**4. High dose (total 500–750 minutes)**

## **4.1 Methods**

 A total of 20 fertilized eggs were incubated in the incubator with the mobile phone in silent mode with the vibration mode disabled. The distance of all the eggs from the mobile phone was maintained within one wavelength (approximately 16.5 cm) of the emitting 1800 MHz frequency electromagnetic waves [21]. The mobile phone was rung from another mobile phone for 5 minutes, 10 times daily with an exposure-free period in between the calls. No calls were made at night. The total daily exposure duration was 50 minutes in each 24 hours starting from day 1. The eggs were sacrificed at day 10 (maximum exposure time 500 minutes) and day 15 (total exposure time 750 minutes). For the control groups, another 20 eggs were placed in the incubator, and 10 eggs were sacrificed at day 10 and 15 each.

Control group's 20 eggs were incubated at the same conditions in the same incubator. The mobile phone was turned off, battery removed, and placed in the middle of the incubator. The embryos were examined just as in the experimental groups at days 10 and 15.

On the scheduled day of sacrifice, mortality, gross morphology, wet weight, and length of the embryos were measured. Liver was dissected and placed in 10% glutaraldehyde solution and stained by toluidine blue stain for histological and ultrastructural preparation. Another five specimens were fixed in RNAlater solution (Invitrogen) for heat shock protein 70 (HSP70) and messenger ribonucleic acid (mRNA).

#### **4.2 Results**

 The results revealed 14% mortality in the exposed group. Gross morphology of the chick at day 15 showed deformities of the limbs, hemorrhages under the skin, lack of feathers, and few anterior abdominal wall defects (**Figure 6a** and **b**). The wet weight and head to toe length were significantly less in the exposed group at both day 10 and 15 (**Figure 7a** and **b**).

#### *4.2.1 Histology: control group*

 At day 10, hepatocytes were seen with rounded central nucleus and nucleoli. They were lying in rows with spaces in-between to form the sinusoids. Central veins with few RBCs and portal areas were observed. At day 15, typical structure of the liver was apparent. Well-formed hepatic lobules formed by rows of hepatocytes and sinusoids lining with epithelium and large number of RBCs were clearly seen (**Figure 8a** and **b**).

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

**Figure 6.** 

*High dose: (a) day 15: control group showing normal embryo development. (b) Experimental group: embryo was small in size, has marked deformities of the limbs, hemorrhages under the skin, and no growth of the feathers.* 

#### **Figure 7.**

*High dose: (a) wet body weight of the chick embryo: experimental and control groups at days 15 showing significant different (8.84 vs. 7.13 g) (p = 0.29). (b) Head to toe length of the chick embryo in the experimental group was significantly smaller than the control group (6.11 vs. 5.17 cm) at day 15 (p = 0.25).* 

#### *4.2.2 Exposed group*

 At day 10 and day 15, nucleus in many hepatocytes was not seen or pushed to the side and without prominent nucleolus. The hepatocytes were seen in rows with sinusoids in-between; however, marked infiltration of the fat vacuoles was observed in the cytoplasm of hepatocytes. The sinusoids were formed showing lining epithelial cells and RBCs. This signifies the beginning of fatty change (**Figure 8c** and **d**).

#### *4.2.3 Electron microscopy: Control group*

 At day 10 and 15, the control group showed hepatocytes arranged in rows forming the hepatic lobules and sinusoids in-between the rows lined by simple squamous epithelium (**Figure 9a**). There was a big central rounded nucleus with central chromatin, surrounded by double layered nuclear membrane with pores. Cytoplasm contains many mitochondria with well-arranged cristae, rough endoplasmic reticulum, ribosomes, and some glycogen vacuoles. Sinusoids were formed lined by single layer of simple squamous epithelium, and few Kupffer cells were also found lining the sinusoidal wall. Spindle-shaped RBCs with oval nucleus were present in the sinusoids. The canaliculi were seen clearly in between the hepatocytes (**Figures 9a** and **10a**).

#### **Figure 8.**

*Control group: (a) day 10 showing developing hepatocytes and sinusoids in between with RBCs and (b) day 15 showing well-formed hepatic lobules, normal hepatocytes arranged in rows, forming central vein, sinusoids lined by epithelial cells, and scattered well-formed RBCs inside the sinusoids. Exposed group: (c) day 10 showing infiltration of few lipid vacuoles in the hepatocytes and few necrotic hepatocytes and (d) day 15 showing marked infiltration of lipids causing necrosis of the hepatocytes.* 

#### *4.2.4 Exposed group*

At day 10, marked increase in the number of mitochondria can be observed (**Figure 9b**). Some of the mitochondria were swollen and surrounded by rough endoplasmic reticulum. The nucleus was in the center and circular in shape. Sinusoids were filled with red blood cells (RBC) and lined with simple squamous epithelium and Kupffer cells. At day 15, cytoplasm showed numerous lipid filled vacuoles

#### **Figure 9.**

*Day 10, (a) in control group, polygonal hepatocytes arranged in row with large central round nucleus and showing sinusoids with multiple red blood cells, and canaliculi can also be seen in-between the hepatocytes. (b) Exposed group revealed marked proliferation of mitochondria and widening of the canaliculi.* 

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

#### **Figure 10.**

*Day 15, (a) control group showing hepatocytes arranged in rows, with few lipid vacuoles and normal looking canaliculi and (b) exposed group revealed mark infiltration of lipid vacuoles in the cytoplasm, and widening of the canaliculi was apparent.* 

#### **Figure 11.**

*(a and b) Day 15, exposed group: mitochondria showed change in shape, some become rounded, while others dumbbell shaped and surrounding by rough endoplasmic reticulum. Cristae were not clearly visible, and some areas of degeneration were apparent.* 

 (**Figure 10b**). At day 15, mitochondria became electron dense, some were swollen, while others were elongated and dumbbell shaped. At places, mitochondria was degenerated and cristae invisible (**Figure 11a** and **b**). A prominent layer of rough endoplasmic layer around the mitochondria could be seen which was interrupted. Myelin-like figures can also be seen in the cytoplasm at day 15 in exposed groups. The canaliculi in-between the hepatocytes have been widened (**Figure 9b**).

#### **4.3 Biochemical analysis**

#### *4.3.1 Heat shock protein 70*

Homogenized sample using an automated homogenizer in a phosphate buffer at pH 7.6 was ultracentrifugated to separate contaminants and soluble proteins and cells. Messenger RNA (mRNA) was extracted from 40 tissue samples using the Qiagen RNeasy Mini Kit (QIAGEN, CA, USA) according to the manufacturer's instructions.

Next, DNAse treatment was given to the extracted mRNA using DNA-free™ DNA Removal Kit (Thermo fisher) according to the manufacturer's protocol. After which, cDNA was synthesized using *DNase treated RNA with the help of* High-Capacity cDNA Reverse Transcription Kit (Applied BioSystems, Austin, TX).

 *Quantitative RT-PCR was performed for the analysis of cDNA obtained from the 40 samples.* TaqMan reagents are used to perform qRT-PCR according to manufacturer's protocol. The relative expression level of *hsp70* was calculated using the comparative delta Ct method by normalizing the cycle threshold values of *hsp70* with those of GAPDH.

Protein extraction was carried out through differential centrifugation to obtain separate subcellular fractions of protein from hepatocytes.

Extracted proteins were analyzed through SDS-PAGE and one step ELISA. HSP70—ab187399 Simple Step ELISA® Kit was used for the detection of HSP70 protein in the control and experimental samples.
