| With the rapid development of mobile phone communication industry, mobile phone users increased sharply. Therefore the growing radiofrequency electromagnetic fields (RF EMF) exposure emitted by mobile phone in the range of 800 to 2000 MHz, mainly 900 and 1800 MHz, has raised public concerns about its potential health hazard. Clarifying the underlying health effects of mobile phone radiation has become one of extremely important public health issues.Population-based epidemiological studies and animal studies imply there may be certain relationship between mobile phone use and certain cancer risk increase. Due to the close relationship between the carcinogenesis and DNA damage, studies at the cellular level over the effects of mobile phone radiation on DNA damage becomes a hot point in bioelectromagnetics field. It was reported RF EMF exposure could induce DNA damage in certain cell lines, while some research groups reported negative results. Therefore it was still controversial whether RF EMF exposure could induce DNA damage. At present, most laboratories adopt comet assay to detect DNA damage. Recently, the relationship between γH2AX and DNA double-strand breaks (DSBs) has gradually draw researchers' attention. Histone H2AX is phosphorylated (denoted as γH2AX) in early response to DSBs induced by many stimuli. γH2AX recruit many DNA repair proteins, e.g. BRCA1 53BP1, Rad50 and NBS1, and form γH2AX foci.It was demonstrated that the number of yH2AX foci is quantitatively the same as DSBs induced by various stimuli, and the disappearance of yH2AX foci is exactly consistent with the repair of DNA damages. Thus, yH2AX foci formation is proposed to be a potential biomarker for DSBs.In the present study, we used yH2AX immunofluorescence assay to investigate the effects of 1800 MHz RF EMF on DNA damage in cells.Part. I Effects of 1800 MHz RF EMF on DNA damage in Chinese hamster lung cells (CHL)We used yH2AX, as an early indicator of DNA double-strand breaks to study the DNA damage effects of CHL cells exposed to 1800 MHz RF EMF. Cells were divided into following groups: (1) sham exposure group; (2) RF EMF exposure for 1 h group; (3) RF EMF exposure for 24 h group; (4) positive control group (20 fig/ml 2-acetaminofluorene (AAF) exposure for 2 h). Cells were sham-exposed or intermittently exposed (5 min on /10 min off) to 1800 MHz RF EMF at the average specific absorption rate (SAR) of 3 W/kg. After exposure, cells were fixed using 4% paraformaldehyde and processed for yH2AX immunofluorescence measurement. The primary antibody used for immunofluorescence was mouse monoclonal antibody against γH2AX and secondary antibody was fluorescein isothiocyanate (FITC) -conjugated goat anti-mouse IgG. Nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI). The samples were examined with an Olympus AX70 fluorescent microscope. For each sample, at least 100 cells were selected to detect γH2AX foci. Cells were classified as positive when more than five yH2AX foci were detected. The percentage of yH2AX foci positive cells was adopted as the index for evaluating DNA damage. The results showed that RF EMF exposure for 24 h or AAF exposure induced more yH2AX focus formations compared to the sham exposure. There was statistically significant difference in the percentage of γH2AX foci positive cells between RF EMF 24 h or AAF exposure and sham exposure (P < 0.05). However, there was no statistically significant difference between the RF EMFexposure for 1 h and sham exposure. The percentage of cells with 0, 1-10, 11-20 or more than 20 yH2AX foci was further analyzed. Compared to sham exposure, more cells with 1-10 γH2AX foci were found in AAF exposure. There was statistically significant difference in the percentage of cells with 1-10 γH2AX foci between AAF exposure and sham exposure (P < 0.05). However, there was no statistically significant difference in the percentage of cells with 1-10 γH2AX foci between RF EMF exposure for 24 h or 1 h and sham exposure. Compared to sham exposure, more cells with 11-20 γH2AX foci were found in RF EMF exposure for 24 h or AAF exposure, and there was statistically significant difference in the percentage of cells with 11-20 γH2AX foci (P < 0.05). There was no statistically significant difference in the percentage of cells with more than 20 yH2AX foci between any exposure group and sham exposure. In conclusion, our preliminary finding shows that 1800 MHz RF EMF at the SAR of 3 W/kg for 24 h could induce DNA damage in CHL cells, but exposure for 1 h does not induce DNA damage under current experimental conditions.Part. II Effects of 1800 MHz RF EMF on DNA damage in human lens epithelial cells (hLECs)hLECs were divided into following groups: (1) sham exposure group; (2) RF EMF exposure for 2 h group; (3) positive control group (0.25 umol/L 4-nitroquinoline-l -oxide (4NQO) exposure for 3 h). Cells were sham-exposed or continously exposed to 1800 MHz RF EMF at the average SARs of 2, 3, 4 W/kg. After exposure, DNA damage was assessed by the methods of alkaline comet assay, yH2AX immunofluorescence assay and flow cytometry. The results showed that there was no statistically significant difference in tail length and tail moment between RF EMF exposure under any conditions and sham exposure. The percentage of yH2AX foci positive cells exhibiting more than one yH2AX focus formation was adopted as the index of DNA damage determined by yH2AX immunofluorescence assay. Compared to sham exposure, 4NQO exposure induced more yH2AX foci formation. The percentage of yH2AX foci positive cells was statistically different between positivecontrol and sham exposure (P < 0.05). However, there was no statistically significant difference in the percentage of γH2AX foci positive cells between RF EMF exposure under any condition and sham exposure. After further analyzing the percentage of cells with 0, 1-20, 11-20 or more than 20 yH2AX foci, more cells with 11-20 or more than 20 yH2AX foci were observed in 4NQO exposure than in sham exposure, and the difference was statistically different (P < 0.05). Compared to sham exposure, about 75% cells in RF EMF exposure didn't exhibit any γH2AX focus, and the percentage of cells with 1-10 or more yH2AX foci was less than 25% in RF EMF exposure. There was no statistically significant difference in the percentage of cells with 0, 1-10, 11-20 or more than 20 γH2AX foci between RF EMF exposure group under any conditions and sham exposure. Statistically significant difference was found in the percentage of positive cells determined by flow cytometry between 4NQO exposure and sham exposure (P < 0.05). However, no significant difference in the percentage of positive cells determined by flow cytometry between RF EMF exposure group and sham exposure. In conclusion, 1800 MHz RF EMF at the SARs of 2, 3 or 4 W/kg for 2 h does not induce DNA damage in human lens epithelial cells under current experimental conditions.
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