Extremely low-frequency magnetic fields (ELF-MF) under 50/60 Hz, which are generated by sources, such as electrical appliances or power lines, have created social concerns about possible adverse effects on human health. However, the carcinogenic potential of ELF-MF has been assessed experimentally in various model systems with inconsistent outcomes. Reviews of
HT22 mouse hippocampus neuronal cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco-In-vitrogen, Paisley, Scotland), and supplemented with 10% fetal bovine serum (HyClone, Thermo Fisher Scientific, Logan, UT, USA) at 37℃ in an incubator with a humidified atmosphere of 95% air and 5% CO2.
Cells (7.0 × 105) in 100 mm cell culture dishes were placed in the exposure chamber and subsequently exposed to a 60-Hz ELF-MF at 0 mT or 2 mT. Over the exposure time, the temperature in the chamber was maintained at 37℃±0.3℃ by circulating water, and the temperature of the culture medium was monitored at 2-hour intervals. Positive controls were exposed to gamma radiation doses (10 Gy as a single dose), which were generated by a 137Cs gamma-ray source (MDS Nordion, Ottawa, ON, Canada) at a dose rate of 5 Gy/min. Control cells were treated in the other incubator under the same experimental procedures, with the exception of ELF-MF exposure.
After ELF-MF exposure, equal amounts of protein (100 μg) were dissolved in a lysis buffer. The samples were boiled for 5 minutes, and proteins were separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred to nitrocellulose membranes. After blocking with 5% skim milk in phosphate-buffered saline with Tween-20 (PBS-T), the membranes were incubated with
The CometAssay (Trevigen Inc., Gaithersburg, MD, USA) was performed according to the manufacturer’s protocol. HT22 cells were seeded at a density of 7 × 105 cells/dish in 100-mm cell culture dishes. The 2-mT ELF-MF exposure times were 4 hours and 16 hours. Cells were resuspended in ice-cold PBS at a concentration of 1 × 105 cells/mL. An aliquot of 25 μL of cells (1 × 105 cells/mL) was added to 250 μL of 1% low-melting agarose, which was kept at 42℃. One hundred microliter aliquots were immediately pipetted and evenly spread over an area of the comet slides. The slides were incubated in the dark for 10 minutes at 4℃ to accelerate gelling of the agarose discs, and then were transferred to a pre-chilled lysis solution (Trevigen Inc.) where they were kept for 60 minutes at 4℃. A denaturation step was performed in an alkali solution (300 mM NaOH, 1 mM EDTA, pH > 13) at 4℃ for 10 minutes, in the dark. The slides were then transferred to a pre-chilled alkaline electrophoresis solution with pH > 13 (300 mM NaOH, 1 mM EDTA) and subjected to electrophoresis at 1 V/cm and 300 mA for 30 minutes. At the end of the electrophoresis, the slides were washed with a neutralizing buffer (0.4 M Tris-HCl, pH = 7.4), immersed in ice-cold 70% ethanol at room temperature for 5 minutes, and air dried. The DNA was stained with ethidium bromide for 20 minutes in the refrigerator, after which the slides were analyzed immediately using the TriTek CometScore Freeware v1.5 image analysis software. Detection was made for Olive tail moments, where the tail moment is defined as the product of the tail length and the fraction of total DNA in the tail. A tail moment incorporates a measure of both the smallest detectable size of migrating DNA (reflected in the comet tail length) and the number of relaxed/broken pieces (represented by the intensity of DNA in the tail). Each assay was performed in triplicate and in at least three independent experiments.
Cell cycle distributions were analyzed using propidium iodide (Sigma-Aldrich, St. Louis, MO, USA) staining, followed by flow cytometry. Cells were seeded at a density of 1 × 106 cells/dish in 100-mm cell culture dishes and incubated overnight. After exposure to a 2-mT ELF-MF for 6, 12, and 24 hours, cells were collected by trypsinization and harvested by centrifugation at 1,300 rpm for 3 minutes. Next, cells were fixed with 70% cold ethanol at –20℃ overnight, washed with PBS, treated with RNase A (1 mg/mL; Sigma-Aldrich), and stained with propidium iodide (50 μg/mL). Samples were analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA); and the data were analyzed with BD CellQuest Pro Software (BD Biosciences).
HT22 cells were seeded at a density of 7 × 104 cells/dish in 60-mm cell culture dishes. After 12 hours, cells were exposed to a 2-mT ELF-MF. Exposure times were 6 hours and 12 hours. Cell numbers were counted after trypan blue staining. Control cells were treated in the other incubator under the same experimental procedures, with the exception of the ELF-MF exposure.
8. Statistical Analysis
Data are expressed as the mean±standard deviation (SD). Statistical significance was determined using Student’s
Numerous experimental studies have attempted to examine the carcinogenic potential of ELF-MFs. Even though a number of studies have reported DNA damage effects from ELF-MFs, the overall results remain inconclusive [9,10,15–19]. The main reason for this inconsistency was the use of different cell lines.
Previously, we investigated the effects of ELF-MFs on DNA damage responses, such as cellular transformation , comet tail moments , micronuclei formation , aneuploidy formation , and
[Fig. 2.] Effects of extremely low-frequency magnetic field (ELF-MF) exposure on γH2AX expression and comet tail formation. (a) HT22 cells were exposed to a 2 mT ELF-MF. After 6 hours and 12 hours, cells were harvested and Western blotting was performed, using a specific antibody against γH2AX (left). The relative band intensity of γH2AX expression was calculated from densitometric scans of the blots with the control values set at 1. Protein expression was normalized to β-actin. Data are expressed as mean±SD from four independent experiments (right). (b) Cells (7.0× 105) were placed in 100 mm cell culture dishes. HT22 cells were exposed to a 2-mT of ELF-MF. After 4 hours and 16 hours, cells were harvested. For a positive control, the cells were exposed to 10 Gy ionizing radiation (IR). Data are expressed as mean±SD from four independent experiments.
Distribution of various cell cycle stages in HT22 cells after 2 mT ELF-MF exposure
[Fig. 3.] Cell number of HT22 cells after extremely low-frequency magnetic field (ELF-MF) exposure. HT22 cells were exposed to a 2-mT ELF-MF. After 6 hours and 12 hours, cells were counted using trypan blue. Data are expressed as mean±SD from three independent experiments.
Our previous study showed that exposure to a 1-mT ELF-MF for a maximum of 24 hours did not produce any increase in MN formation or comet tail production in several cell lines, including lung epithelial and fibroblast cells. Moreover, when we examined
Different sensitivity for induction of
The magnetic flux intensity (2 mT) was selected on the basis of Korean exposure guidelines, and this magnetic flux intensity was 2–10 times higher than the reference levels proposed by the International Commission on Non-Ionizing Radiation Protection (2010) for occupational exposure (1 mT) and for exposure of the general public (200 μT), respectively.
From the results, we suggest that future experiments should be carried out in a systematic manner to guarantee the reliability of the results.