**5.3 Gross malformations**

 In the low dose group, the most significant finding in this study was growth retardation of the embryo at day 10 only; body weight, CR length, and eye diameter were all significantly decreased at day 10 in the exposure group. However, they were indistinguishable from the control group at both day 5 and day 15. No gross malformation was observed except subcutaneous hemorrhagic areas under the skin at day 10.

Cox et al. reported no malformations when he exposed the fertilized eggs to 50 Hz [16]. Al Qusdi et al. using 900–1800 MHz electromagnetic waves and by ringing 4 times for 15 minutes/day (60 minutes/day) reported significant increase in body weight and length at day 10, which could not be sustained at day 14 [17]. Gross malformation in the brain and retina was also reported; however, the incubator used in that study was not an egg incubator, which could have negative effects on the development of the embryo [17].

In high dose group in this study, 14% mortality, limb deformities, and changes in the skin feathers were observed. There was growth retardation of the embryo at both day 10 and day 15. Other studies also reported growth retardation [18]. Other malformations reported by Lahijani et al. such as spina bifida, monophthalmia,

 microphthalmia, anophthalmia, exencephalic embryos, and embryos with asymmetrical faces, crossed or shorter beak, and gastroschisis were not found in this study [18]. In an extensive study done by Farrell et al. at five different laboratories and using 60 Hz magnetic field (pulsed and sinusoidal by a Tenma function generator) over 2500 chick embryos, did not show significant difference in the mortality for 48 hours; however, over the period of time, malformations were 6.8% in the exposed group compared to 1.8% in the control; most common anomaly reported was the neural tube defects [19].

Ubeda et al. observed in a study using 100 Hz and electromagnetic field intensity between 0.4 and 104 microTeslas (μT) that the chick embryo is sensitive to electromagnetic fields at extremely low frequency and intensity. He further stated that pulse shape may influence the development of embryo development [20].

 Effects of electromagnetic waves on living cells are dose and duration dependent [21]. It is likely that growth retardation of the chick embryo in exposed groups was a result of interference in the proliferation of the multiplying embryonic cells due to the RFW exposure. Development of the embryo is a complicated process which includes cell proliferation, differentiation, relocation, and programed cell death. These events involve endogenous ionic currents and electric fields which could be disturbed by the RFW exposure. Growth retardation in the exposed group is most likely due to the adverse effects of RFW on the DNA. Production of heat shock proteins is stimulated to repair this damage. This also increases reactive oxygen species production and apoptosis [17, 22, 23]. DNA damage if not repaired would most likely result in cell death [23]. This cell self-repair depends on the intensity of the initial injury by the radio waves. Thus at day 10, the chick embryo cells proliferation and multiplication declined due to DNA damage by the RFW exposure, hence reducing the wet body weight of the embryo as compared to the control group. At day 15, the wet body weight, CR length, and eye diameter did not show significant difference from the control in low dose because the cells were able to repair the damage; however, in the high dose group, the damage could not be repaired and significant difference persisted at day 15.

#### **5.4 Histology and electron microscopy**

In this study, an increase number of mitochondria were observed in the exposed group at day 10 and day 15. It has been reported that oxidative stress induced by H2O2 leads to increase number of mitochondria and mtDNA in human lung [24]. At day 10 in the exposed group, some of the mitochondria were swollen. It has been reported that permeability of mitochondrial membrane is dependent on interaction between Ca+2 and reactive oxygen species (ROS) system. Increased sympathetic activity is considered a primary cause of REW-induced calcium influx into the mitochondria [26]. ROS when stimulated on inner surface of mitochondria will produce free O+2 which invade thiol protein and cause transition pores in the membrane to open thus increasing its permeability and causing mitochondria swelling [26]. Mitochondrial swelling was also observed by Atti [27] in rat hepatocytes in response to electromagnetic field exposure.

 At day 15, in the exposed group, most of the mitochondria were elongated, dumbbell shaped, and in the process of degeneration. They were surrounded by fragments of rough endoplasmic reticulum and lots of free ribosomes. It was reported that mitochondria is the source of free radicals produced in response to electromagnetic wave exposure in human sperms [22]. It further reported potential causative mechanism of electron leakage from the mitochondrial electron transport chain which causes oxidative DNA damage.

The next most significant alteration in the hepatocytes was the increase number of lipid filled vacuoles in the exposed groups. It was reported that this marked

#### *Effects of Electromagnetic Field on the Development of Chick Embryo: An In Vivo Study DOI: http://dx.doi.org/10.5772/intechopen.84704*

increase in the cytoplasmic vacuolation is due to disturbances in lipid inclusions and fat metabolisms in pathological conditions, blockage of gluconeogenesis due to free radicals changing the nature of lipids, protein groups, and cell damage [26]. It seems that rough endoplasmic reticulum surrounding the mitochondria is damaged and fragmented thus contributing in increase in fat droplets.

 Electromagnetic waves caused fatty change in the hepatocytes of the developing chick embryos. In fatty liver, there is an increase in lipid droplets in the cytoplasm of the hepatocytes suggesting that cells are under oxidative stress when exposed to electromagnetic waves [28–30]. Lahijani et al. had similar results showing abnormal lipid droplets in the hepatocyte cytoplasm and pushing the nuclei to one side [26]. Similar results were reported in rats and rabbits [22, 31]. The breakdown of fat in the liver may be disrupted by radiation exposure, which may be similar to alcoholism, malnutrition, poisoning, and pregnancy. Fatty change is the beginning of injury to the hepatocytes, showing an increase in vacuoles filled with triglyceride fat, a sign of abnormal metabolism which may be due to the production of oxygen radicles species in the hepatocytes [29–31].

### **5.5 Biochemical test**

Heat shock protein70 (HSP70) is a multifaceted chaperon protein that is conserved across species and is involved in many cellular metabolic processes. Its prime role as an anti-stress agent has been exploited heavily in an attempt to further clarify its mode of action and cellular distribution [33]. In this study, Hsp70 was also investigated for its role and degree of expression in stress conditions induced by electromagnetic waves. Previous studies have demonstrated the overexpression of Hsp70 mRNA under stress stimuli but have not elucidated the magnitude and the subcellular compartment localization of the protein [34, 35].

In this study, we observed significant increase in levels of mRNA and Hsp70 in the exposed groups at both day 10 and 15. This increase in mRNA was 14% at day 10 and became 39% at day 15 (**Figure 13**). Levels of mRNA and HSP70 in the liver tissue are demonstrating an important rule liver plays in combating cellular stress using HSP70 as one of the approaches to attenuate the stress stimuli (**Figure 12**).

Mobile phone radiation induces reactive oxygen species (ORS) and DNA damage as seen by increase in the HSP70 and its mRNA and which causes metabolic, immunological, and carcinogenic effects [2, 36–38]. ORS was also reported in human sperm, which affects genes, cell membrane function, and signal transduction [28, 39, 40]. Rao et al. recently provided new evidence supporting the theory that radio waves affect the plasma membrane [39]. Radio waves also induce NADH oxidase enzyme, which might play a key role in the various cellular adverse effects [36, 40]. Various cellular and physiological processes can be affected as a consequence of increased levels of free radicals, including gene expression, cell growth, apoptosis, and release of calcium from intracellular storage sites [32, 36, 41–49].
