The effect of prenatal exposure to 1800 MHz electromagnetic field on calcineurin and bone development in rats

PURPOSE: To investigated the effects of exposure to an 1800 MHz electromagnetic field (EMF) on bone development during the prenatal period in rats. METHODS: Pregnant rats in the experimental group were exposed to radiation for six, 12, and 24 hours daily for 20 days. No radiation was given to the pregnant rats in the control group. We distributed the newborn rats into four groups according to prenatal EMF exposure as follows: Group 1 was not exposed to EMF; groups 2, 3, and 4 were exposed to EMF for six, 12, and 24 hours a day, respectively. The rats were evaluated at the end of the 60th day following birth. RESULTS: Increasing the duration of EMF exposure during the prenatal period resulted in a significant reduction of resting cartilage levels and a significant increase in the number of apoptotic chondrocytes and myocytes. There was also a reduction in calcineurin activities in both bone and muscle tissues. We observed that the development of the femur, tibia, and ulna were negatively affected, especially with a daily EMF exposure of 24 hours. CONCLUSION: Bone and muscle tissue development was negatively affected due to prenatal exposure to 1800 MHz radiofrequency electromagnetic field.


Introduction
Even though there are attractive technological developments to improving human life, the exposure to environmental pollution and radiation may affect prenatal development in both humans and animals 1 .Various studies have reported that an electromagnetic field (EMF) may have side effects in various tissues and cells [2][3][4] .Although some studies have shown tissues with proliferative epithelial cells during and after pregnancy, reproductive cells and tissues of the hematopoietic system seem potentially sensitive to EMF exposure [5][6][7] .However, there are still few studies that have investigated prenatal bone development.According to recent studies, bone cells, including osteoblasts, osteocytes, and osteoclasts are considered radiosensitive and could die early in the postradiational period 8 .
EMF is used in the treatment of osteoporosis and musculoskeletal disorders 9 .The stimulatory mechanisms that underlie EMF have been investigated in vitro in mesenchymal stem cells, osteoblasts, and osteoblast-like cells.Furthermore, EMF has been related to bone morphogenetic protein 2, transforming growth factor beta (TGF-β), insulin-like growth factor II, prostaglandins, nitric oxide synthase phosphorylation, and mitogen-activated protein kinase 10 .
Calcineurin (CN) is a protein phosphatase known as serine/threonine phosphatase and is present in all eukaryotes.
In mammals, calcineurin has a role in the regulation of enzyme secretion.Alternatively, it is activated physiologically by Ca 2+ / calmodulin during the differentiation of skeletal muscles and the development of osteoclasts in bone.The normal distribution of CN in tissues and its immunoreactivity have been investigated in various rat organs 11 .It has a widely proven distribution in the brain, kidney, testes, skeletal muscle, cardiac valves, splenic lymphocytes, and bone 12,13 .The development of the first bone cells starts early in the fetal stage.Some studies have shown that CN activation occurs in bone cells during the prenatal period.Additionally, CN activity has been reported to be required for biochemical and morphological differentiation of skeletal muscle myocytes 14 .Even though there are many studies that have investigated the effect of high EMF on organs and tissues, no studies have investigated the EMF effect on the development of bone trabeculae, bone marrow, and muscle tissue surrounding bones and joints during the fetal period.
In this study, we investigated the positive and negative effects of 6-, 12-, and 24-hour daily exposures to 1800 MHz radiofrequencies during the entire embryogenesis period on the histologic development of bone and musculoskeletal tissues.
Furthermore, we also examined the effects of EMF exposure on CN levels.

Methods
This study was conducted in our University School of Medicine with the permission of the Local Ethics Committee of Experimental Animals on approval number: 2014/16).All procedures were performed according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
Fifteen female and three male Sprague Dawley rats weighing 620-300g were used in this study.All of the animals were placed into special care units and were fed ad-libitum at a temperature of 20-23 ± 2°C and 50-55% humidity with a 12-hour dark: 12-hour light cycle.The 15 female rats were examined with vaginal smears during the estrous period.The rats were placed into three cages with each cage containing five females and one male.The next day, the male rats were taken out of the cages.The presence of sperm, as determined by the vaginal smear method, was detected with light microscopy 15 .Animals found to have sperm were considered pregnant at the first day.While 12 pregnant rats were exposed to EMF radiation during pregnancy via a digital signal generator (Anritsu MG3670 B type, Japan), four pregnant rats were not exposed to radiation.
The carrier frequency of the device was managed to be 1800 MHz, the modulation frequency was 217 Hz with a pulse width of 577 µsec and a maximum power of 2 W. Female rats delivered 120 pups, 40 of these newborn rats were males.The randomly selected groups were divided into Groups 1, 2, 3, and 4. The groups were designed as follows: Group 1 contained the newborn male rats (n = 10) from females in the control group that were not exposed to EMF during pregnancy; Group 2 contained newborn male rats (n = 10) from females exposed to 6 hours of daily EMF during pregnancy; Group 3 contained newborn male rats (n = 10) from females exposed to 12 hours of daily EMF during pregnancy; and Group 4 contained newborn male rats (n = 10) from females exposed to 24 hours of daily microwave radiation during pregnancy.The rats were cared for and fed in our unit for 60 days following birth without forming any special environment.
At the end of 60th day, the body weights were 130-180 grams and their development was considered complete.Injections of 6 mg/ kg of 2% xylazine hydrochloride (Rompun) followed by 75 mg/kg of ketamine hydrochloride (Ketalar) were given intraperitoneally to induce a deep anesthesia, the rats were then sacrificed using the intracardiac perfusion method (4% formaldehyde).

Histopathological methods
After weighing all of the rats, their bone tissue (femur, tibia, and ulna) and muscle tissue (quadriceps) were extracted and given code numbers before being incubated in a 10% formaldehyde solution.After standing in a fixative material for 24 hours, the bones were put in a Müller solution for 24-48 hours to be decalcified.Decalcification was completed by the addition of a 3% Müller solution.After standing in a fixative material for 24 hours, the muscle tissue was combined with the bone tissue and washed for 1-2 hours with running distilled water.They were then washed with an ethanol-xylene series (50-100%) before performing automated tissue processing (Citadel 2000, Thermo Fisher Scientific Shandon, England).The tissues were then embedded separately in paraffin.Tissues were cut into 4-6 μm thicknesses for routine hematoxylin-eosin staining.During light microscopic examinations with varying magnifications, suitable areas were photographed.
Sections that were cut into 4-6 μm thicknesses for immunohistochemical staining were incubated in xylene for 20 minutes, they were then allowed to stand for 10 minutes in an alcohol series (50-100%) before being put in a 3% H 2 O 2 solution.
After being washed with phosphate-buffered saline (PBS), the samples were heated with 700W in a citrate buffer solution for (heated four minutes (repeated five times) minutes and allowed to stand in a secondary blocker substance for 20 minutes.For every preparation to be stained with Anti-CN B (Code: ab136526, Abcam plc, Cambridge CB4 0FL UK), they were put into various dilutions (Anti-CN B 1/100) for 75 minutes.Diaminobenzidine (DAB) solution was used as a chromogen and Mayer's hematoxylin was used as a counterstain for 5 minutes.PBS was used as a negative controller.The preparations were photographed after being covered with a suitable substance.The results of the immunohistochemical stained tissues were subdivided into four categories according to the percentage of immunopositivity reaction as follows: mild (+), moderate (++), severe (+++), and very severe (++++).Two histologists and one pathologist blindly scored and evaluated the preparations with regard to statistical analysis.

Bone measurement methods
After being sacrificed, the animal's internal organs were extracted and the animal was fixed on an Xray board to clearly display their skeletal system.The anteroposterior Xrays were evaluated using a G.E. X-R 6000 device.They were then photographed before being sent to a digital photography program (Akgun PACS Viewer 4.0) to perform the measurements.The femur, tibia, quadriceps muscle, and sciatic nerve were extracted bilaterally for histologic evaluation.The means (with fallibility ± 0.01 mm) of the following distances were measured bilaterally using the Akgun PACS Viewer Program.Femur head diameters were measured from the physeal cartilage level, femur lengths were measured between the femur head and femur condyle, tibia lengths were measured between anterior eminence and ankle, ulna lengths were measured between the olecranon and wrist, and were measured the cortical thickness and medullary cavity in the distal area of the minor trochanter on radiographs.

Statistical analysis was performed using SPSS version 18
(SPSS Inc., Chicago, IL, USA).All the data were evaluated and while nonparametric values are reported as medians ± standard deviation (SD), parametric values are reported as means ± standard deviation (SD).One-way analysis of variance (ANOVA) was used to determine the significant differences among the groups.As a post hoc test, multiple comparisons were performed using Tukey's HSD and LSD.For all the comparisons p<0.05 was considered statistically significant.

Results
The histopathological investigation of the bones of the control group revealed they had a normal histologic appearance for cartilage, bone trabeculae, and bone marrow.The cellular nuclei of chondroblasts, osteoblasts, chondrocytes, and osteocytes were rounded and flat.The mitotic activity of the isogenous groups had a normal appearance (Figure 1A).The histopathologic investigation of the bones of the group 2 exposed for six hours revealed that both the upper zone (resting cartilage) and lower zone (proliferative and hypertrophic cartilage) regions were relatively shorter than in the control group.There were decreasing bone thickness and loss of proteoglycan in the bone trabeculae.
In the group 2, there was intensive eosinophilic infiltration surrounding chondrocytes of hyaline cartilage.There were no significant changes in the epiphyseal plate of the control group and 6-hour application group.The shortness found in the application group was not statistically significant.In the application group, bone marrow spaces were wider, the intensity of the trabecular structure was decreased and mildly eosinophilically stained.
There was no change for osteocytes within the lacuna of bone trabeculae and these were observed to preserve their morphologic structure.While there were no decreases in the volume of both the medial femoral condyle and lateral femoral condyle of the application groups, there were scattered chondrocyte necrosis with few apoptotic cells due to the loss of proteoglycan (Figure 1B).The histopathologic investigation of both the upper zone (resting cartilage) and lower zone (proliferative and hypertrophic cartilage) regions of the bones of the 12-hour application group revealed lower distances than the control group with decreased growth distances.Additionally, shedding of the chondroblasts was observed on the surface of the cartilage.There was scattered thickness in the trabeculae.In the application group, hyaline cartilage and the surroundings of the chondrocytes of the isogenous groups were intensively eosinophilically stained, and there were intensive apoptotic cell clusters.There were no obvious changes between the epiphyseal plate of the control group and the 12-hour application group.The shorted distance of the upper zone and the lower zone in the application group was not statistically significant.In the application group, bone marrow spaces were wider, the amount of the adipocytes of the bone marrow were decreased, and the amount of the megakaryocytes were increased.
The structures of the trabeculae were eosinophilically stained due to the increase in bone density.Even though osteocytes within the lacuna of bone trabeculae were basophilically stained, there was no decrease in their amount and their morphologic structures were preserved (Figure 1C).The histopathologic investigation of the 24-hour application group revealed a slimming of the upper zone (resting cartilage), whereas the distances of lower zone regions (proliferative and hypertrophic cartilage) were similar to the controls as well as the 6-and 12hour application groups.The volumes of the isogenous groups in the proliferative zone were decreased.However, the formation of structures of four and five chondrocytes was continued.Apoptosis was intensively increased due to the accumulation of glycogen in the chondrocytes within the hypertrophic zone of the controls and 6-hour application group.However, these levels were similar to the 12-hour application group.While the number of trabeculae increased, the thickness of the trabeculae was not distributed homogenously in the bone matrix.Hyaline cartilage and chondrocytes in the upper zone areas near the joint surface were intensively eosinophilic in the application group.There was a minimal insignificant difference in the epiphyseal plate between the controls and the 24-hour application group.Additionally, the distance increase in the application group was not statistically significant.In the application group, bone marrow spaces were wider, hematopoietic cells intensity was normal, and the intensity of the bone trabeculae was decreased.There was no decrease for osteocytes within the lacuna of bone trabeculae and they preserved their morphologic structure (Figure 1D).The histopathologic investigation of the bone tissues revealed that the resting cartilages of Groups 2, 3, and 4 were obviously decreased compared with the control group.The values of the proliferative hypertrophic cartilage and epiphyseal plate of the four groups were similar.Proteoglycan loss and chondrocytic apoptosis of Groups 3 and 4 were obviously higher than in Groups 1 and 2 (Table 1).Bone CN levels of Groups 2 and 3 were lower than in Group1, its level of Group 4 was lower than the other three groups (Figure 2).2).Muscle levels of CN in Groups 3 and 4 were lower than in Groups 1 and 2 (Figure 4).The weight of Group 4 rats was lower than the other groups.Except for the femoral head, all femur measurements of Group 4 were lower than the other groups (Figure 5).Tibia measurements were not obviously different among the groups.The length of the ulna of Group 4 was lower than the other groups.All these results are shown in Table 3.   otherwise.On the other hand, some authors have argued that high dosages of EMF have a positive effect on bone development [16][17][18] .
Two studies that investigated EMF exposure on ovariectomized rats with osteoporosis found that EMF positively affected bone development and obviously decreased bone loss 16,17 .EMF has been reported to stimulate the differentiation of cartilage cells and to have positive effects on bone tissue by increasing the level of bone alkaline phosphate 19 .However, van der Jagt et al. 10 investigated the effects of EMF on osteoporotic and nonosteoporotic rats; they found no positive effects of EMF on the development of cancellous and trabecular bone in osteoporotic rats.Atay et al. 4 did not find an important effect of 1800 MHz radiation on bone mineral density levels of the spine and femur.Conversely, Yildiz et al. 18 26,27 .However, the positive or negative effects of both the exposure to high frequency and the duration of this exposure on bone and muscle tissues during the prenatal period have not been investigated.In contrast to previous studies, in our study, which investigated bone and muscle tissue damage by both the exposure and duration to high frequencies during the prenatal period, there was intensive damage to myocytes and an obvious increase in apoptosis.EMF increases the development of chondrocytes by stimulating the release of TGF-β, then decreases the release of cytokines like interleukin-1β, and inhibits matrix metalloproteinase 28,29 .In our study, even though CN levels were low, chondrocyte development was similar to the control group due to the direct effects of EMF.However, an increase in chondrocyte cell apoptosis might suggest that the exposure to EMF during the prenatal period had harmful effects, rather than benefits, on EMF exposure during the prenatal period may lead to obvious damage to muscle, bone, and cartilage tissues.

FIGURE 1 -
FIGURE 1 -Histopathologic examination of bone tissue by light microscopy, hematoxylin and eosin staining.(A) Group 1 (not exposed to electromagnetic field), u: upper zone of the femur head region, d: lower zone of the femur head region, arrowhead; apoptotic cells; (B) Group 2 (exposed to electromagnetic field 6 hours a day), u: upper zone of the femur head region, d: lower zone of the femur head region, arrowhead; apoptotic cells; (C) Group 3 (exposed to electromagnetic field 12 hours a day), u: upper zone of the femur head region, d: lower zone of the femur head region, 1: adipocytes, 2: megakaryocytes, arrowhead; apoptotic cells; (D) Group 4 (exposed to electromagnetic field 24 hours a day), u: upper zone of the femur head region, d: lower zone of the femur head region, arrowhead; apoptotic cells.

FIGURE 5 -
FIGURE 5 -Femur bone measurements of the all groups by radiologically.(A) Group 1 (not exposed to electromagnetic field); (B) Group 2 (exposed to electromagnetic field 6 hours a day); (C) Group 3 (exposed to electromagnetic field 12 hours a day); (D) Group 4 (exposed to electromagnetic field 24 hours a day).
chondrocyte cells.CN stimulates apoptosis through various pathways.However, in our study, while chondrocyte apoptosis was increased, CN levels were low.EMF leads to oxidative stress and DNA damage.EMF increases intracellular calcium, which stimulates apoptosis through a direct effect30 .Therefore, we may have found an increase in apoptosis due to a toxic dosage of EMF during the prenatal period.EMF stimulates the development of chondrocytes and conversely, it stimulates chondrocyte apoptosis and tissue damage by its toxic effects.In contrast to the adult stage, in the prenatal period, EMF may decrease osteoblastic activity and suppressed the development of myocytes by suppressing CN in bone tissues.ConclusionsFetal chondrocytes of bone tissue and myocytes of muscle tissue are more susceptible to electromagnetic field (EMF) exposure during the prenatal period.EMF may lead to the formation of oxygen radicals by a direct toxic effect, and increase apoptosis and excessive osteoblast inhibition by suppressing CN.

TABLE 1 -
The evaluation of histopathologic results of bone tissues.

TABLE 2 -
The evaluation of histopathologic results of muscle tissues.Group 1, control; Group 2, 6 h EMF applied; Group 3, 12 h EMF applied; Group 4, 24 h EMF applied.a,b,c p<0.05 vs group I; d,e p<0.05 v. group II; f p<0.05 vs group III.

TABLE 3 -
The evaluation of bone tissue morphology of femur, tibia and ulna.
25ported that bone mineral density levels of the spine and femur were decreased due to exposure to 900 MHz and 1800 MHz In the literature, there is only one study that has investigated the effect of EMF on CN25.This study reported increases in CN activity in hippocampal tissue due to EMF exposure25.We found CN activity to be decreased in both bone and muscle tissues and bone development was decelerated due to EMF exposure during the prenatal period.In our study, high dosages of EMF exposure during the prenatal period and the investigation of CN activity in bone and muscle tissues may explain the different results between this study and the study ofManikonda et al.
15Exposure to high dosages of EMF during the fetal period may suppress CN activity, however, further studies are needed to clarify this relationship.It has been reported that EMF exposure during the prenatal period may lead to teratogenic effects, cranial and thoracic skeletal anomalies, DNA1and chromosome anomalies, and negative effects on male and female reproductive systems. Mastudies have reported that low frequency EMF may have healing effects on bone tissue and stimulatory effects on chondrocytes