Effect of 50-Hz Magnetic Fields on Argyrophilic Nucleolar Organizer Regions in Bone Marrow Cells

Background: Exposure to an increasing amount of extremely low-frequency magnetic fields (MFs) causes some adverse effects. Considering a direct association between the sizes and numbers of argyrophilic nucleolar organizer regions (AgNORs) and cell proliferation, this study was conducted to evaluate the effect of 50-Hz MFs on the AgNORs of bone marrow (BM) cells. Materials and Methods: Eighty male rats were randomly divided into four exposed groups and one control group. The exposed groups received 50-Hz MFs with magnetic flux densities of 1, 100, 500, and 2000 μT two hours a day for two months. BM cells were aspirated from sacrificed rats’ femoral bones, smeared on glass slides, and then stained with silver nitrate for AgNORs. The area (AA), length (AL), and number (AN) of AgNORs were calculated by a microscope equipped with a camera and Scion Image software in 100 BM cells of each rat. The mean of AA, AL, and AN was computed for each group. Results: AA, AL, and AN significantly reduced in the 1 μT group compared with 2000 μT and control groups. Eventually, there was a nonlinear relationship between the effect of 50-Hz MFs and magnetic

density for one day (4 hours, as an acute-exposed group) and 45 days (4 h/day, as a long-term-exposed group). Their results revealed that MI in BM cells significantly decreased in acute-and long-term-exposed groups compared with controls. However, there was no significant change between the above-mentioned groups (11). Another in vitro study addressed various magnetic flux densities of ELF-MFs on the proliferation of human breast cancer cells , and the results indicated a nonlinear decrease in cell proliferation (3). In contrast, a study investigated the effect of continuous whole-body exposure of newborn rats to 50-Hz MF with 500 μT magnetic flux density and reported a nearly three-fold increase in the MI of the exposed BM cells (4).
Therefore, this study aimed to evaluate the AgNORs (i.e., area, length, and number) of BM cells following exposure to various flux densities of 50-Hz MFs.

Animals
Eighty albino Wistar eight-week-old male rats were purchased from the animal facilities of Hamadan University of Medical Sciences, Hamadan, Iran. The rats were randomly separated into four exposed groups and one control group each containing 16 rats. The rats were kept in plastic cages in an air-conditioned lab at a temperature of 22 • C and relative humidity of about 60%. After one week of adaptation, the exposed groups were treated in the middle of solenoids producing 1, 100, 500, and 2000 µT magnetic flux densities at 50-Hz MFs from 8-10 am for two months. The controls were also placed in a solenoid when it was unplugged. To activate BM cells, all the rats received human serum albumin. The rats were fed with standard food pellets and drank tap water ad libitum (12)(13)(14)(15)(16). The protocol of the study was compliant with the national guidelines for using animals and was approved by the Ethics Committee of Hamadan University of Medical Sciences, Hamadan, Iran (Code: UMSHA/P/16/35/9/6829).

50-Hz MF Exposure Systems
Exposure units (solenoids) were designed using 200 cm length and 20 cm radius of polyvinyl chloride cylinders by an electrical engineer (Microsystem Company Hamadan, Iran). The cylinders were coiled with two millimeters (in diameter) of copper wires. The solenoids could produce magnetic flux densities of 1, 100, 500, and 2000 µT by adjusting different electric currents (0.2, 0.7, 2, and 3 A), electrical voltages (2.7, 6, 37, and 88 V), and turns of wires (600, 600, 1200, and 1200), respectively. All systems were connected with the 50-Hz frequency of power supplies. The rats were placed in the middle of the solenoids in their plastic cages. An ELF-MF Survey Meter (HI-3604, Holaday Ind., USA) was used to measure magnetic flux densities. The background magnetic flux densities in the animal and experimental labs were 0.07 ± 0.03, and 0.09 ± 0.04 µT, respectively (12)(13)(14)(15)(16).

AgNOR Staining
All the rats were weighed and sacrificed, and femoral bones were separated from the rats' legs and washed in normal saline. BM cells were extracted and smeared on glass slides. The smears were then labeled and fixed with methanol. Silver nitrate (AgNO 3 ) staining of nucleolar organizer regions was performed as described before (17). Briefly, two volumes of aqueous silver nitrate 50% were mixed with one volume of gelatin 2% (prepared in formic acid 1% v/v) to make silver nitrate staining solution. The samples were stained with freshly prepared staining solution for 15 minutes at 37°С and in a dark place. After staining, the slides were rinsed with deionized water, dried, and mounted with Canada balsam (17).

Image Analysis
A light microscope equipped with a high-resolution lens (magnification = 1000X) and a digital camera (Euromex, Holland) was used to capture the images of BM cells. All the captured images were saved as tagged image file format with the same sizes and resolutions ( Figure 1). The area, length, and number of AgNORs were calculated by Scion Image Software (Version Beta 4.0.2, Scion Corporation, USA) in 100 BM cells for each rat. AA and AL were presented as pixels ranging from 0 (for white) to 255 (for black). Then, the mean of AA, AL, and AN was computed for each group (17,18).

Statistical Analysis
The results are presented as a mean ± standard deviation. A one-way analysis of variance (ANOVA) test was used to compare the mean of AA, AL, and AN among the groups. Further, the least significant difference (LSD) test was applied to compare the AA, AL, and AN between the two groups. A regression analysis was performed to calculate the relationship between the densities and the amounts of AA, AL, and AN. A P < 0.05 was considered significant, and finally, SPSS (version 16) was used for data analysis.
Regression analysis also showed that the amount of the AgNOR index (i.e., AA, AL, and AN) sharply decreased in the 1 μT group. However, the effect was neutralized and reached the level of control in 2000 μT ( Figure 2) by an increase in magnetic flux density. Therefore, it seems that exposure of rats to the magnetic flux density of 1 µT of 50-Hz MFs suppresses the proliferation of BM cells. However, magnetic flux densities of 100, 500, and 2000 µT did not affect AgNORs compared to controls.

Discussion
The results of this study revealed that exposure of rats to 50-Hz MFs with different magnetic flux densities (i.e., 1, 100, 500, and 2000 µT) two hours a day for two months only decreased AA, AL, and AN in the group of 1 µT.
AA, AL, and AN, which are indicators of the size and number of AgNORs, sharply decreased in 1 µT and then increased at magnetic flux densities of 100 to 2000 µT and reached the control level, representing that the BM proliferation is inhibited in 1 µT. There are some in vivo and in vitro studies about the effect of MFs on the AgNORs and proliferation of cells (3,4,7,11). Similarly, Okudan et al investigated the effect of ELF-MFs on the AgNORs in 120 Swiss albino mice. They exposed mice to 1, 2, 3, 4, and 5 μT of 50-Hz MFs for 40 days. Based on the results, a significant decline was observed in the AgNORs of the 1 μT group while there was no statistically significant difference in the groups that were exposed to higher magnetic flux densities compared with the controls (7). In another in vivo study, Erdal et al exposed Wistar rats to 50-Hz -ELF-MFs with 1000 μT magnetic flux density for four hours or 45 days and found a significant decrease in the MI of BM cells in both exposed groups compared with the controls (11).
Likewise, Park et al performed another experiment with similar results to this work and concluded that the effects of ELF-MFs on cell proliferation were inversely related to magnetic flux density, and the impact could be neutralized in the upper magnetic flux density. They evaluated the in vitro effect of intermediate frequency (200 Hz-10 kHz) on the proliferation of BT-20. BT-20 cell cultures were exposed to a range of magnetic flux densities from 3 to 7 mT for 72 hours. They found that the cell proliferation rate decreased by about 40% due to 5-mT exposure. Nevertheless, the exposed cells to 7 mT did not reveal any reduction of proliferation (3). However, Rageh et al. studied the effect of whole-body exposure of newborn rats to 50-Hz MF with a density of 500 μT for 30 days on the MI of BM cells observed about three-fold higher MI in the BM cells of exposed animals compared with the controls (4).
According to the results of this study and some other reports, there is a reduction in the size and count of AgNORs in 1 μT of 50-Hz MFs which is closer to magnetic flux density that we may be exposed to in our ordinary life compared with 100, 500, and 2000 μT (19). Based on the findings of Eroz et al, no change in AgNORs in higher magnetic flux density groups can be due to the protective roles of AgNOR. They reported that all living cells protect their conditions by AgNOR proteins that have protective roles in the structure and function toward dangerous agents (9).
According to some studies (20)(21)(22), a possible mechanism that may describe the difference between high and low magnetic flux densities of 50-Hz -MF on BM cell proliferation is that in higher densities, cells sense 50-Hz MFs as a robust stressor and express stress proteins such as heat shock proteins (HSPs). These proteins act as molecular chaperones and protect the cells from apoptosis. For example, HSP70 is one of the powerful anti-apoptotic proteins that can be expressed during stress. In one in vitro experiment, exposure of murine femoral BM cells to 50-Hz MFs with a magnetic flux density of 80 mT showed suppression in cell division and differentiation of granulocyte-macrophage progenitors. Therefore, it can be proposed that hemopoietic cells might be a target for the detrimental effect of 50-Hz MFs (23). In another in vitro study, Huang et al attempted to determine the molecular mechanism of cell division inhibition by ELF-MF. To this end, they exposed a human immortalized epidermal keratinocyte cell line to a magnetic flux density of 1.5 mT of 60-Hz MF for 144 hours. Protein expression analysis demonstrated a G1 phase arrest of the cells, an increased activity of the ATM/Chk2 signaling, and a higher level of p21 protein (6).
Accordingly, 50-Hz MFs decrease AgNORs at the magnetic flux density of 1 µT, which may represent the inhibition of proliferation in BM cells. However, 50-Hz MFs with higher magnetic flux densities of 100, 500, and 2000 µT did not affect the AgNORs and proliferation of BM cells. Thus, the impact of 50-Hz MFs on BM cell proliferation could be inversely associated with the density of the magnetic field. Sciences (Grant No. 9404091983) and approved by its Ethics Committee (Code: UMSHA/P/16/35/9/6829).