Free
Lens  |   May 2008
Effect of Superposed Electromagnetic Noise on DNA Damage of Lens Epithelial Cells Induced by Microwave Radiation
Author Affiliations
  • Ke Yao
    From the Eye Center, Affiliated Second Hospital, the
  • Wei Wu
    From the Eye Center, Affiliated Second Hospital, the
  • Yibo Yu
    From the Eye Center, Affiliated Second Hospital, the
  • Qunli Zeng
    Bioelectromagnetics Laboratory, and the
  • Jiliang He
    Institute of Occupational and Environmental Health, College of Medicine, Zhejiang University, Hangzhou, China.
  • Deqiang Lu
    Bioelectromagnetics Laboratory, and the
  • Kaijun Wang
    From the Eye Center, Affiliated Second Hospital, the
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 2009-2015. doi:10.1167/iovs.07-1333
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Ke Yao, Wei Wu, Yibo Yu, Qunli Zeng, Jiliang He, Deqiang Lu, Kaijun Wang; Effect of Superposed Electromagnetic Noise on DNA Damage of Lens Epithelial Cells Induced by Microwave Radiation. Invest. Ophthalmol. Vis. Sci. 2008;49(5):2009-2015. doi: 10.1167/iovs.07-1333.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

purpose. To investigate the influence of the 1.8-GHz radiofrequency fields (RFs) of the Global System for Mobile Communications on DNA damage, intracellular reactive oxygen species (ROS) formation, cell cycle, and apoptosis in human lens epithelial cells (hLECs) and whether the effects induced by RF could be blocked by superposing of electromagnetic noise.

methods. After 24-hour intermittent exposure at the specific absorption rate of 1 W/kg, 2 W/kg, 3 W/kg, and 4 W/kg, the DNA damage of hLECs was examined by alkaline comet assay and immunofluorescence microscope detection of the phosphorylated form of histone variant H2AX (γH2AX) foci, respectively. ROS production was quantified by the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). Cell cycle and cell apoptosis were determined by flow cytometry.

results. DNA damage examined by alkaline comet assay was significantly increased after 3 W/kg and 4 W/kg radiation (P < 0.05), whereas the double-strand breaks (DSBs) evaluated by γH2AX foci were significantly increased only after 4 W/kg radiation (P < 0.05). Significantly elevated intracellular ROS levels were also detected in the 3-W/kg and 4-W/kg groups (P < 0.05). After exposure to 4 W/kg for 24 hours, hLECs exhibited significant G0/G1 arrest (P < 0.05). There was no detectable difference in cell apoptosis between the microwave radiation and sham exposure groups (P > 0.05). All the effects mentioned were blocked when the RF was superposed with 2 μT electromagnetic noise.

conclusions. Microwave radiation induced hLEC DNA damage after G0/G1 arrest does not lead to cell apoptosis. The increased ROS observed may be associated with DNA damage. Superposed electromagnetic noise blocks microwave radiation-induced DNA damage, ROS formation, and cell cycle arrest.

The ever-increasing applications of radiofrequency (RF) electromagnetic fields (EMFs; 10–300 GHz) in, for example, mobile phones, microwave ovens, radios, and radar, have raised public concerns about the potential health hazards associated with nonionizing radiation. The microwave radiation (300 MHz-300 GHz) included in RF EMFs is also widespread, especially the 1.8-GHz microwaves of the Global System for Mobile Communications (GSM) used in mobile phones. 
Given that positive 1 2 3 4 and negative 5 6 7 results are published in the literature, whether microwave radiation does induce DNA damage is still controversial. Previously, we reported that 1.8-GHz RF EMF (specific absorption rate [SAR], 3 W/kg) radiation could induce repairable DNA damage in hLECs after 2-hour exposure 8 and DNA damage in Chinese hamster lung cells after 24-hour exposure. 9 However, exposure to 1.8-GHz RF EMF (SAR, 3 W/kg) for 2 hours did not induce human lymphocyte DNA damage in vitro but could enhance DNA damage effects induced by mitomycin C (MMC; DNA cross-linker) and 4-nitroquinoline-1-oxide (4NQO; UV-mimetic agent). 10 It may be associated with the different sensitivity of cell line exposure to EMF. Because the energy of microwaves is ostensibly too weak to break DNA directly, the mechanism is unclear. Previous studies have shown that EMF increased the formation of reactive oxygen species (ROS), 11 12 which have been shown to induce DNA damage. It is possible the RF-induced DNA damage may be associated with the overproduction of ROS. 
Eyeballs are hot spots of radiofrequency field radiation (RFR). 13 Many investigations have demonstrated that microwave radiation can induce cataracts. 14 15 The ocular lens is sensitive to microwave radiation because of its nonvascularity, noninnervation, and high percentage of water. The oxidative damage of hLECs is the main mechanism of cataract formation 16 17 ; DNA damage to hLECs is also associated with cataracts. 18  
The biophysical mechanisms by which cells respond to electromagnetic exogenous fields are still unknown. These fields are several orders of magnitude weaker than the random thermal noise fields generated by the thermal motion of the ions in and around the cells. To resolve the signal-to-noise problem, Litovitz et al. 19 20 21 proposed that living cells were affected only by EMF that were temporally and spatially coherent, whereas endogenous thermal noise fields are normally temporally and spatially incoherent. They suggested that the bioeffects induced by EMF would be interfered with when superposed with spatially coherent but temporally incoherent “noise” fields. This suggestion has been subsequently supported by findings from several experiments. Simultaneous exposure to noise significantly attenuated the bioeffects caused by microwave radiation, such as enhancement of ornithine decarboxylase activity in L929 cells 22 and spatial learning deficits in rats. 23 In our previous studies, superposition of noise magnetic field (MF) could inhibit epidermal growth factor (EGF) receptor clustering in Chinese hamster lung fibroblasts induced by GSM 1800 MHz RF EMF 24 and could block MF-induced gap junction intercellular communication suppression in mouse fibroblast cells. 25  
A localized specific absorption rate (head and trunk) of 2 W/kg is recommended by the International Commission on Non-Ionizing Radiation Protection (ICNIRP) as the basic restriction in terms of RF EMF (10 MHz-10 GHz) for general public exposure. 26 According to these criteria, the safety limit for mobile phones in most European countries is 2 W/kg, though the safety limit for mobile phone emissions in the USA is 1.6 W/kg according to the Institute of Electrical and Electronics Engineers standard established in 1999. 27 Furthermore, in the 1998 guidelines of the ICNIRP, 26 the available experimental evidence indicated that the threshold for irreversible effects in even the most sensitive tissues was greater than 4 W/kg, which is recommended as the occupational whole-body exposure restriction with a traditional safety factor of 10, indicating 0.4 W/kg. Therefore, in this experiment, we used an sXc-1800 RF exposure system to investigate whether exposure to 1.8 GHz RF (217 Hz amplitude-modulated) at the SAR of 1, 2, 3, and 4 W/kg for 24 hours intermittently (5 minutes fields on/10 minutes fields off) would cause DNA damage or ROS formation. Cell cycle arrest and cell apoptosis, the events that follow DNA damage, were examined by flow cytometry. The effects of microwave radiation superposed with a noise MF or noise MF alone were also examined. 
Materials and Methods
Cell Culture
Human lens epithelial cell line SRA01/04 was purchased from the Riken Cell Bank (Ibaraki, Japan) and was cultured in Dulbecco modified Eagle medium (DMEM; Gibco, Grand Island, NY) with 20% heat-inactivated fetal bovine serum (HIFBS; Hyclone Laboratories Inc., Logan, UT) at 37°C in an atmosphere of 95% air and 5% CO2. The cells were divided into four groups: a sham exposure group; microwave radiation group at the SAR of 1, 2, 3, or 4 W/kg; a 2-μT noise MF group, and a microwave radiation superposed of noise MF, for 24 hours, respectively. Exponentially dividing cells were cultured in 35-mm-diameter dishes (Nunc, Roskilde, Denmark) in a total volume of 2 mL for radiation or sham exposure. 
Exposure Systems
sXc-1800 equipment producing a GSM signal, designed by the Foundation for Information Technologies in Society (Zurich, Switzerland) and described in detail by the designer and other groups, 4 28 was used as a microwave source. It consists primarily of an RF generator, an arbitrary function generator, a narrow band amplifier, and two rectangular waveguides operating at a frequency of 1.8 GHz. The two waveguides, one for exposure and the other for sham exposure, are placed inside a conventional incubator to ensure constant environmental conditions (37°C, 5% CO2/95% air atmosphere). The increased temperatures of the cells within the culture dish exposed to the SAR of 1 W/kg, 2 W/kg, 3 W/kg, and 4 W/kg were 0.027°C, 0.054°C, 0.081°C, and 0.108°C, respectively. A dish holder inside the waveguide guarantees that the dishes are placed exactly in the H-field maximum of the standing wave and are exposed simultaneously in E polarization inside a waveguide. The system enables the exposure of a monolayer of cells with less than 30% nonuniformity of SAR. Six Petri dishes can be exposed simultaneously in one exposure waveguide. The entire setup is computer controlled, enabling the automated control of the exposure parameters, including exposure strength (SAR), exposure time, and exposure pattern. The RF EMF-simulating GSM 1.8-GHz signal is amplitude modulated by a rectangular pulse with a repetition frequency of 217 Hz and a duty cycle of 1:8. The hLECs were intermittently (5 minutes fields on/10 minutes fields off) exposed or sham-exposed to RF EMF for 24 hours at an average SAR of 1, 2, 3, or 4 W/kg, respectively. 
To generate a noise MF, both sides of the waveguides of sXc-1800 system were wrapped with two rectangular Helmholtz coils. The center distance of two Helmholtz coils is 24 cm. The direction of the coils is the same as the circular wires in the RF waveguides, and the direction of the noise MF is consistent with the magnetic field of microwave radiation. The coils were provided with a 30- to 90-Hz white noise signal (generated through software designed by Luis M. Penafiel, Catholic University of America). The amplitude of the noise MF was 2 μT in the experiment. 
Comet Assay
The alkaline (pH >13) single-cell gel electrophoresis assay was performed by a modified method of Singh et al. 29 The solution, 0.65% normal melting agarose (NMA) and 0.65% low melting agarose (LMA), was prepared in Ca2+, Mg2+-free phosphate-buffered saline (PBS). Cells were suspended in LMA, and 75 μL LMA-cell suspension were pipetted onto a frosted glass microscope slide precoated with a 100-μL layer of 0.65% NMA. The third layer of 75 μL of 0.65% LMA was finally added. Then the slides were immersed in freshly prepared ice-cold lysis solution (1% N-lauroylsarcosine sodium salt, 2.5 M NaCl, 100 mM Na2 EDTA, 10 mM Tris-HCl, 1% Triton X-100, and 10% DMSO [pH 10]) to lyse the cell proteins and allow DNA unfolding. After at least 1 hour at 4°C in the dark, the slides were covered with fresh buffer (1 mM Na2 EDTA, 300 mM NaOH [pH >13]) in a horizontal electrophoresis unit. The slides were allowed to sit in this buffer for 20 minutes to allow for DNA unwinding. Then the DNA was electrophoresed at 20 V and 300 mA for 20 minutes. Unwinding and electrophoresis were performed at 4°C. The slides were washed gently to remove alkali and detergent in a neutralization buffer (0.4 M Tris HCl [pH 7.5]) and fixed in methanol for 3 minutes, and then were stained with 50 μL ethidium bromide (20 μg/mL). All the steps described were conducted under yellow light or in the dark to prevent additional DNA damage. Pictures of 300 cells per treatment sample (100 cells/slide; three replicate slides per experiment per SAR group) were taken individually at 400× magnification under a fluorescence microscope (BX51; Olympus, Tokyo, Japan) equipped with a 530-nm excitation filter, a 590-nm emission filter, and a digital camera (DP50; Olympus). Nuclear width and extent of migration of DNA fragments, mean tail length, and mean tail moment were analyzed using the Image-Pro Plus program (Media Cybernetics Inc.). 
Immunofluorescence Microscope Detection of γH2AX Foci
After radiation, cells were fixed in 4% paraformaldehyde for 15 minutes, washed with PBS, and permeabilized in 0.2% Triton X-100. After treatment with blocking serum (Zhongshan Biotechnology Co., Beijing, China) for 2 hours, samples were incubated with a 1:1000 mouse monoclonal anti-γH2AX antibody (Upstate Technology, Lake Placid, NY) for 2 hours, followed by 1:500 FITC-conjugated goat anti-mouse IgG secondary antibody (Zhongshan Biotechnology Co.) for 1 hour. To stain the nuclei, DAPI was added to the cells and incubated for another 15 minutes. The coverslip was then removed from the plate, mounted onto a glass slide, and observed under a fluorescence microscope (AX70; Olympus), and the γH2AX foci in each cell were counted (Image Pro Plus; Media Cybernetics). 
Intracellular ROS Detection
ROS production was quantified by the DCFH-DA method 30 based on the ROS-dependent oxidation of DCFH-DA to DCF. Cells were incubated for 30 minutes at 37°C with DCFH-DA solution with a final concentration of 50 μM. After incubation, cells were rinsed twice with PBS, collected with trypsin-EDTA solution (0.25% trypsin-0.02% ethylenediamine tetra acetic acid solution; Gibco, Grand Island, NY). After centrifugation at 1500 rpm for 5 minutes, the supernatant was discarded. The pellet was suspended with PBS. Fluorescence of the samples was monitored at an excitation wavelength of 485 nm and an emission wavelength of 538 nm. The ROS level was expressed as OD per milligram protein. 
Cell Cycle Assay
After treatment, approximately 2 × 106 cells were washed twice with PBS, then fixed in 75% ethanol for 24 hours at 4°C. After three washes with cold PBS, cells were stained with solution containing 50 μg/mL propidium iodide (Sigma, St. Louis, MO) and 10 μg/mL RNase (Sigma). After incubation at 37°C for 30 minutes, cells were analyzed by flow cytometry (FACSCalibur; Becton Dickinson, Mountain View, CA) using specialized software (CellQuest 3.1f; BD Biosciences, San Jose, CA). 
Cell Apoptosis Assay
Apoptosis assay was performed by flow cytometry (FACSCalibur; Becton Dickinson), using an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit (Sigma) in which Annexin V bound to the exposed phosphatidylserine on the plasma membrane of the apoptotic cells. Statistical analysis was performed using specialized software (CellQuest 3.1f; BD Biosciences). The percentage of the early apoptosis was estimated by counting cells that were Annexin V positive but PI negative, whereas the percentage of late apoptosis plus necrosis was estimated by counting cells that were both Annexin V and PI positive. 
Statistical Analysis
Statistical comparisons were conducted with one-way ANOVA multiple-comparison followed by the Dunnett test. P < 0.05 was considered statistically significant. 
Results
DNA Damage Induced by Microwaves in Comet Assay
Figure 1shows the mean tail length and mean tail moment results of the alkaline comet assay on hLECs after exposure to 1.8 GHz microwave radiation for 24 hours. No significant difference in DNA damage was observed between 1 W/kg or 2 W/kg microwave exposure and sham exposure (P > 0.05). However, 1.8 GHz microwave exposure at the SAR of 3 W/kg and 4 W/kg for 24 hours induced significantly increased DNA damage compared with sham exposure (P < 0.05). Moreover, DNA damage induced by 4 W/kg microwave radiation was significantly higher than that induced by 3 W/kg microwave radiation (P < 0.05). DNA damage induced by 3 W/kg and 4 W/kg microwave radiation was blocked by superposing of electromagnetic noise (P > 0.05). 
DSBs Induced by Microwaves in γH2AX Foci Formation Test
Statistically significant difference of DSBs in the γH2AX foci formation test was observed between the sham exposure and 4 W/kg microwave exposure (P < 0.05). No significant increase in γH2AX foci (P > 0.05) was found at the SAR of 1-, 2-, or 3-W/kg groups and also not in the microwave radiation superposed with the noise MF group (Fig. 2A)
Figure 2Bshows that 70% to 80% of cells in sham exposure and microwave radiation at the SAR of 1, 2, or 3 W/kg did not contain any γH2AX foci, whereas the percentage of cells lacking γH2AX foci in the 4-W/kg group was 57.34% ± 5.73% (P < 0.05). The percentage of cells with 1 to 10 γH2AX foci was 36.65% ± 4.90%, and the percentage with more than 20 γH2AX foci was 4.58% ± 1.28% in 4-W/kg group; these were significantly higher than the percentages in the sham exposure group (21.42% ± 2.98% and 1.75% ± 1.00%, respectively; P < 0.05). 
Intracellular ROS Increase after Microwave Radiation
ROS production in exposed samples was expressed as a percentage of the sham-exposed ones. SAR of 3 W/kg and 4 W/kg microwave radiation induced significantly increased intracellular ROS (P < 0.05), whereas no significant changes were found at the SAR of the 1-W/kg and 2-W/kg groups compared with sham exposure (P > 0.05). Electromagnetic noise blocked the increased ROS induced by microwaves (P > 0.05; Fig. 3 ). 
Microwave Radiation Blocks hLEC Cell Cycle in the G0/G1 Phase
Figure 4shows the effects of 3 W/kg and 4 W/kg microwave radiation on the cell cycle, as determined by flow cytometry. The hLECs were arrested in the G0/G1 phase of the cell cycle after microwave radiation at the SAR of 4 W/kg after S-phase and G2/M-phase decreases (P < 0.05). Changes in the cell cycle induced by 4 W/kg microwave radiation were blocked by superposing of a noise MF compared with sham exposure (P > 0.05). There was no significant modification on the cell cycle in 3-W/kg radiation-treated cells (P > 0.05). 
Microwave Radiation Does Not Induce Significant Cell Apoptosis
Percentages of early apoptosis were estimated by counting cells that were Annexin V positive but PI negative, whereas the percentage of late apoptosis plus necrosis was estimated by counting cells that were both Annexin V and PI positive. There were no significant differences in cell apoptosis between the microwave radiation and sham exposure groups (P > 0.05). 
Electromagnetic Noise Does Not Induce Bioeffects
Exposure to electromagnetic noise alone did not lead to DNA damage, intracellular ROS formation, cell cycle arrest, or cell apoptosis compared with sham exposure (P > 0.05). 
Discussion
The eyes, despite their small volume, absorb a considerable amount of electromagnetic radiation. A specific absorption rate map of a versatile eccentric-sphere model of the human head, designed by Moneda et al., 13 revealed the existence of hot spots in the eyes and near the center of the brain. The RFR emitted by mobile phones, which are held close to the head and thus are in close proximity to eyeballs when in use, has provoked concern about the potential cataractogenesis that develops as a result of such exposure. The lens is an encapsulated, avascular, transparent tissue susceptible to damage. It contains a single layer of epithelial cells, and their functional disorders often lead to the formation of cataracts. In this study, we used a human lens epithelial cell line to investigate the mechanism of microwave radiation-induced cataracts. Although the immortalized human lens epithelial cells SRA 01/04 retain some of the human lens-specific characteristics, 31 they do not undergo differentiation into lens fibers in long-term culture. 32 33  
The assessment of direct and indirect effects on DNA is one of the worldwide subjects of interest in terms of the RFR domain. 34 The alkaline comet assay (pH >13) is considered a sensitive assay for detecting DNA damage, such as single-strand breaks (SSB), double-strand breaks (DSB), and alkali labile sites (ALS), and incomplete excision repair sites, especially the SSB. 35 36 The phosphorylated form of histone variant H2AX (γH2AX) plays an important role in the recruitment of DNA repair and checkpoint proteins such as the Mre11/Rad50/Nbs1 complex, BRCA1, and 53BP1, to sites of DNA damage, particularly at DSBs. 37 38 39 40 41 42 43 A few minutes after DSB formation, H2AX is phosphorylated by members of the phosphatidylinositol 3-kinase family and forms localized foci at DSB sites. 44 45 46 47 48 The number of γH2AX foci, which is quantitatively the same as that of DSBs, 49 has been used as an indicator for DSBs in many studies. 50 51 52 53  
The results of alkaline comet assay on hLECs in the present study indicated that DNA damage induced by microwave exposure (SAR; both 3 W/kg and 4 W/kg) was significantly higher than that induced by sham exposure. On the other hand, there was a significant difference in DNA damage examined by comet assay between the 4-W/kg group and 3-W/kg group; DSBs detected by the γH2AX foci formation test only were observed in the 4-W/kg group. Results of the comet assay and the γH2AX foci formation test showed that DNA damage induced by 4-W/kg microwave radiation was significantly higher than that induced by 3-W/kg microwave radiation. We suppose that the G0/G1 phase arrest in the 4-W/kg radiation-treated cells may be related to higher DNA damage, which needs more time for repair. Although 3 W/kg and 4 W/kg microwave radiation could induce DNA damage of hLECs, the results of cell apoptosis indicated that DNA damage induced by microwave radiation did not result in apoptosis; it is possible that DNA damage is repairable. 
The mechanism by which microwaves induce DNA damage is still unclear. As is well known, ROS are reactive and readily damage biological molecules, including DNA. ROS are generated as a byproduct of normal mitochondrial activity in aerobic cells. The overproduction of ROS reportedly causes severe damage to cellular macromolecules, especially the DNA. 54 Stopczyk et al. 11 found that oxidative stress after exposure to microwaves may be the reason for many adverse changes in cells. The study of Moustafa et al. 12 indicated that acute exposure to the radiofrequency fields of commercially available cellular phones may modulate the oxidative stress of free radicals by enhancing lipid peroxidation and reducing the activation of SOD and GSH-Px, which are free radical scavengers. Balci et al. 55 reported that mobile phone radiation leads to oxidative stress in corneal and lens tissues. We also detected elevated intracellular ROS levels of hLECs after mobile phone radiation at the SAR of 3 W/kg and 4 W/kg. We speculate that the surplus ROS produced by microwaves disturbs the balance between the oxidation and reduction systems, leading to DNA damage indirectly. The DNA lesions caused by ROS include oxidized bases, sugar lesions, abasic sites, DNA–protein cross-links, SSBs, and DSBs. 56 In addition, the oxidation of proteins and lipids may also generate intermediates that attack DNA. 57  
These bioeffects of microwave radiation may be attributed to nonthermal mechanisms because they were blocked in the microwave radiation/electromagnetic noise group, the temperature of which was the same as for the microwave radiation group. Litovitz et al. 19 20 21 22 have proposed that an EMF has to be coherent to exert bioeffects. Coherent is defined by certain characteristic parameters, such as frequency, amplitude, waveform, and pattern, which have to remain constant over a set period (more than approximately 10 seconds). Incoherent EMFs do not trigger a biological response. Furthermore, superposing an incoherent EMF, such as electromagnetic noise, on a coherent EMF, such as microwave radiation, can make the combined field become incoherent, thus blocking the biological effects of the coherent field. 23 The results of our research demonstrate that superposition of a noise MF over RF blocked RF-induced DNA damage and increased ROS and cell cycle arrest, thus supporting the temporal-and-spatial coherency hypothesis of Litovitz 19 20 21 22 and providing a potential approach to preventing the hazards of microwave radiation. 
 
Figure 1.
 
Results of alkaline comet assay on hLECs induced by 1.8 GHz microwave radiation with or without the superposition of electromagnetic noise. (A) Mean tail length and mean tail moment. Significantly increased DNA damage induced by 3 W/kg and 4 W/kg microwave radiation was blocked by the superposing of electromagnetic noise. (B) Representative images of alkaline comet assay. *P = 0.024; **P = 0.005; #P < 0.0001.
Figure 1.
 
Results of alkaline comet assay on hLECs induced by 1.8 GHz microwave radiation with or without the superposition of electromagnetic noise. (A) Mean tail length and mean tail moment. Significantly increased DNA damage induced by 3 W/kg and 4 W/kg microwave radiation was blocked by the superposing of electromagnetic noise. (B) Representative images of alkaline comet assay. *P = 0.024; **P = 0.005; #P < 0.0001.
Figure 2.
 
The γH2AX foci formation in hLECs induced by 1.8 GHz microwave radiation with or without the superposition of electromagnetic noise. (A) Percentage of positive cells. Microwave radiation (4 W/kg) induced more γH2AX foci than sham exposure, which was blocked by the superposition of electromagnetic noise. (B) Quantitative analyses of γH2AX foci formation (foci/cell). More percentages of cells with 1 to 10 γH2AX foci and with more than 20 γH2AX foci were observed in the 4-W/kg group. (C) Representative images of γH2AX foci. *P < 0.0001; #P = 0.004.
Figure 2.
 
The γH2AX foci formation in hLECs induced by 1.8 GHz microwave radiation with or without the superposition of electromagnetic noise. (A) Percentage of positive cells. Microwave radiation (4 W/kg) induced more γH2AX foci than sham exposure, which was blocked by the superposition of electromagnetic noise. (B) Quantitative analyses of γH2AX foci formation (foci/cell). More percentages of cells with 1 to 10 γH2AX foci and with more than 20 γH2AX foci were observed in the 4-W/kg group. (C) Representative images of γH2AX foci. *P < 0.0001; #P = 0.004.
Figure 3.
 
Intracellular ROS formation in hLECs induced by 1.8 GHz microwave radiation with or without the superposition of electromagnetic noise. Significant increases in intracellular ROS induced by 3 W/kg and 4 W/kg microwave radiation were blocked by the superposition of electromagnetic noise. *P = 0.035; #P = 0.036.
Figure 3.
 
Intracellular ROS formation in hLECs induced by 1.8 GHz microwave radiation with or without the superposition of electromagnetic noise. Significant increases in intracellular ROS induced by 3 W/kg and 4 W/kg microwave radiation were blocked by the superposition of electromagnetic noise. *P = 0.035; #P = 0.036.
Figure 4.
 
Flow cytometry analysis of the cell cycle of hLECs after 1.8 GHz microwave radiation with or without the superposition of electromagnetic noise. The hLECs of the 4-W/kg group were arrested in the G0/G1 phase, which was blocked by the superposition of electromagnetic noise. *P < 0.0001; #P = 0.002.
Figure 4.
 
Flow cytometry analysis of the cell cycle of hLECs after 1.8 GHz microwave radiation with or without the superposition of electromagnetic noise. The hLECs of the 4-W/kg group were arrested in the G0/G1 phase, which was blocked by the superposition of electromagnetic noise. *P < 0.0001; #P = 0.002.
Supplementary Materials
Retraction - (PDF) 
This research was conducted primarily in the Bioelectromagnetics Laboratory in Zhejiang University. The authors thank Zhengping Xu and Huai Jiang of the Bioelectromagnetics Laboratory for providing the 1.8-GHz microwave and electromagnetic noise sources. They also thank Ji-liang He of the Institute of Occupational and Environmental Health and Li-hong Xu of the Department of Biochemistry and Molecular Biology in Zhejiang University for providing equipment and techniques of comet assay and ROS detection, respectively. 
GarajVV, HorvatD, KorenZ. The relationship between colony-forming ability, chromosome aberrations and incidence of micronuclei in V79 Chinese hamster cells exposed to microwave radiation. Mutat Res. 1991;263(3)143–149. [CrossRef] [PubMed]
LaiH, SinghNP. Acute low-intensity microwave exposure increases DNA single-strand breaks in rat brain cells. Bioelectromagnetics. 1995;16(3)207–210. [CrossRef] [PubMed]
LaiH, SinghNP. Single- and double-strand DNA breaks in rat brain cells after acute exposure to radiofrequency electromagnetic radiation. Int J Radiat Biol. 1996;69(4)513–521. [CrossRef] [PubMed]
DiemE, SchwarzC, AdlkoferF, JahnO, RüdigerH. Non-thermal DNA breakage by mobile-phone radiation (1800 MHz) in human fibroblasts and in transformed GFSH-R17 rat granulosa cells in vitro. Mutat Res. 2005;583(2)178–183. [CrossRef] [PubMed]
MalyapaRS, AhernEW, BiC, et al. DNA damage in rat brain cells after in vivo exposure to 2450 MHz electromagnetic radiation and various methods of euthanasia. Radiat Res. 1998;149(6)637–645. [CrossRef] [PubMed]
HookGJ, ZhangP, LagroyeI, et al. Measurement of DNA damage and apoptosis in Molt-4 cells after in vitro exposure o radiofrequency radiation. Radiat Res. 2004;161(2)193–200. [CrossRef] [PubMed]
BelyaevIY, KochCB, TereniusO, et al. Exposure of rat brain to 915 MHz GSM microwaves induces changes in gene expression but not double stranded DNA breaks or effects on chromatin conformation. Bioelectromagnetics. 2006;27(4)295–306. [CrossRef] [PubMed]
LixiaS, YaoK, KaijunW, et al. Effects of 1.8 GHz radiofrequency field on DNA damage and expression of heat shock protein 70 in human lens epithelial cells. Mutat Res. 2006;602(1–2)135–142. [CrossRef] [PubMed]
ZhangDY, XuZP, ChiangH, LuDQ, ZengQL. Effects of GSM 1800 MHz radiofrequency electromagnetic fields on DNA damage in Chinese hamster lung cells. Zhonghua Yu Fang Yi Xue Za Zhi. 2006;40(3)149–152. [PubMed]
BaohongW, JiliangH, LifenJ, et al. Studying the synergistic damage effects induced by 1.8 GHz radiofrequency field radiation (RFR) with four chemical mutagens on human lymphocyte DNA using comet assay in vitro. Mutat Res. 2005;578(1–2)149–157. [CrossRef] [PubMed]
StopczykD, GniteckiW, BuczyńskiA, KowalskiW, BuczyńskaM, KrocA. Effect of electromagnetic field produced by mobile phones on the activity of superoxide dismutase (SOD-1)–in vitro researches. Ann Acad Med Stetin. 2005;51(suppl 1)125–128. [PubMed]
MoustafaYM, MoustafaRM, BelacyA, et al. Effects of acute exposure to the radiofrequency fields of cellular phones on plasma lipid peroxide and antioxidase activities in human erythrocytes. J Pharm Biomed Anal. 2001;26(4)605–608. [CrossRef] [PubMed]
MonedaAP, IoannidouMP, ChrissoulidisDP. Radio-wave exposure of the human head: analytical study based on a versatile eccentric spheres model including a brain core and a pair of eyeballs. IEEE Trans Biomed Eng. 2003;50(6)667–676. [CrossRef] [PubMed]
LipmanRM, TripathiBJ, TripathiRC. Cataract induced by microwave and ionizing radiation. Surv Ophthalmol. 1988;33(3)200–210. [CrossRef] [PubMed]
YeJ, YaoK, LuD, WuR, JiangH. Low power density microwave radiation induced early changes in rabbit lens epithelial cells. Chin Med J (Engl). 2001;114(12)1290–1294. [PubMed]
TruscottRJ. Age-related nuclear cataract-oxidation is the key. Exp Eye Res. 2005;80(5)709–725. [CrossRef] [PubMed]
SpectorA. Oxidative stress-induced cataract mechanism of action. FASEB J. 1995;9(12)1173–1182. [PubMed]
KleimanNJ, SpectorA. DNA single strand breaks in human lens epithelial cells from patients with cataract. Curr Eye Res. 1993;12(5)423–431. [CrossRef] [PubMed]
LitovitzTA, KrauseD, MullinsJM. Effect of coherence time of the applied magnetic field on ornithine decarboxylase activity. Biochem Biophys Res Commun. 1991;178(3)862–865. [CrossRef] [PubMed]
LitovitzTA, MontroseCJ, DoinovP, BrownKM, BarberM. Superimposing spatially coherent electromagnetic noise inhibits field-induced abnormalities in developing chick embryos. Bioelectromagnetics. 1994;15(2)105–113. [CrossRef] [PubMed]
LitovitzTA, KrauseD, MontroseCJ, MullinsJM. Temporally incoherent magnetic fields mitigate the response of biological systems to temporally coherent magnetic fields. Bioelectromagnetics. 1994;15(5)399–409. [CrossRef] [PubMed]
LitovitzTA, PenafielLM, FarrelJM, KrauseD, MeisterR, MullinsJM. Bioeffects induced by exposure to microwaves are mitigated by superposition of ELF noise. Bioelectromagnetics. 1997;18(6)422–430. [CrossRef] [PubMed]
LaiH. Interaction of microwaves and a temporally incoherent magnetic field on spatial learning in the rat. Physiol Behav. 2004;82(5)785–789. [CrossRef] [PubMed]
XieL, JiangH, SunWJ, FuYT, LuDQ. GSM 1,800 MHz radiofrequency electromagnetic fields induced clustering of membrane surface receptors and interference by noise magnetic fields. Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2006;24(8)461–464. [PubMed]
ZengQ, KeX, GaoX, et al. Noise magnetic fields abolish the gap junction intercellular communication suppression induced by 50 Hz magnetic fields. Bioelectromagnetics. 2006;27(4)274–279. [CrossRef] [PubMed]
International Commission on Non-Ionizing Radiation Protection. Guidelines for limiting exposure to time-varying electric, magnetic, and electromagnetic fields (up to 300 GHz). Health Phys. 1998;74(4)494–522. [PubMed]
IEEE Standard for Safety Levels with Respect to Human Exposure to Radio Frequency Electromagnetic Fields, 3 KHz to 300 GHz. Std C95.1. 1999;IEEE
SchönbornF, PokovićK, BurkhardtM, KusterN. Basis for optimization of in vitro exposure apparatus for health hazard evaluations of mobile communications. Bioelectromagnetics. 2001;22(8)547–559. [CrossRef] [PubMed]
SinghNP, McCoyMT, TiceRR, SchneiderEL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res. 1988;175(1)184–191. [CrossRef] [PubMed]
LawlerJM, SongW, DemareeSR. Hindlimb unloading increases oxidative stress and disrupts antioxidant capacity in skeletal muscle. Free Radic Biol Med. 2003;35(1)9–16. [CrossRef] [PubMed]
IbarakiN, ChenSC, LinLR, OkamotoH, PipasJM, ReddyVN. Human lens epithelial cell line. Exp Eye Res. 1998;67(5)577–585. [CrossRef] [PubMed]
AritaT, LinLR, SusanSR, ReddyVN. Enhancement of differentiation of human lens epithelium in tissue culture by changes in cell-substrate adhesion. Invest Ophthalmol Vis Sci. 1990;31(11)2395–2404. [PubMed]
AritaT, MurataY, LinLR, TsujiT, ReddyVN. Synthesis of lens capsule in long-term culture of human lens epithelial cells. Invest Ophthalmol Vis Sci. 1993;34(2)355–362. [PubMed]
BrusickD, AlbertiniR, McReeD, et al. Genotoxicity of radiofrequency radiation: DNA/Genetox Expert Panel. Environ Mol Mutagen. 1998;32(1)1–16. [CrossRef] [PubMed]
FairbairnDW, OlivePL, O’NeillKL. The comet assay: a comprehensive review. Mutat Res. 1995;339(1)37–59. [CrossRef] [PubMed]
SinghNP. Microgel electrophoresis of DNA from individual cells.PfeiferGP eds. Technologies for Detection of DNA Damage and Mutations. 1996;3–24.Plenum Press, NY New York.
RedonC, PilchD, RogakouE, SedelnikovaO, NewrockK, BonnerW. Histone H2A variants H2AX and H2AZ. Curr Opin Genet Dev. 2002;12(2)162–169. [CrossRef] [PubMed]
Fernandez-CapetilloO, LeeA, NussenzweigM, NussenzweigA. H2AX: the histone guardian of the genome. DNA Repair (Amst). 2004;3(8–9)959–967. [CrossRef] [PubMed]
PilchDR, SedelnikovaOA, RedonC, CelesteA, NussenzweigA, BonnerWM. Characteristics of gamma-H2AX foci at DNA double-strand breaks sites. Biochem Cell Biol. 2003;81(3)123–129. [CrossRef] [PubMed]
WardIM, MinnK, JordaKG, ChenJ. Accumulation of checkpoint protein 53BP1 at DNA breaks involves its binding to phosphorylated histone H2AX. J Biol Chem. 2003;278(22)19579–19582. [CrossRef] [PubMed]
Fernandez-CapetilloO, ChenHT, CelesteA, et al. DNA damage-induced G2-M checkpoint activation by histone H2AX and 53BP1. Nat Cell Biol. 2002;4(12)993–997. [CrossRef] [PubMed]
FurutaT, TakemuraH, LiaoZY, et al. Phosphorylation of histoneH2AXand activation of Mre11, Rad50, and Nbs1 in response to replication-dependent DNA double-strand breaks induced by mammalian DNA topoisomerase I cleavage complexes. J Biol Chem. 2003;278(22)20303–20312. [CrossRef] [PubMed]
Fernandez-CapetilloO, CelesteA, NussenzweigA. Focusing on foci: H2AX and the recruitment of DNA-damage response factors. Cell Cycle. 2003;2(5)426–427. [PubMed]
RogakouEP, PilchDR, OrrAH, IvanovaVS, BonnerWM. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem. 1998;273(10)5858–5868. [CrossRef] [PubMed]
PaullTT, RogakouEP, YamazakiV, KirchgessnerCU, GellertM, BonnerWM. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr Biol. 2000;10(15)886–895. [CrossRef] [PubMed]
MacPhailSH, BanáthJP, YuTY, ChuEH, LamburH, OlivePL. Expression of phosphorylated histone H2AX in cultured cell lines following exposure to X-rays. Int J Radiat Biol. 2003;79(5)351–358. [CrossRef] [PubMed]
StiffT, O'DriscollM, RiefN, IwabuchiK, LöbrichM, JeggoPA. ATM and DNA-PK function redundantly to phosphorylate H2AX after exposure to ionizing radiation. Cancer Res. 2004;64(7)2390–2396. [CrossRef] [PubMed]
WangH, WangM, WangH, BöckerW, IliakisG. Complex H2AX phosphorylation patterns by multiple kinases including ATM and DNA-PK in human cells exposed to ionizing radiation and treated with kinase inhibitors. J Cell Physiol. 2005;202(2)492–502. [CrossRef] [PubMed]
RothkammK, LöbrichM. Evidence for a lack of DNA double-strand break repair in human cells exposed to very low X-ray doses. Proc Natl Acad Sci U S A. 2003;100(9)5057–5062. [CrossRef] [PubMed]
BanáthJP, OlivePL. Expression of phosphorylated histone H2AX as a surrogate of cell killing by drugs that create DNA double-strand breaks. Cancer Res. 2003;63(15)4347–4350. [PubMed]
TanejaN, DavisM, ChoyJS, et al. Histone H2AX phosphorylation as a predictor of radiosensitivity and target for radiotherapy. J Biol Chem. 2004;279(3)2273–2280. [CrossRef] [PubMed]
AlbinoAP, HuangX, JorgensenE, et al. Induction of H2AX phosphorylation in pulmonary cells by tobacco smoke: a new assay for carcinogens. Cell Cycle. 2004;3(8)1062–1068. [PubMed]
GallmeierE, WinterJM, CunninghamSC, KahnSR, KernSE. Novel genotoxicity assays identify norethindrone to activate p53 and phosphorylate H2AX. Carcinogenesis. 2005;26(10)1811–1820. [CrossRef] [PubMed]
BarzilaiA, YamamotoK. DNA damage responses to oxidative stress. DNA Repair (Amst). 2004;3(8–9)1109–1115. [CrossRef] [PubMed]
BalciM, DevrimE, DurakI. Effects of mobile phones on oxidant/antioxidant balance in cornea and lens of rats. Curr Eye Res. 2007;32(1)21–25. [CrossRef] [PubMed]
DizdarogluM. Mechanisms of free radical damage to DNA.AruomaO HalliwellB eds. DNA and Free Radicals. 1998;3–26.OICA International London.
MarnettLJ. Oxyradicals and DNA damage. Carcinogenesis. 2000;21(3)361–370. [CrossRef] [PubMed]
Figure 1.
 
Results of alkaline comet assay on hLECs induced by 1.8 GHz microwave radiation with or without the superposition of electromagnetic noise. (A) Mean tail length and mean tail moment. Significantly increased DNA damage induced by 3 W/kg and 4 W/kg microwave radiation was blocked by the superposing of electromagnetic noise. (B) Representative images of alkaline comet assay. *P = 0.024; **P = 0.005; #P < 0.0001.
Figure 1.
 
Results of alkaline comet assay on hLECs induced by 1.8 GHz microwave radiation with or without the superposition of electromagnetic noise. (A) Mean tail length and mean tail moment. Significantly increased DNA damage induced by 3 W/kg and 4 W/kg microwave radiation was blocked by the superposing of electromagnetic noise. (B) Representative images of alkaline comet assay. *P = 0.024; **P = 0.005; #P < 0.0001.
Figure 2.
 
The γH2AX foci formation in hLECs induced by 1.8 GHz microwave radiation with or without the superposition of electromagnetic noise. (A) Percentage of positive cells. Microwave radiation (4 W/kg) induced more γH2AX foci than sham exposure, which was blocked by the superposition of electromagnetic noise. (B) Quantitative analyses of γH2AX foci formation (foci/cell). More percentages of cells with 1 to 10 γH2AX foci and with more than 20 γH2AX foci were observed in the 4-W/kg group. (C) Representative images of γH2AX foci. *P < 0.0001; #P = 0.004.
Figure 2.
 
The γH2AX foci formation in hLECs induced by 1.8 GHz microwave radiation with or without the superposition of electromagnetic noise. (A) Percentage of positive cells. Microwave radiation (4 W/kg) induced more γH2AX foci than sham exposure, which was blocked by the superposition of electromagnetic noise. (B) Quantitative analyses of γH2AX foci formation (foci/cell). More percentages of cells with 1 to 10 γH2AX foci and with more than 20 γH2AX foci were observed in the 4-W/kg group. (C) Representative images of γH2AX foci. *P < 0.0001; #P = 0.004.
Figure 3.
 
Intracellular ROS formation in hLECs induced by 1.8 GHz microwave radiation with or without the superposition of electromagnetic noise. Significant increases in intracellular ROS induced by 3 W/kg and 4 W/kg microwave radiation were blocked by the superposition of electromagnetic noise. *P = 0.035; #P = 0.036.
Figure 3.
 
Intracellular ROS formation in hLECs induced by 1.8 GHz microwave radiation with or without the superposition of electromagnetic noise. Significant increases in intracellular ROS induced by 3 W/kg and 4 W/kg microwave radiation were blocked by the superposition of electromagnetic noise. *P = 0.035; #P = 0.036.
Figure 4.
 
Flow cytometry analysis of the cell cycle of hLECs after 1.8 GHz microwave radiation with or without the superposition of electromagnetic noise. The hLECs of the 4-W/kg group were arrested in the G0/G1 phase, which was blocked by the superposition of electromagnetic noise. *P < 0.0001; #P = 0.002.
Figure 4.
 
Flow cytometry analysis of the cell cycle of hLECs after 1.8 GHz microwave radiation with or without the superposition of electromagnetic noise. The hLECs of the 4-W/kg group were arrested in the G0/G1 phase, which was blocked by the superposition of electromagnetic noise. *P < 0.0001; #P = 0.002.
Retraction
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×