The Effects Of Electromagnetic Fields On Genome Integrity In Cells With Different Genetic Backgrounds

With the rapid development of electric power and wireless communication technologies over the last several decades,electromagnetic fields?EMFs?have become one of the fast growing environmental factors that have raised great public concerns for the possible health hazards on humans.Although large scale in vivo and in vitro studies have investigated the health and biological effects of EMFs,unfortunately,the results are inconsistent.Possible causes of this situation are that different groups:1)adopted different biological models;2)applied different exposure systems and/or exposure parameters;and/or 3)employed different protocols to detect the same endpoint.To address these concerns,our group has proposed the adoption of a systematic research approach in order to faithfully elucidate the effects of RF-EMFs.We have employed an internationally well-accepted exposure system and the ?H2AX foci formation assay to compare the effects of 1800 MHz RF-EMF on DNA damage in six different cell types.Our data show that different cells respond differently to the exposure,and the exposure only induces ?H2AX foci formation in human skin fibroblasts and Chinese hamster lung fibroblasts.However,the slight increase in DNA damage does not result in significant DNA fragmentation and abnormal cellular behaviour,suggesting that the RF-EMF-induced DNA damage might be repaired or compensated for by the cells.These findings prompted us to investigate whether the difference in genetic backgrouds of various cell types is one of the reasons for the reported inconsistent data of RF-EMF on DNA damage,and adopt a biological system with a deficiency in DNA repair in order to more sensitively and more accurately reflect the impact of RF-EMF exposure on genome stability.1.The effect of EMFs on DNA damage in human choriocarcinoma JAR and JEG-3 cell lines.JAR and JEG-3 are both human choriocarcinoma cell lines but from individuals with different genetic backgrounds.The effect of 50 Hz EMF on DNA damage in JAR and JEG-3First,to visualize the total DNA fragmentation in cells,alkaline comet assay was applied in this study.The results showed that,compared to sham exposure group,DNA fragmentation in JAR were not significantly changed after exposure to 2.0 mT 50 Hz EMF for 1 or 12 h,but significantly increased when the exposure durations were extended to 24 or 36 h.In JEG-3 cells,under the same exposure conditions,DNA fragmentation were significantly decreased after 1-h-exposure,but not affected after prolonged 12-,24-,or 36-h-exposure.Then,to determine whether 50 Hz EMF induce DNA double strand breaks?DSBs?,we used yH2AX foci formation assay.The results showed that,compared to sham exposure group,yH2AX foci formation in both JAR and JEG-3 were not significantly affected after exposure to 2.0 mT 50 Hz EMF for 1 or 24 h.The effect of 1800 MHz EMF on DNA damage in JAR and JEG-3In alkaline comet assay,the data showed that,compared to sham exposure group,total DNA fragmentation in JAR were significantly decreased after 1-h-exposure to 4.0 W/kg 1800 MHz EMF,but no obvious changes were observed when the exposure time were prolonged to 24 h.In JEG-3 exposed to the same EMF,DNA fragmentation were not significantly affected after 1-h-exposure,but significantly increased after 24-h-exposure,compared to sham exposure group.In yH2AX foci formation assay,the data showed that,compared to sham exposure group,yH2AX foci formation in JAR were not significantly affected after 1-h-exposure to 4.0 W/kg 1800 MHz EMF,but decreased after 24-h-exposure.In JEG-3 treated with the same exposure,yH2AX foci formation were also not changed after 1-h-exposure but significantly increased after 24-h-exposure.To determine whether the effect of 50 Hz or 1800 MHz on DNA damage would result in abnormalities in cell behaviours,we detected the cell cycle and viability.The data showed that,compared to sham exposure group,the distribution of JAR cells in G2/M phase was significantly increased after exposure to 2.0 mT 50 Hz EMF for 24 h,and the cell viability showed a significant inhibition 24 h later,but no significantly changes were observed in 4.0 W/kg-RF-EMF-exposed groups.In JEG-3,neither 50 Hz nor 1800 MHz EMF had significant effects on cell cycle progression or cell viability.In this part,we found that,under current experimental conditions,2.0 mT 50 Hz EMF induce DNA single strand breaks?SSBs?and result in abnormalities of cell behaviours in JAR but not JEG-3,and 1800 MHz EMF reduce DNA damage in JAR but increase breaks in JEG-3.2.The effect of 50 Hz and 1800 MHz EMFs on DNA damage in wide-type(Atm^+/+)and Atm deficient(Atm^-/-)MEFs.Ataxia telangiectasia mutated?ATM?is a large protein?350 KDa?of PI3K-like protein kinase?PIKK?family,and plays a central role in DNA damage repair.ATM has been regarded as the primary activator of the cellular response to DNA double-strand breaks?DSBs?,and its deficiency would lead to inherent destabilization of chromosomal integrity,ATM deficient cells or animals have been reported to be more sensitive to ionizing radiation,oxidative stress and other insults.In this study,we chose both Atm^+/+ and Atm^-/-MEFs to investigat the effects of EMFs on DNA damage,hoping to reveal if DNA repair deficiency would result in higher sensitivity in reflecting the impact of RF-EMF exposure on genome stability.The effect of 50 Hz EMF on Atm^+/+,and Atm^-/-MEFsIn alkaline comet assay,the results showed that,compared to sham exposure group,total DNA fragmentation in Atm^+/+ MEF were not significantly changed after 1-or 24-h-exposure to 2.0 mT 50 Hz EMF.In Atm^-/-MEF,under the same exposure conditions,DNA fragmentation were also not significantly affected.To determine whether 50 Hz EMF inuduce early DNA DSBs,yH2AX foci formation assay was used.The results showed that,compared to sham exposure group,?H2AX foci formation in neither Atm^+/+ nor Atm^-/-MEF were significantly affected by exposure to 2.0 mT 50 Hz EMF for 1 or 24 h.Then,we detected cell cycle and viability of Atm^+/+ and Atm^-/-MEFs to determine whether 50 Hz EMFs would affect cell behaviours without inducing DNA damage.The data showed that,compared to sham exposure group,the cell cycle progression and cell viability were not significantly changed in either Atm^+/+ or Atm^-/-MEFs after exposure to 2.0 mT 50 Hz EMF.The effect of 1800 MHz EMF on Atm^+/+ and Atm^-/-MEFsThe results of alkaline comet assay showed that,compared to sham exposure group,total DNA fragmentation in Atm^+/+M EV were significantly increased after 1-h-exposure to 4.0 W/kg 1800 MHz EMF,but no significant changes were observed after 12-or 24-h-exposure,and even decreased to lower than the sham exposure group's level after 36-h-exposure.Under the same exposure conditions,DNA fragmentation in Atm^-/-MEF were not significantly affected after 1-h-exposure but increased after 12-h-exposure,and then significantly reduced to lower than the sham exposure group's level after 24-or 36-h-exposure.To distinguish if there is DSBs existing,we applied neutral comet assay to analyze the same samples.The results showed that,compared to sham exposure group,DNA fragmentation in Atm^+/+ MEF were not significantly changed after 1-,12-,24-,or 36-h-exposure to 4.0 W/kg 1800 MHz EMF.While,in Atm^-/-MEFs,under the same exposure conditions,DNA fragmentation were significantly increased after 12-and 24-h-exposure,but,as observerd in alkaline comet assay,the DNA fragmentation were also reduced to lower than the sham exposure group's level when the exposure duration was prolonged to 36 h,suggesting 1800 MHz EMF can induce DNA DSBs in Atm^-/-but not Atm^+/+ MEFs,and the breaks can also be repaired.To further confirm the DNA damage effects we observed in the 1800-MHz-EMF exposed cells,we analysed the marker molecules of different DNA damage repair pathways in Atm^+/+ and Atm^-/-MEF.X-ray repair cross-complementing protein 1?XRCC1?plays an important role in DNA SSB repair pathway,and yH2AX foci formation is regarded as an early marker of D SB repair.The results of Western blotting showed that,compared to shmn exposure group,XRCC1 expression and its phosphorylation were significantly increased in Atm^+/+ MEF after 1-h exposure and in Atm^-/-MEF after 12-h-exposure to 4.0 W/kg 1800 MHz EMF.The data of yH2AX foci formation assay showed that,compared to sham exposure group,?yH2AX foci formation in Atm^+/+ MEF were not significantly changed after 1-,12-,24-,or 36-h-exposure to 4.0 W/kg 1800 MHz EMF.While,in Atm^-/-MEFs,?yH2AX foci formation were only significantly increased in 12-h-exposed cells.The results suggest that corresponding DNA repair pathways were activated when DNA damage were induced by 1800 MHz EMF in both Atm^+/+ and Atm^-/-MEFs.To determine whether cell behaviours were affected by 1800 MHz EMF in Atm^+/+and Atm^-/-MEFs,we also detected their cell cycles and cell viabilities.The data showed that neither cell cycle progression nor cell viability were significantly changed after exposure.To make the genotoxicity of 1800 MHz EMF observed in this study more understandable,we compared the effect of 1800 MHz EMF to well studied DNA damaging agents?4NQO and H₂O₂?.The results showed that both 4NQO and H₂O₂ have similar effect on DNA damage and cell behaviours in Atm^+/+ and Atm^-/-MEFs when the dose is sufficiently low.In this part,we found that,50 Hz EMF has no effect on DNA damage and cell behaviours in either Atm^+/+ or Atm^-/-MEF;1800 MHz EMF can induce SSBs in both Atm^+/+ and Atm^-/-MEF,and even DSBs in Atm^-/-MEF.And,we also found that the effect of 1800 MHz EMF on DNA damage is similar to the effect of low-dose toxins or stimulus which has been regarded as hormesis.Conclusions:under current experimental conditions,1)the genotoxicity of 50 Hz or 1800 MHz EMF on human choriocarcinoma cell lines with different genetic backgrounds,or MEFs with sufficient or deficient DSB repair pathway are different,suggesting cells with different genetic backgrounds respond differentially to the same EMF,and different frequency EMFs have different effects on DNA damage in the same cells;2)JAR not only responds to 50 Hz EMF exposure,but also shows obvious cell damage,i.e.,resulting in G2/M retardation and viability decrease;3)1800 MHz EMF induces DSBs in Atm^-/-but not Atm^+/+ MEF,indicating DNA repair deficient cells are more sensitive to 1800 MHz EMF exposure;4)1800 MHz EMF induces hormesis-like effect on DNA damage in Atm^+/+ and Atm^-/-MEFs.Innovations:to the best of our knowledge,this is the first study to investigate the genotoxicity of 50 Hz and 1800 MHz EMFs in human choriocarcinoma cell lines with different genetic backgrounds,and MEFs with sufficient or deficient DSB repair pathway,and we found:1)under the same exposure conditions,cells with different genetic background respond differently to EMFs;2)Atm^-/-MEFs are more sensitive to 1800 MHz EMF compared to wide type ones;3)1800 MHz EMF might have hormesis-like effect.