric field. cAMP or cGMP agonists completely abolished directional migration of fragments, but had no effect on parental cells. The inhibition effects were prevented by pre-incubating with cAMP and cGMP antagonists. Blocking cAMP and cGMP downstream signaling by inhibition of PKA and PKG also recovered fragment galvanotaxis. Both perturbations confirmed that the inhibitory effect was mediated by cAMP or cGMP signaling. Inhibition of cathode signaling with PI3K inhibitor LY294002 also prevented the effects of cAMP or cGMP agonists. Our results suggest that cAMP and cGMP are essential for galvanotaxis of cell fragments, in contrast to the signaling mechanisms in parental cells.

KCNJ15/Kir4.2 couples with polyamines to sense weak extracellular electric fields in galvanotaxis.

Weak electric fields guide cell migration, known as galvanotaxis/electrotaxis. The sensor(s) cells use to detect the fields remain elusive. Here we perform a large-scale screen using an RNAi library targeting ion transporters in human cells. We identify 18 genes that show either defective or increased galvanotaxis after knockdown. Knockdown of the KCNJ15 gene (encoding inwardly rectifying K(+) channel Kir4.2) specifically abolishes galvanotaxis, without affecting basal motility and directional migration in a monolayer scratch assay. Depletion of cytoplasmic polyamines, highly positively charged small molecules that regulate Kir4.2 function, completely inhibits galvanotaxis, whereas increase of intracellular polyamines enhances galvanotaxis in a Kir4.2-dependent manner. Expression of a polyamine-binding defective mutant of KCNJ15 significantly decreases galvanotaxis. Knockdown or inhibition of KCNJ15 prevents phosphatidylinositol 3,4,5-triphosphate (PIP3) from distributing to the leading edge. Taken together these data suggest a previously unknown two-molecule sensing mechanism in which KCNJ15/Kir4.2 couples with polyamines in sensing weak electric fields.

Power frequency magnetic fields induced reactive oxygen species-related autophagy in mouse embryonic fibroblasts.

Power frequency magnetic fields (PFMF) have been reported to affect several cellular functions, such as cell proliferation and apoptosis. In this study, we investigated the effects of PFMF on mouse embryonic fibroblasts (MEF) autophagy. After cells were exposed to 50 Hz PFMF at 2 mT for 0.5 h, 2 h, 6 h, 12 h, and 24 h, we observed a significant increase in autophagic markers at 6 h, including (i) higher microtubule-associated protein 1 light chain 3-II (LC3-II), (ii) the increased formation of GFP-LC3 puncta, and (iii) increased numbers of autophagic vacuoles under transmission electron microscope. Moreover, we provide convincing evidence using chloroquine (CQ) that the increase of autophagic markers was the result of enhanced autophagic flux and not the suppression of lysosomal function. In a search for molecular mechanisms underlying PFMF-mediated autophagy, we observe that the autophagic process involved reactive oxygen species (ROS) and was independent of the mammalian target of rapamycin (mTOR) signaling pathway.

Promotion of p53 expression and reactive oxidative stress production is involved in zerumbone-induced cisplatin sensitization of non-small cell lung cancer cells.

p53 signaling plays an important role in cell death. Zerumbone, a natural cyclic sesquiterpene, has shown cytotoxic activity against many cancers. This study was done to investigate the anticancer effects of zerumbone on non-small cell lung cancer (NSCLC) cells and explored the involvement of p53 signaling. Cell viability was assessed by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium assay. Apoptosis was confirmed by annexin-V/propidium iodide staining and caspase activity assay. Mitochondrial membrane potential (Δφm) and reactive oxygen species (ROS) production were measured by flow cytometry. Depletion of p53 was achieved by transfection of specific small interfering RNA. Gene expression changes were determined by Western blot analysis. Zerumbone treatment caused a dose-dependent inhibition of A549 and H460 NSCLC cell viability. Zerumbone-induced mitochondrial apoptosis of NSCLC cells, evidenced loss of Δφm, release of mitochondrial cytochrome c, and activation of caspase-9 and -3. There was increased p53 and Bax expression and ROS production in zerumbone-treated cells. Downregulation of p53 or scavenging ROS interfered with the pro-apoptotic action of zerumbone. Combinational treatment with zerumbone and cisplatin significantly accelerated apoptosis and promoted p53 expression and ROS production in NSCLC cells, compared with each alone. These findings demonstrate that zerumbone induces mitochondrial apoptosis and enhances the susceptibility to cisplatin in NSCLC cells, which are, at least partially, mediated through activation of p53 signaling and promotion of ROS generation. This study may provide a rationale for the potential clinical application of zerumbone as a chemotherapeutic agent against NSCLC.

Weak power frequency magnetic field acting similarly to EGF stimulation, induces acute activations of the EGFR sensitive actin cytoskeleton motility in human amniotic cells.

In this article, we have examined the motility-related effects of weak power frequency magnetic fields (MFs) on the epidermal growth factor receptor (EGFR)-sensitive motility mechanism, including the F-actin cytoskeleton, growth of invasive protrusions and the levels of signal molecules in human amniotic epithelial (FL) cells. Without extracellular EGF stimulation, the field stimulated a large growth of new protrusions, especially filopodia and lamellipodia, an increased population of vinculin-associated focal adhesions. And, an obvious reduction of stress fiber content in cell centers was found, corresponding to larger cell surface areas and decreased efficiency of actin assembly of FL cells in vitro, which was associated with a decrease in overall F-actin content and special distributions. These effects were also associated with changes in protein content or distribution patterns of the EGFR downstream motility-related signaling molecules. All of these effects are similar to those following epidermal growth factor (EGF) stimulation of the cells and are time dependent. These results suggest that power frequency MF exposure acutely affects the migration/motility-related actin cytoskeleton reorganization that is regulated by the EGFR-cytoskeleton signaling pathway. Therefore, upon the MF exposure, cells are likely altered to be ready to transfer into a state of migration in response to the stimuli.

Optimizing imaging quality and radiation dose by the age-dependent setting of tube voltage in pediatric chest digital radiography.

OBJECTIVE: The quality and radiation dose of different tube voltage sets for chest digital radiography (DR) were compared in a series of pediatric age groups. MATERIALS AND METHODS: Forty-five hundred children aged 0-14 years (yr) were randomly divided into four groups according to the tube voltage protocols for chest DR: lower kilovoltage potential (kVp) (A), intermediate kVp (B), and higher kVp (C) groups, and the fixed high kVp group (controls). The results were analyzed among five different age groups (0-1 yr, 1-3 yr, 3-7 yr, 7-11 yr and 11-14 yr). The dose area product (DAP) and visual grading analysis score (VGAS) were determined and compared by using one-way analysis of variance. RESULTS: The mean DAP of protocol C was significantly lower as compared with protocols A, B and controls (p < 0.05). DAP was higher in protocol A than the controls (p <0.001), but it was not statistically significantly different between B and the controls (p = 0.976). Mean VGAS was lower in the controls than all three protocols (p < 0.001 for all). Mean VGAS did not differ between protocols A and B (p = 0.334), but was lower in protocol C than A (p = 0.008) and B (p = 0.049). CONCLUSION: Protocol C (higher kVp) may help optimize the trade-off between radiation dose and image quality, and it may be acceptable for use in a pediatric age group from these results.

Cell type-dependent induction of DNA damage by 1800 MHz radiofrequency electromagnetic fields does not result in significant cellular dysfunctions.

BACKGROUND: Although IARC clarifies radiofrequency electromagnetic fields (RF-EMF) as possible human carcinogen, the debate on its health impact continues due to the inconsistent results. Genotoxic effect has been considered as a golden standard to determine if an environmental factor is a carcinogen, but the currently available data for RF-EMF remain controversial. As an environmental stimulus, the effect of RF-EMF on cellular DNA may be subtle. Therefore, more sensitive method and systematic research strategy are warranted to evaluate its genotoxicity. OBJECTIVES: To determine whether RF-EMF does induce DNA damage and if the effect is cell-type dependent by adopting a more sensitive method γH2AX foci formation; and to investigate the biological consequences if RF-EMF does increase γH2AX foci formation. METHODS: Six different types of cells were intermittently exposed to GSM 1800 MHz RF-EMF at a specific absorption rate of 3.0 W/kg for 1 h or 24 h, then subjected to immunostaining with anti-γH2AX antibody. The biological consequences in γH2AX-elevated cell type were further explored with comet and TUNEL assays, flow cytometry, and cell growth assay. RESULTS: Exposure to RF-EMF for 24 h significantly induced γH2AX foci formation in Chinese hamster lung cells and Human skin fibroblasts (HSFs), but not the other cells. However, RF-EMF-elevated γH2AX foci formation in HSF cells did not result in detectable DNA fragmentation, sustainable cell cycle arrest, cell proliferation or viability change. RF-EMF exposure slightly but not significantly increased the cellular ROS level. CONCLUSIONS: RF-EMF induces DNA damage in a cell type-dependent manner, but the elevated γH2AX foci formation in HSF cells does not result in significant cellular dysfunctions.

Electrically guiding migration of human induced pluripotent stem cells.

A major road-block in stem cell therapy is the poor homing and integration of transplanted stem cells with the targeted host tissue. Human induced pluripotent stem (hiPS) cells are considered an excellent alternative to embryonic stem (ES) cells and we tested the feasibility of using small, physiological electric fields (EFs) to guide hiPS cells to their target. Applied EFs stimulated and guided migration of cultured hiPS cells toward the anode, with a stimulation threshold of <30 mV/mm; in three-dimensional (3D) culture hiPS cells remained stationary, whereas in an applied EF they migrated directionally. This is of significance as the therapeutic use of hiPS cells occurs in a 3D environment. EF exposure did not alter expression of the pluripotency markers SSEA-4 and Oct-4 in hiPS cells. We compared EF-directed migration (galvanotaxis) of hiPS cells and hES cells and found that hiPS cells showed greater sensitivity and directedness than those of hES cells in an EF, while hES cells migrated toward cathode. Rho-kinase (ROCK) inhibition, a method to aid expansion and survival of stem cells, significantly increased the motility, but reduced directionality of iPS cells in an EF by 70-80%. Thus, our study has revealed that physiological EF is an effective guidance cue for the migration of hiPS cells in either 2D or 3D environments and that will occur in a ROCK-dependent manner. Our current finding may lead to techniques for applying EFs in vivo to guide migration of transplanted stem cells.

Benzo[a]pyrene induces complex H2AX phosphorylation patterns by multiple kinases including ATM, ATR, and DNA-PK.

H2AX is phosphorylated (γH2AX) by members of the phosphatidylinositol 3-kinase (PI3K) family, including ataxia telangiectasia-mutated (ATM), ATM- and Rad3-related (ATR) and DNA-PK in response to DNA damage. While it has been reported that benzo[a]pyrene (BaP) cannot induce γH2AX alone in several cell lines, we have shown that BaP alone could induce γH2AX in human amnion FL cells. Thus, we further examined the ability of BaP to induce γH2AX in different cell systems. It was shown that BaP-induced γH2AX in HeLa cells in a time- and dose-dependent manner. BaP also induced γH2AX in ATM(-/-) mouse fibroblasts, DNA-PKcs(-/-) mouse fibroblasts, and a genetically modified human osteosarcoma U2OS cell line. PI3K inhibitors caffeine and wortmannin were then used in an effort to identify the kinase(s) responsible for BaP-induced γH2AX. Unexpectedly, in BaP-treated HeLa cells, caffeine pretreatment did not inhibit but rather increased γH2AX level. On the other hand, caffeine or wortmannin can inhibit BaP-induced γH2AX in either U2OS, DNA-PKcs(-/-) or ATM(-/-) cells. Taken together, these data suggest that BaP alone can induce H2AX phosphorylation in certain cell systems, and that members of the PI3K family, including ATM, ATR, and DNA-PK can participate in the phosphorylation of H2AX in the various cell types.