Huai Chiang

50-Hz magnetic field induces EGF-receptor clustering and activates RAS

Purpose: In a previous study, we found that exposure to a 50 Hz magnetic field (MF) could activate stress-activated protein kinase (SAPK) and P38 mitogen-activated protein (MAP) kinase (P38 MAPK) in Chinese hamster lung (CHL) fibroblast cells, and simultaneous exposure to a ‘noise’ MF of the same intensity inhibited these effects. In order to explore the possible target sites and upstream signal transduction molecules of SAPK and P38 MAPK, and further validate the interference effects of ‘noise’ MF on 50 Hz MF, the effects of MF exposure on clustering of epidermal growth factor (EGF) receptors and Ras protein activation were investigated. Materials and methods: CHL cells were exposed to a 50 Hz sinusoidal MF at 0.4 mT for different durations, and clustering of EGF receptors on cellular membrane and Ras protein activation were analyzed using immunofluorescence confocal microscopy and co-precipitation technology. EGF treatment served as the positive control. Results: The results showed that, compared with sham-exposed cells, exposure to a 50 Hz MF at 0.4 mT for 5 min slightly induced EGF receptor clustering, whereas exposure for 15 min enhanced receptor clustering significantly. Corresponding to receptor clustering, Ras protein was also activated after exposure to the 50 Hz MF. Exposure to a ‘noise’ MF (with frequency ranges from 30 – 90 Hz) at the same intensity and durations, did not significantly affect EGF receptor clustering and Ras protein. However, by superimposing the ‘noise’ MF, receptor clustering and Ras activation induced by 50 Hz MF were inhibited. Conclusion: The results suggested that membrane receptors could be one of the most important targets where extremely low frequency (ELF) MF interacts with cells, and Ras may participate in the signal transduction process of 50 Hz MF. Furthermore, a ‘noise’ MF could inhibit these effects caused by ELF-MF.

An incoherent magnetic field inhibited EGF receptor clustering and phosphorylation induced by a 50-Hz magnetic field in cultured FL cells.

Previously, we found that exposure to a 50-Hz magnetic field (MF) at 0.4 mT could induce epidermal growth factor (EGF) receptor clustering in Chinese hamster lung (CHL) fibroblast cells and superposition of an incoherent MF with the same intensity could inhibit the effect. In the present experiment, we investigated the effects of 50-Hz MF exposure at different intensities on EGF receptor clustering and phosphorylation in human amniotic cells (FL), and explored the interaction effect of an incoherent MF. Clustering and phosphorylation of EGF receptors on cellular membrane surface were analyzed using immunofluorescence assessed by confocal microscopy and western blot technology, respectively. EGF treatment served as a positive control. The results showed that, compared with sham exposure, exposure to a 50-Hz MF at 0.1, 0.2 or 0.4 mT for 15 min could significantly induce EGF receptor clustering and enhance phosphorylation on tyrosine-1173 residue in FL cells, whereas exposure to a 0.05 mT field for 15 min did not caused a significant effect. Exposure to an incoherent MF (frequency range between 30 to 90 Hz) at 0.2 mT for the same time neither induced EGF receptor clustering nor enhanced phosphorylation of EGF receptor in FL cells. When superposed, the incoherent MF at 0.2 mT completely inhibited EGF receptor clustering and phosphorylation induced by a 50-Hz MF at 0.1 and 0.2 mT. However, the incoherent MF could not completely eliminate the effects induced by a 0.4 mT 50-Hz MF. Based on the results of this experiment, we conclude that membrane receptors could be one of the main targets where extremely-low frequency (ELF) MF interacts with cells, and the intensity threshold, in the case of EGF receptors, is between 0.05 and 0.1 mT. An incoherent MF could completely inhibit the effects induced by an ELF-MF of equal or lower intensity.

EXPOSURE TO 50 HZ ELECTROMAGNETIC FIELDS INDUCES THE PHOSPHORYLATION AND ACTIVITY OF STRESS-ACTIVATED PROTEIN KINASE IN CULTURED CELLS

Protein phosphorylation is one of the important processes of cell signal transduction pathways. To study the effects of 50 Hz electromagnetic field (EMF) on the cell signal transduction process, the phosphorylation of stress-activated protein kinase (SAPK/JNK) extracted from Chinese hamster lung (CHL) cells exposed to 0.4 and 0.8 mT 50 Hz EMF for various durations was measured. A solid-phase kinase assay was used to measure the enzymatic activity of SAPK extracted from cells exposed to 50 Hz EMF at the same magnetic flux density and for only 15 min. The results showed that both 0.4 and 0.8 mT could induce the phosphorylation of SAPK, the phosphorylation of SAPK presented a time-dependent course, and there was a difference between the two intensities. The phosphorylated SAPK enhanced its enzymatic activity. All the data indicated that 50 Hz EMF could activate SAPK in a time- and intensity-dependent manner. The biological effects caused by 50 Hz EMF maybe related to the SAPK signal transduction pathway.

Using model organism Saccharomyces cerevisiae to evaluate the effects of ELF-MF and RF-EMF exposure on global gene expression.

The potential health hazard of exposure to electromagnetic fields (EMF) continues to cause public concern. However, the possibility of biological and health effects of exposure to EMF remains controversial and their biophysical mechanisms are unknown. In the present study, we used Saccharomyces cerevisiae to identify genes responding to extremely low frequency magnetic fields (ELF-MF) and to radiofrequency EMF (RF-EMF) exposures. The yeast cells were exposed for 6 h to either 0.4 mT 50 Hz ELF-MF or 1800 MHz RF-EMF at a specific absorption rate of 4.7 W/kg. Gene expression was analyzed by microarray screening and confirmed using real-time reverse transcription-polymerase chain reaction (RT-PCR). We were unable to confirm microarray-detected changes in three of the ELF-MF responsive candidate genes using RT-PCR (P > 0.05). On the other hand, out of the 40 potential RF-EMF responsive genes, only the expressions of structural maintenance of chromosomes 3 (SMC3) and aquaporin 2 (AQY2 (m)) were confirmed, while three other genes, that is, halotolerance protein 9 (HAL9), yet another kinase 1 (YAK1) and one function-unknown gene (open reading frame: YJL171C), showed opposite changes in expression compared to the microarray data (P < 0.05). In conclusion, the results of this study suggest that the yeast cells did not alter gene expression in response to 50 Hz ELF-MF and that the response to RF-EMF is limited to only a very small number of genes. The possible biological consequences of the gene expression changes induced by RF-EMF await further investigation.

A 1.8-GHz radiofrequency radiation induces EGF receptor clustering and phosphorylation in cultured human amniotic (FL) cells

Purpose: Many studies have shown that exposure to radiofrequency radiation (RFR) could activate cellular signal transduction pathways. In the present research, we investigated the effects of exposure to a 1.8-GHz RFR at different intensities on epidermal growth factor (EGF) receptor clustering and phosphorylation in human amniotic (FL) cells. Materials and methods: Receptor clustering on cellular membrane surface was analyzed using immunofluorescence assessed by confocal microscopy, and phosphorylation of EGF receptors was measured by western blot technology. EGF treatment served as a positive control. Results: The results showed that, compared with sham exposure, exposure to RFR at specific absorption rate (SAR) of 0.5, 1.0, 2.0, or 4.0 W/kg for 15 min significantly induced EGF receptor clustering and enhanced phosphorylation on the tyrosine-1173 residue in FL cells, whereas exposure to a SAR 0.1 W/kg radiation for 15 min did not cause a significant effect. Conclusion: Based on the results of this experiment, we conclude that membrane receptors could be one of the main targets that RFR interacts with cells, and the dose-rate threshold, in the case of EGF receptors, is between SAR of 0.1 and 0.5 W/kg. The results indicate a sigmoid dependence of RFR effects on intensity.

STUDY ON GAP JUNCTIONAL INTERCELLULAR COMMUNICATION INHIBITION BY ELF MAGNETIC FIELDS USING FRAP METHOD

Inhibition of gap junctional intercellular communication (GJIC) is an important event in the multistage process of carcinogenesis. Our previous study showed that extremely low frequency (ELF) magnetic fields (MFs) inhibit GJIC, and enhance the suppression of GJIC induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) using a microinjection technique. In the present study, the inhibition of GJIC by ELF MFs and its threshold were further studied with fluorescence recovery after photobleaching (FRAP) technique. The results indicated that the FRAP technique is more sensitive in detecting the changes of GJIC than microinjection, and the threshold level is 0.4 mT for GJIC suppression by 50 Hz MFs. In addition, 0.2 mT, or more than 0.2 mT ELF can enhance the inhibition of GJIC induced by TPA. We concluded that MFs thus might act as a cancer promoter or work in synergy with other cancer promoters. The data also provide grounds to revise the reference standard of ELF MFs exposure.

Superposition of an incoherent magnetic field inhibited EGF receptor clustering and phosphorylation induced by a 1.8 GHz pulse-modulated radiofrequency radiation

Abstract Purpose: The present study was conducted to investigate the effect of a temporally incoherent (‘noise’) magnetic field (MF) on radiofrequency radiation (RFR)-induced epidermal growth factor (EGF) receptor clustering and phosporylation in cultured cells. Materials and methods: Human amniotic epithelial (FL) cells were exposed for 15 min to either a 1.8 GHz RFR (modulated at 217 Hz), a 2 μT incoherent MF, or concurrently to the RFR and incoherent MF. Epidermal growth factor treatment severed as the positive control. Epidermal growth factor receptor clustering on cellular membrane surface was analyzed using confocal microscopy after indirect immunofluorescence staining, and phosphorylation of EGF receptors was measured by western blot technology. Results: Exposure of FL cells to the 1.8 GHz RFR at SAR (specific absorption rate) of 0.5, 1.0, 2.0, or 4.0 W/kg for 15 min induced EGF receptor clustering and enhanced phosphorylation on tyrosine-1173 residue, whereas exposure to RFR at SAR of 0.1 W/kg for 15 min did not significantly cause these effects. Exposure to a 2 μT incoherent MF for 15 min did not significantly affect clustering and phosphorylation of EGF receptor in FL cells. When superimposed, the incoherent MF completely inhibited EGF receptor clustering and phosphorylation induced by RFR at SAR of 0.5, 1.0, and 2.0 W/kg, but did not inhibit the effects induced at SAR of 4.0 W/kg. Conclusion: Based on the data of the experiment, it is suggested that membrane receptors could be one of the main targets by which RFR interacts with cells. An incoherent MF could block the interaction to a certain extent.

Superimposition of an Incoherent Magnetic Field Eliminated the Inhibition of Hormone Secretion Induced by a 50-Hz Magnetic Field in Human Villous Trophoblasts in vitro

The effects of exposure to a sinusoidal 50-Hz magnetic field (MF) and an incoherent (noise) MF on hormone secretion in human first trimester villous trophoblasts were investigated. Trophoblasts were isolated from more than 30 first trimester human chorionic villi of 8-10 weeks gestation. They were cultured and exposed to MFs for different durations. The concentrations of human chorionic gonadotropin (hCG) and progesterone in culture medium were measured by electrochemiluminescence immunoassay. The results showed that exposure of the villous trophoblasts to a 50-Hz MF at 0.4 mT (milli Tesla) for 72 hrs could significantly inhibit the secretion of hCG and progesterone, whereas exposure to an incoherent MF (frequency range between 30 to 90 Hz) with the same conditions did not significantly affect the secretion. However, when the incoherent MF was superimposed on the 50-Hz MF and the cells were exposed to both fields simultaneously, no significant change in hormone secretion was observed. Based on these results, we concluded that 50-Hz MF exposure for 72 hrs could inhibit the hormone secretion of trophoblasts, and an incoherent MF of equal intensity could completely eliminate the effects induced by the 50-Hz MF.

EFFECTS OF 50 Hz MAGNETIC FIELD EXPOSURE ON PROTEIN TYROSINE PHOSPHORYLATION IN CULTURED CELLS

Protein phosphorylation is an extremely important and widely used mechanism of cellular regulation. Here, the effects of 50 Hz magnetic fields (MFs) on tyrosine phosphorylation were studied. A Chinese hamster lung (CHL) cell line was exposed to 50 Hz magnetic fields at two intensities (0.4 mT and 0.8 mT) for different exposure durations, and western blot analysis was used to measure the degree of tyrosine phosphorylation of cellular proteins. Results showed that both 0.4 mT and 0.8 mT 50 Hz magnetic fields could affect the protein tyrosine phosphorylation in cultured cells. Both intensities could affect the tyrosine phosphorylation of 38 and 97.4 kDa proteins. In addition, 0.4 mT could affect tyrosine phosphorylation of 61.7, 105, and 112 kDa proteins, and 0.8 mT affected the tyrosine phosphorylation of 79 and 150 kDa proteins. Moreover, all the tyrosine phosphorylation changes of these proteins were time-dependent. The findings from this study demonstrated that under these experimental conditions, there was evidence that protein tyrosine phosphorylation was a possible process for ELF-EMF producing bioeffects.

EXPOSURE TO 50 Hz MAGNETIC FIELDS DOES NOT INDUCE THE PHOSPHORYLATION OF SEK1/MKK4 IN CULTURED CELLS

In our previous studies, we found that 50 Hz magnetic fields (MFs) could induce the phosphorylation of stress-activated protein kinase (SAPK) and enhance its enzymatic activity. In order to clarify the relationship between MF exposure and the SAPK pathway clearly, we studied the effects of 50 Hz MF exposure on phosphorylation (activation) of SEK1/MKK4 (the upstream kinase of SAPK). A Chinese hamster lung (CHL) cell line was exposed to 50 Hz MFs at two intensities (0.4 and 0.8 mT) for different durations, and Western blot analysis was used to measure the degree of phosphorylation (activation), and nonphosphorylation (non-activation) of SEK1/MKK4 with corresponding antibodies. The results showed that the exposures at both intensities could not induce the phosphorylation of SEK1/MKK4. However, treatment with high osmotic pressure NaCl could induce the phosphorylation of SEK1/MKK4 in cultured cells. It is suggested that 50 Hz MFs may activate the SAPK through a kinase other than SEK1/MKK4.