*growth index. 8CPD,cumulativepopulationdoubling.*

*7GI,* 

#### **Table 2.**

*The growth parameters, percentages of dead cells, and percentages of Caspase-3 positive cells.*

### *Square-Wave Electric Impulses of 10 ms and 100 V/cm of Field Force, Produced… DOI: http://dx.doi.org/10.5772/intechopen.90506*

**Monolayer**

**Cell treatment**

**124**

Untreated control

 24 h

GI7 GI inh. %

% of dead cells

% of Casp.3 pos. cells

CPD8

> 72 h

GI GI inh. %

% of dead cells

% of Casp.3 pos. cells

CPD

GI GI inh. %

% of dead cells

% of Casp.3 pos. cells

CPD

GI GI inh. %

% of dead cells

% of Casp.3 pos. cells

CPD

GI

0.29

 0.02

 0.56

 0.05

 0.88

 0.07

 1.04

 0.09

 0.28

 0.02

 1.35

 0.12

 1.97

 0.17

3 impulses

24 h

2.19

 0.19

5.71 34.7

 23.5

1.13

 0.11

 0.99

 0.09

 0.45

 0.04

 0.32

 0.02

0.15

 0.01

 2.47

 0.22

 2.33

 0.21

12.7

8.6

7.7

26.1

7.6

2.1

1.2

11.4

38.4

11.2

3.2

1.8

26.7

35.7

25.1

16.1

85.6

36.4

 2.16

 0.19

 1.78

 0.16

 1.5

 1.97

 0.17

 5.16

 0.46

 5.21

 0.46

 0.13

72 h

1.49

 0.13

0.40

 0.03

0.43

 0.03

0.38

 0.03

2.51

 0.22

 0.40

 0.03 0.89

 0.08

0.35

 0.03

74.4

9.2 5.8

6.9

5.3

9.1

4.5

1.6

1.7

11.2

8.6

14.7

7.4

2.7

2.9

31.5

37.2

38.8

78

27

59.1

 0.81

 0.07

 0.96

 0.08

 0.91

 0.08

 0.38

 0.03

 1.19

 0.1

 1.9

 0.17

1 impulse

24 h

2.32

 0.2

0 5.2

 2.02

1.21

 0.11

 1.44

 0.13

 1.09

 0.09

 0.73

 0.06

 0.28

 0.02

 1.43

 0.12

 1.9

 0.17

3.7 1.66

2.20

3.06

3.28

2.29

3.01

4.9

6.8

7.3

5.1

6.7

0

0

0

0

0

0

 2.95

 0.2

 2.77

 0.2

 2.00

 0.18

 2.67

 0.24

 2.78

 0.25

 3.82

 0.34

1.38

 0.12

0 4.5

 2.34

0.47

 0.04

 1.37

 0.12

 0.23

 0.02

 0.32

 0.02

0.34

 0.03

0.1

 0.009 0.22

 0.02

3.9 2.02

2.70

3.22

2.70

2.18

3.17

*Electromagnetic Field Radiation in Matter*

5.2

6.2

5.2

4.2

6.1

0

0

0

0

0

0

 1.19

 0.11

 1.52

 0.13

 1.5

 1.73

 0.15

 0.94

 0.08

 1.19

 0.11

 0.13

 **Time of treatment**

**Chicken fibroblasts**

 **MDBK1**

 **cells**

**Vero2**

**MRC-53**

**HeLa4**

**FB15**

**K 5626**

**Suspension**

 **cells**

Chicken fibroblasts: after one impulse 5.7% and after three impulses 57.7%.

MRC-5 cells: after one impulse 25.1% and after three impulses this percentage

HeLa cells: after one impulse it was 16.1% and after three impulses 46.9%.

The evaluation of the percentages of dead cells after one impulse or three impulses shows the following data for different monolayer cultures: In Chicken fibroblasts the percentage of dead cells after one impulse was 34.7% and after three impulses was 63.5%. In Vero cells after one impulse, there is 11.4% of dead cells and after three impulses 38.9%. In the cells MDBK, after one impulse there is 12.7% of dead cells and after three impulses 15.1%. In the case of MRC-5 cells, after one impulse there is 38.4% of dead cells and after three impulses 70.1%. In the case of HeLa cells, after one impulse there were 11.2% of dead cells, and after three

In the evaluation of the trend of GI inhibition percentage, the following comparison was found: In most of the cells, Chicken fibroblasts, MDBK cells, Vero cells, MRC-5 cells, and HeLa cells have higher values after three impulses than after one impulse. The exception is the Vero cells, where the percentage of GI inhibition is a bit lower after three impulses. The analysis about the data of the percentage of dead cells shows the following: The highest percentage of dead cells was 63.5% after three impulses in Chicken fibroblasts. Also was higher percentage of dead cells after three impulses of 38.9. The lowest percentage of dead cells was in MDBK cells after three impulses which were 16.1%. In addition, surprisingly, in the case of MRC-5 cells, again the percentage of dead cells after three impulses is 70.1%. Moreover, the lowest percentage of dead cells was in the case of HeLa cells where it was 14.5%.

*3.1.3 Percentage of Caspase-3 positive cells after treating of different cells growing in monolayer, with one or three impulses and cultivated for 24 h or 72 h*

During the experiments, different monolayer cells put into suspension were treated with one or three impulses with the adsorbed dose (AD) of one impulse was 0.163 J/g and of three impulses was 0.490 J/g. After 24 h or 72 h of cultivation, the cells were trypsinized, sedimented by the low speed centrifugation, and analyzed for detection of the percentage of Caspase-3-positive cells **(Figure 5)**. In the Chicken embryonic fibroblasts, the percentage of Caspase-3-positive cells after 24 h and one impulse was 23.5% and after three impulses was 14.5%. After 72 h of incubation and one impulse, the percentage of Caspase-3 positive cells was 23.2%. After three impulses, the percentage increased to 44.7%. In the case of Vero cells, after 24 h of incubation and one impulse, the percentage of Caspase-3 positive cells was 7.7%. After three impulses, the percentage increases to 19.3%. After 72 h of incubation and treatment with one impulse, the percentage of Caspase-3 positive cells was 7.5%. After three impulses, this increased to 30.5%. In the case of MDBK cells, after 24 h of incubation and treatment with one impulse, the percentage of Caspase-3 positive cells was 8.5%. After three impulses, this increased to 9.9%. After 72 h of incubation and one impulse, the percentage was 8.6%. After three impulses, this percentage increased to 11.7%. When the MRC-5 cells were analyzed, after 24 h of incubation and one impulse, the percentage of Caspase-3-positive cells was 26.1%; after three impulses, the percentage was 26.5%. After 72 h of incubation and one impulse, the percentage was 26.1%. After three impulses, the percentage highly increased to 55.9%. In the case of HeLa cells after 24 h of incubation and one

MDBK cells: after one impulse 26.7% and after three impulses 48.3%.

Vero cells: after one impulse are 37.7% of GI inhibitions.

*Square-Wave Electric Impulses of 10 ms and 100 V/cm of Field Force, Produced…*

is 37.7%.

**127**

impulses, there were 14.5% of dead cells.

*DOI: http://dx.doi.org/10.5772/intechopen.90506*

**Figure 3.**

*Growth parameters and percentage of different monolayer dead cells after 24 h of incubation.*

#### **Figure 4.**

*The Growth parameters and percentage of different monolayer dead cells after 72 h of incubation.*

*Square-Wave Electric Impulses of 10 ms and 100 V/cm of Field Force, Produced… DOI: http://dx.doi.org/10.5772/intechopen.90506*

Chicken fibroblasts: after one impulse 5.7% and after three impulses 57.7%. Vero cells: after one impulse are 37.7% of GI inhibitions. MDBK cells: after one impulse 26.7% and after three impulses 48.3%. MRC-5 cells: after one impulse 25.1% and after three impulses this percentage is 37.7%. HeLa cells: after one impulse it was 16.1% and after three impulses 46.9%.

The evaluation of the percentages of dead cells after one impulse or three impulses shows the following data for different monolayer cultures: In Chicken fibroblasts the percentage of dead cells after one impulse was 34.7% and after three impulses was 63.5%. In Vero cells after one impulse, there is 11.4% of dead cells and after three impulses 38.9%. In the cells MDBK, after one impulse there is 12.7% of dead cells and after three impulses 15.1%. In the case of MRC-5 cells, after one impulse there is 38.4% of dead cells and after three impulses 70.1%. In the case of HeLa cells, after one impulse there were 11.2% of dead cells, and after three impulses, there were 14.5% of dead cells.

In the evaluation of the trend of GI inhibition percentage, the following comparison was found: In most of the cells, Chicken fibroblasts, MDBK cells, Vero cells, MRC-5 cells, and HeLa cells have higher values after three impulses than after one impulse. The exception is the Vero cells, where the percentage of GI inhibition is a bit lower after three impulses. The analysis about the data of the percentage of dead cells shows the following: The highest percentage of dead cells was 63.5% after three impulses in Chicken fibroblasts. Also was higher percentage of dead cells after three impulses of 38.9. The lowest percentage of dead cells was in MDBK cells after three impulses which were 16.1%. In addition, surprisingly, in the case of MRC-5 cells, again the percentage of dead cells after three impulses is 70.1%. Moreover, the lowest percentage of dead cells was in the case of HeLa cells where it was 14.5%.

#### *3.1.3 Percentage of Caspase-3 positive cells after treating of different cells growing in monolayer, with one or three impulses and cultivated for 24 h or 72 h*

During the experiments, different monolayer cells put into suspension were treated with one or three impulses with the adsorbed dose (AD) of one impulse was 0.163 J/g and of three impulses was 0.490 J/g. After 24 h or 72 h of cultivation, the cells were trypsinized, sedimented by the low speed centrifugation, and analyzed for detection of the percentage of Caspase-3-positive cells **(Figure 5)**. In the Chicken embryonic fibroblasts, the percentage of Caspase-3-positive cells after 24 h and one impulse was 23.5% and after three impulses was 14.5%. After 72 h of incubation and one impulse, the percentage of Caspase-3 positive cells was 23.2%. After three impulses, the percentage increased to 44.7%. In the case of Vero cells, after 24 h of incubation and one impulse, the percentage of Caspase-3 positive cells was 7.7%. After three impulses, the percentage increases to 19.3%. After 72 h of incubation and treatment with one impulse, the percentage of Caspase-3 positive cells was 7.5%. After three impulses, this increased to 30.5%. In the case of MDBK cells, after 24 h of incubation and treatment with one impulse, the percentage of Caspase-3 positive cells was 8.5%. After three impulses, this increased to 9.9%. After 72 h of incubation and one impulse, the percentage was 8.6%. After three impulses, this percentage increased to 11.7%. When the MRC-5 cells were analyzed, after 24 h of incubation and one impulse, the percentage of Caspase-3-positive cells was 26.1%; after three impulses, the percentage was 26.5%. After 72 h of incubation and one impulse, the percentage was 26.1%. After three impulses, the percentage highly increased to 55.9%. In the case of HeLa cells after 24 h of incubation and one

**Figure 3.**

*Electromagnetic Field Radiation in Matter*

**Figure 4.**

**126**

*Growth parameters and percentage of different monolayer dead cells after 24 h of incubation.*

*The Growth parameters and percentage of different monolayer dead cells after 72 h of incubation.*

**Figure 5.** *The percent of Caspase-3-positive cells after 24 h and 72 h of incubation of different types of monolayer cells.*

after the treatment with one impulse from 4.2% in the control fell down to 2.7%. After three impulses, this percentage fell down to 1.3%. It is unusual that GI

*The Growth parameters and percentage of dead cells after 24 h of incubation and in suspension growing cells*

*Square-Wave Electric Impulses of 10 ms and 100 V/cm of Field Force, Produced…*

*DOI: http://dx.doi.org/10.5772/intechopen.90506*

For the Lymphoblast cells K562, the GI increase was even higher after one impulse, and it was 59.1%. Unexpectedly, after three impulses it fell down to 3.7%. When the percentages after one impulse to 1.7% after three impulses. In general, the GI increase in percentage was higher after one impulse than after

*3.2.2 The growth parameters and percentage of dead cells after 72 h of incubation*

When the cells growing in suspension were treated with one or three impulses with an AD of 0.163 J/g for one impulse and 0.490 J/g for three impulses, after 72 h of incubation **(Figure 7),** the GI increases, which was much higher in the case of lymphoblast cells FB1 after one impulse, which was 86.5%. Again, it fell down to 10.1% after three impulses. In the case of Lymphoblast cells K562, the situation is opposite, namely, after one impulse the percentage fell down to 36.4% and after

The percentage of dead cells shows in lymphoblast cells FB1 the following trend:

from the control value of 5.1 to 3.2% after one impulse and to 2.2% after three impulses. In the case of Lymphoblast cells K562, from the xontrol value of 6.7 to 1.8% after one impulse to 1.2% after three impulses. Interestingly, the trend is the

*3.2.3 Percentage of Caspase-3-positive cells after one or three impulses on suspension*

When the in suspension growing Lymphoblast cells FB1 or K562 were treated with one or three impulses with an AD for one impulse of 0.163 J/g and for three impulses of 0.490 J/g, after the 24 h of incubation were analyzed for percent of Caspase-3 positive cells **(Figure 8)**. These values were compared with the

increase also fell down after the three impulses.

three impulses even lower to only 2.3%.

*lymphoblast cells FB1 or K562 for 24 h*

three impulses.

**Figure 6.**

*treated with one or three impulses.*

same in both cells.

**129**

impulse, the percentage of Caspase-3-positive cells was 7.6%. After three impulses, the percentage was 8.2%. After 72 h of incubation and one impulse, the percentage of Caspase-3 positive cells was 7.6%. After three impulses, this percentage increased to 11.4%.

The comparison of the data on **Figure 5** show the following: (a) The pattern of Caspase-3-positive cells after 72 h and three impulses was the highest in MRC-5 cells 55.1% followed by chicken fibroblasts with 44.7%, Vero cells with 30.5%, MDBK cells with 11.7%, and finally in HeLa cells with 21.3%. (b) The increase of percentage of Caspase-3 positive cells after 72 h of incubation and three impulses was statistically significant in comparison to 24 h of incubation and three impulses. For Chicken fibroblasts, this was 14.5% versus 44.7%. For MRC-5 cells, this was 26.5 versus 55.9%. For Vero cells, this was 19.3% versus 30.5%. (c) For MDBK cells this was 9.9% versus 11.7% and for HeLa cells, this was 8.2% versus 21.3%. For both cell lines, the increase was not statistically significant.

#### **3.2 The growth parameters, percentages of dead cells, and percentages of Caspase-3 positive cells of the in suspension growing cells**

#### *3.2.1 The growth parameters and percentage of dead cells after 24 h of incubation*

After the in suspension growing cell's treatment with one or three impulses having an adsorbed dose for one impulse of 0.163 J/g and for three impulses 0.490 J/ g, the main effect was the GI increase expressed in percentage. In comparison, the percentage of dead cells was determined after the treatment with one or three impulses. The detailed results **(Table 2** and **Figure 6)** show the following: For the Lymphoblast cells FB1, the GI increase after one impulse was 27.1%, which fell down to 13.6% after three impulses. In comparison, the percentage of dead cells

*Square-Wave Electric Impulses of 10 ms and 100 V/cm of Field Force, Produced… DOI: http://dx.doi.org/10.5772/intechopen.90506*

#### **Figure 6.**

impulse, the percentage of Caspase-3-positive cells was 7.6%. After three impulses, the percentage was 8.2%. After 72 h of incubation and one impulse, the percentage of Caspase-3 positive cells was 7.6%. After three impulses, this percentage increased

*The percent of Caspase-3-positive cells after 24 h and 72 h of incubation of different types of monolayer cells.*

The comparison of the data on **Figure 5** show the following: (a) The pattern of Caspase-3-positive cells after 72 h and three impulses was the highest in MRC-5 cells 55.1% followed by chicken fibroblasts with 44.7%, Vero cells with 30.5%, MDBK cells with 11.7%, and finally in HeLa cells with 21.3%. (b) The increase of percentage of Caspase-3 positive cells after 72 h of incubation and three impulses was statistically significant in comparison to 24 h of incubation and three impulses. For Chicken fibroblasts, this was 14.5% versus 44.7%. For MRC-5 cells, this was 26.5 versus 55.9%. For Vero cells, this was 19.3% versus 30.5%. (c) For MDBK cells this was 9.9% versus 11.7% and for HeLa cells, this was 8.2% versus 21.3%. For both cell

**3.2 The growth parameters, percentages of dead cells, and percentages of**

*3.2.1 The growth parameters and percentage of dead cells after 24 h of incubation*

After the in suspension growing cell's treatment with one or three impulses having an adsorbed dose for one impulse of 0.163 J/g and for three impulses 0.490 J/ g, the main effect was the GI increase expressed in percentage. In comparison, the percentage of dead cells was determined after the treatment with one or three impulses. The detailed results **(Table 2** and **Figure 6)** show the following: For the Lymphoblast cells FB1, the GI increase after one impulse was 27.1%, which fell down to 13.6% after three impulses. In comparison, the percentage of dead cells

**Caspase-3 positive cells of the in suspension growing cells**

lines, the increase was not statistically significant.

to 11.4%.

**128**

**Figure 5.**

*Electromagnetic Field Radiation in Matter*

*The Growth parameters and percentage of dead cells after 24 h of incubation and in suspension growing cells treated with one or three impulses.*

after the treatment with one impulse from 4.2% in the control fell down to 2.7%. After three impulses, this percentage fell down to 1.3%. It is unusual that GI increase also fell down after the three impulses.

For the Lymphoblast cells K562, the GI increase was even higher after one impulse, and it was 59.1%. Unexpectedly, after three impulses it fell down to 3.7%. When the percentages after one impulse to 1.7% after three impulses. In general, the GI increase in percentage was higher after one impulse than after three impulses.

#### *3.2.2 The growth parameters and percentage of dead cells after 72 h of incubation*

When the cells growing in suspension were treated with one or three impulses with an AD of 0.163 J/g for one impulse and 0.490 J/g for three impulses, after 72 h of incubation **(Figure 7),** the GI increases, which was much higher in the case of lymphoblast cells FB1 after one impulse, which was 86.5%. Again, it fell down to 10.1% after three impulses. In the case of Lymphoblast cells K562, the situation is opposite, namely, after one impulse the percentage fell down to 36.4% and after three impulses even lower to only 2.3%.

The percentage of dead cells shows in lymphoblast cells FB1 the following trend: from the control value of 5.1 to 3.2% after one impulse and to 2.2% after three impulses. In the case of Lymphoblast cells K562, from the xontrol value of 6.7 to 1.8% after one impulse to 1.2% after three impulses. Interestingly, the trend is the same in both cells.

#### *3.2.3 Percentage of Caspase-3-positive cells after one or three impulses on suspension lymphoblast cells FB1 or K562 for 24 h*

When the in suspension growing Lymphoblast cells FB1 or K562 were treated with one or three impulses with an AD for one impulse of 0.163 J/g and for three impulses of 0.490 J/g, after the 24 h of incubation were analyzed for percent of Caspase-3 positive cells **(Figure 8)**. These values were compared with the

The main conclusion is that the percentage of Caspase-3 positive cells correlates with the percentage of dead cells. In the Lymphoblast cells, K562, in the cell control, there was 3.17% of positive cells. After one impulse, this fell down to 1.2%, and after

*Square-Wave Electric Impulses of 10 ms and 100 V/cm of Field Force, Produced…*

*3.2.4 The Percentage of Caspase-3-positive cells after one or three impulses on in*

It is interesting that the Lymphoblast cells K562 have one-third higher percentages of dead cells than Lymphoblast cells FB1. In the cell control, there were 6.7% of dead cells. After one impulse, it fell down to 1.8% and after three impulses to 1.2%.

*suspension growing lymphoblast cells FB1 or K562 growing in suspension, after 72 h*

The in suspension growing Lymphoblast cells FB1 or K562 were treated with one or three impulses with an AD for one impulse of 0.163 J/g and for three impulses of 0.490 J/g after 72 h of incubation and were analyzed for the percentage of

Caspase-3 positive cells (**Figure 9**). In comparison, the same cells were analyzed for the percentage of dead cells. In the case of lymphoblast cells FB1 in the cell control, there were 4.8% of Caspase-3 positive cells, which fell down to 2.1% after one impulse and further fell down to 1.7% after three impulses. The comparison of the percentage of dead cells shows the following: In the cell control, there were 4.6% of dead cells, which fell down to 1.2% after one impulse and further to 0.9% after three

The value for the percentage of Caspase-3-positive cells correlates with the percentage of dead cells. The values for the percentage of dead cells were higher than those for Caspase-3. When the lymphoblast cells K562 were analyzed for the percentage of Caspase-3 positive cells, it was found that in the cell control there were 4.6% of positive cells, which fell down to 1.2% after one impulse and to 0.9% after three impulses. The comparison of the percentage of dead cells shows the following: In the cell control, there were 6.7% of dead cells, which fell down to 1.8%

*The Percentage of Caspase-3-positive cells after one or three impulses on suspension lymphoblast cells FB1 and*

three impulses this increases a little to 1.2%.

*DOI: http://dx.doi.org/10.5772/intechopen.90506*

impulses.

**Figure 9.**

**131**

*K562 after 72 h of incubation.*

*The Growth parameters and percentage of dead cells after 72 h of incubation and in suspension growing cells treated with one or three impulses.*

#### **Figure 8.**

*The Percentage of Caspase-3-positive cells after one or three impulses on in suspension growing lymphoblast cells FB1 and K562 for 24 h.*

percentage of dead cells after 24 h of incubation. In the case of lymphoblast cells FB1 in the cell control, there was 2.18% of Caspase-3 positive cells. After one impulse, this percentage was 2.1%, and after three impulses, it fell down to 1.02%.

The comparison with the percentage of dead cells after 24 h shows the following: In the cell control, there were 4.2% of dead cells. After one impulse, the percentage of dead cells fell down to 2.7%, and after three impulses, it further fell down to 1.3%. *Square-Wave Electric Impulses of 10 ms and 100 V/cm of Field Force, Produced… DOI: http://dx.doi.org/10.5772/intechopen.90506*

The main conclusion is that the percentage of Caspase-3 positive cells correlates with the percentage of dead cells. In the Lymphoblast cells, K562, in the cell control, there was 3.17% of positive cells. After one impulse, this fell down to 1.2%, and after three impulses this increases a little to 1.2%.

It is interesting that the Lymphoblast cells K562 have one-third higher percentages of dead cells than Lymphoblast cells FB1. In the cell control, there were 6.7% of dead cells. After one impulse, it fell down to 1.8% and after three impulses to 1.2%.

#### *3.2.4 The Percentage of Caspase-3-positive cells after one or three impulses on in suspension growing lymphoblast cells FB1 or K562 growing in suspension, after 72 h*

The in suspension growing Lymphoblast cells FB1 or K562 were treated with one or three impulses with an AD for one impulse of 0.163 J/g and for three impulses of 0.490 J/g after 72 h of incubation and were analyzed for the percentage of Caspase-3 positive cells (**Figure 9**). In comparison, the same cells were analyzed for the percentage of dead cells. In the case of lymphoblast cells FB1 in the cell control, there were 4.8% of Caspase-3 positive cells, which fell down to 2.1% after one impulse and further fell down to 1.7% after three impulses. The comparison of the percentage of dead cells shows the following: In the cell control, there were 4.6% of dead cells, which fell down to 1.2% after one impulse and further to 0.9% after three impulses.

The value for the percentage of Caspase-3-positive cells correlates with the percentage of dead cells. The values for the percentage of dead cells were higher than those for Caspase-3. When the lymphoblast cells K562 were analyzed for the percentage of Caspase-3 positive cells, it was found that in the cell control there were 4.6% of positive cells, which fell down to 1.2% after one impulse and to 0.9% after three impulses. The comparison of the percentage of dead cells shows the following: In the cell control, there were 6.7% of dead cells, which fell down to 1.8%

#### **Figure 9.**

*The Percentage of Caspase-3-positive cells after one or three impulses on suspension lymphoblast cells FB1 and K562 after 72 h of incubation.*

percentage of dead cells after 24 h of incubation. In the case of lymphoblast cells FB1 in the cell control, there was 2.18% of Caspase-3 positive cells. After one impulse, this percentage was 2.1%, and after three impulses, it fell down to 1.02%. The comparison with the percentage of dead cells after 24 h shows the following: In the cell control, there were 4.2% of dead cells. After one impulse, the percentage of dead cells fell down to 2.7%, and after three impulses, it further fell down to 1.3%.

*The Percentage of Caspase-3-positive cells after one or three impulses on in suspension growing lymphoblast cells*

*The Growth parameters and percentage of dead cells after 72 h of incubation and in suspension growing cells*

**Figure 7.**

**Figure 8.**

**130**

*FB1 and K562 for 24 h.*

*treated with one or three impulses.*

*Electromagnetic Field Radiation in Matter*

after one impulse and further fell to 1.2% after three impulses. In addition, in this case, the values for the percentage of Caspase-3 positive cells correlate with the percentage of dead cells.

after 72 h of incubation and three impulses was statistically significant in comparison to 24 h of incubation and three impulses. For chicken fibroblasts, the percentage was 14.5% versus 44.7%. For MRC-5 cells, the percentage was 26.5% versus 55.9%. For Vero cells, the percentage was 19.3% versus 30.5%. For MDBK cells, the percentage was 9.9% versus 11.7%, and for HeLa cells, the percentage was 8.2% versus

It is important that our data are in quite good agreement with the data of Zhang

11.4%. For both cell lines, the increase was not statistically significant.

*Square-Wave Electric Impulses of 10 ms and 100 V/cm of Field Force, Produced…*

restricting the cells in G0 phase.

*DOI: http://dx.doi.org/10.5772/intechopen.90506*

neuroendocrine cells.

decrease.

**133**

**cm on the cells that grow in suspensions**

[32] even he worked on human A375 melanoma cells only. He found that the nsPEFs failed to induce apoptosis of A375 melanoma cells, though it induced necrosis. However, the viability and migration of A375 melanoma cells were significantly inhibited by nsPEFs. It also suppressed the proliferation of A375 melanoma cells by

**4.2 The influence of one or three square impulses with a field force of 100 V/**

The Ren et al. [33] in their experiments studied the influence of nanosecond pulsed electric fields on the activation of intrinsic Caspase-dependent and Caspaseindependent cell death in Jurkat cells in vitro. In other world, is the cell dead in Jurkat cells Caspase-dependent or Caspase-independent. Experiments with U937 Lymphoid cell line cultures treated with pulsed electromagnetic field (PEMF) revealed that PEMF decreased the apoptosis of cells [34]. In order to discriminate and clarify mechanisms responsible for cell death after PEMF, cell culture was stimulated with death inducer Puromycin. Puromycin treatment combined with simultaneous single PEMF exposition reduced the death cell rate. Three times PEMF exposition increased the cell viability by about 25%. These data were in agreement with Grassi et al. [35], who found that 50 Hz EMF exposure enhanced proliferation and inhibited Puromycin induced cell death in human Neuroblastoma and rat

In our experiments, cells growing in suspension, like Lymphoid cells FB1 or K562, were treated with one or three impulses having an AD for one impulse of 0.163 J/g and for three impulses of 0.490 J/g. After the treatment with one or three impulses, different cells growing in suspension were analyzed for the percentage of GI increase, percentage of dead cells, and percentage of Caspase-3 positive cell

The pattern of the percentage of GI increase after one or three impulses after 24 h at lymphoblast FB1 shows a GI increase after one impulse of 27.1%, which fell down to 13.6% after cell treatment with three impulses. At lymphoblast K562, the picture is opposite: after one impulse the GI increase was 59.1% versus 3.7% after three impulses. The pattern after 72 h at lymphoblast cells FB1 shows 85.6% after one impulse versus 10.1% after three impulses. In lymphoblast cells K562, the picture is opposite: after one impulse 36.4% and after three impulses 2.3% only. The percentage of dead cells after one or three impulses after 24 h, at Lymphoblast cells FB1, shows the decrease after one impulse from 2.7 to 1.3% after three impulses. The same trend was found in the Lymphoblast cells K562. The decrease of percentage of dead cells was from 2.9% after one impulse to 1.7% after three impulses. After 72 h, we found the same trend: at FB1 cells, the percentage was 3.2% after one impulse versus 2.2% after three impulses. In K562 cells, the percentage

When the pattern of the changes of the Caspase-3 positive cells after one or three impulses was analyzed, the following picture was found after 24 h: in the lymphoblast cells FB1 after one impulse 2.7% which fell to 1.3% after three impulses. It is similar in the picture in the lymphoblast cells K562: from 1.2% after one impulse to

was from 1.8% after one impulse to 1.2% after three impulses.

#### **4. Discussion**

#### **4.1 The influence of one or three square impulses with field force of 100 V/cm on different cells growing in monolayer**

It was found that nanosecond pulsed electric fields (nsPEFs) can induce the direct electric fields and biological effects on the human colon carcinoma cells [29]. The main effect is the reduction of cell number after electrical impulses, together with the increase of apoptosis markers p53. The dead cells occurred through the necrosis. The direct role of Caspase-3 was not clear. In another research [30], it was found that the picosecond pulsed electric field (psPEF) could induce apoptosis in HeLa cells. The treatments with psPEF led to increased cell apoptosis and cell cycle arrest in the G2/M phase. The psPEF also affected the phosphorylation levels of endoplasmic reticulum sensors and upregulated the expression of glucose-regulated protein 78 (GRP78), glucose-regulated protein 94 (GRP94), and CCAAT enhancerbinding protein (C/EBP) homologous protein (CHOP). All changes were accompanied with the elevation of intracellular Ca2+ concentrations. The activation of Caspase-12, Caspase-9, and Caspase-3 led to the release of cytochrome *c* and the upregulation of Bax or the downregulation of Bcl-2, as observed in the HeLa cells. All these data suggest that psPEF is an efficient apoptosis-inducing agent for HeLa cells, which exerts its effects, at least partially, through the endoplasmic reticulum stress and Caspase-dependent signaling pathways. The MTT assay demonstrated that psPEF displayed strong growth inhibitory effects on HeLa cells. The explained cell death and apoptosis by nanosecond pulsed electric fields were found by Beebe et al. [31]. Despite nsPEFs having multiple cellular targets, these studies show that nsPEF affects the cell viability. The cell death depended on the presence of Ca2+. When both events occur, cell death can arise.

In our experiments, different monolayer cells being in suspension were treated with one or three impulses having an AD for one impulse of 0.163 J/g and for three impulses of 0.490 J/g. After the treatment with one or three impulses, different monolayer cells were analyzed for the percentage of growth index inhibition, percentage of dead cells, and percentage of Caspase-3 positive cells. In the evaluation of the trend of percentage of GI inhibition, the following comparison was found: In most of the cells, chicken fibroblasts, MDBK cells, Vero cells, MRC-5 cells, and HeLa cells have higher values after three impulses than after one impulse. The exception is the Vero cells, where the percentage of GI inhibition is a bit lower after three impulses. The analysis about the data of the percentage of dead cells shows the following: The highest was the percentage of dead cells (63.5%) after three impulses in Chicken fibroblasts. Also the percentage of dead cells (38.9%) was higher after three impulses. The lowest was the percentage of dead cells in MDBK cells after three impulses (16.1%). Surprisingly, in the case of MRC-5 cells, again the percentage of dead cells after three impulses is 70.1%. And the lowest was the percentage of dead cells in the case of HeLa where it was 14.5%.

The comparison of the data about the percentage of Caspase-3 positive cells shows the following: (a) The pattern of Caspase-3 positive cells after 24 h and three impulses was the highest in MRC-5 cells with 55.9%, followed by Chicken fibroblasts with 44.7%, Vero cells with 30.5%, MDBK cells with 11.7%, and finally in HeLa cells with 11.4%. (b) The increase of percentage of Caspase-3 positive cells

*Square-Wave Electric Impulses of 10 ms and 100 V/cm of Field Force, Produced… DOI: http://dx.doi.org/10.5772/intechopen.90506*

after 72 h of incubation and three impulses was statistically significant in comparison to 24 h of incubation and three impulses. For chicken fibroblasts, the percentage was 14.5% versus 44.7%. For MRC-5 cells, the percentage was 26.5% versus 55.9%. For Vero cells, the percentage was 19.3% versus 30.5%. For MDBK cells, the percentage was 9.9% versus 11.7%, and for HeLa cells, the percentage was 8.2% versus 11.4%. For both cell lines, the increase was not statistically significant.

It is important that our data are in quite good agreement with the data of Zhang [32] even he worked on human A375 melanoma cells only. He found that the nsPEFs failed to induce apoptosis of A375 melanoma cells, though it induced necrosis. However, the viability and migration of A375 melanoma cells were significantly inhibited by nsPEFs. It also suppressed the proliferation of A375 melanoma cells by restricting the cells in G0 phase.

#### **4.2 The influence of one or three square impulses with a field force of 100 V/ cm on the cells that grow in suspensions**

The Ren et al. [33] in their experiments studied the influence of nanosecond pulsed electric fields on the activation of intrinsic Caspase-dependent and Caspaseindependent cell death in Jurkat cells in vitro. In other world, is the cell dead in Jurkat cells Caspase-dependent or Caspase-independent. Experiments with U937 Lymphoid cell line cultures treated with pulsed electromagnetic field (PEMF) revealed that PEMF decreased the apoptosis of cells [34]. In order to discriminate and clarify mechanisms responsible for cell death after PEMF, cell culture was stimulated with death inducer Puromycin. Puromycin treatment combined with simultaneous single PEMF exposition reduced the death cell rate. Three times PEMF exposition increased the cell viability by about 25%. These data were in agreement with Grassi et al. [35], who found that 50 Hz EMF exposure enhanced proliferation and inhibited Puromycin induced cell death in human Neuroblastoma and rat neuroendocrine cells.

In our experiments, cells growing in suspension, like Lymphoid cells FB1 or K562, were treated with one or three impulses having an AD for one impulse of 0.163 J/g and for three impulses of 0.490 J/g. After the treatment with one or three impulses, different cells growing in suspension were analyzed for the percentage of GI increase, percentage of dead cells, and percentage of Caspase-3 positive cell decrease.

The pattern of the percentage of GI increase after one or three impulses after 24 h at lymphoblast FB1 shows a GI increase after one impulse of 27.1%, which fell down to 13.6% after cell treatment with three impulses. At lymphoblast K562, the picture is opposite: after one impulse the GI increase was 59.1% versus 3.7% after three impulses. The pattern after 72 h at lymphoblast cells FB1 shows 85.6% after one impulse versus 10.1% after three impulses. In lymphoblast cells K562, the picture is opposite: after one impulse 36.4% and after three impulses 2.3% only.

The percentage of dead cells after one or three impulses after 24 h, at Lymphoblast cells FB1, shows the decrease after one impulse from 2.7 to 1.3% after three impulses. The same trend was found in the Lymphoblast cells K562. The decrease of percentage of dead cells was from 2.9% after one impulse to 1.7% after three impulses. After 72 h, we found the same trend: at FB1 cells, the percentage was 3.2% after one impulse versus 2.2% after three impulses. In K562 cells, the percentage was from 1.8% after one impulse to 1.2% after three impulses.

When the pattern of the changes of the Caspase-3 positive cells after one or three impulses was analyzed, the following picture was found after 24 h: in the lymphoblast cells FB1 after one impulse 2.7% which fell to 1.3% after three impulses. It is similar in the picture in the lymphoblast cells K562: from 1.2% after one impulse to

after one impulse and further fell to 1.2% after three impulses. In addition, in this case, the values for the percentage of Caspase-3 positive cells correlate with the

**4.1 The influence of one or three square impulses with field force of 100 V/cm**

It was found that nanosecond pulsed electric fields (nsPEFs) can induce the direct electric fields and biological effects on the human colon carcinoma cells [29]. The main effect is the reduction of cell number after electrical impulses, together with the increase of apoptosis markers p53. The dead cells occurred through the necrosis. The direct role of Caspase-3 was not clear. In another research [30], it was found that the picosecond pulsed electric field (psPEF) could induce apoptosis in HeLa cells. The treatments with psPEF led to increased cell apoptosis and cell cycle arrest in the G2/M phase. The psPEF also affected the phosphorylation levels of endoplasmic reticulum sensors and upregulated the expression of glucose-regulated protein 78 (GRP78), glucose-regulated protein 94 (GRP94), and CCAAT enhancerbinding protein (C/EBP) homologous protein (CHOP). All changes were accompanied with the elevation of intracellular Ca2+ concentrations. The activation of Caspase-12, Caspase-9, and Caspase-3 led to the release of cytochrome *c* and the upregulation of Bax or the downregulation of Bcl-2, as observed in the HeLa cells. All these data suggest that psPEF is an efficient apoptosis-inducing agent for HeLa cells, which exerts its effects, at least partially, through the endoplasmic reticulum stress and Caspase-dependent signaling pathways. The MTT assay demonstrated that psPEF displayed strong growth inhibitory effects on HeLa cells. The explained cell death and apoptosis by nanosecond pulsed electric fields were found by Beebe et al. [31]. Despite nsPEFs having multiple cellular targets, these studies show that nsPEF affects the cell viability. The cell death depended on the presence of Ca2+.

In our experiments, different monolayer cells being in suspension were treated with one or three impulses having an AD for one impulse of 0.163 J/g and for three impulses of 0.490 J/g. After the treatment with one or three impulses, different monolayer cells were analyzed for the percentage of growth index inhibition, percentage of dead cells, and percentage of Caspase-3 positive cells. In the evaluation of the trend of percentage of GI inhibition, the following comparison was found: In most of the cells, chicken fibroblasts, MDBK cells, Vero cells, MRC-5 cells, and HeLa cells have higher values after three impulses than after one impulse. The exception is the Vero cells, where the percentage of GI inhibition is a bit lower after three impulses. The analysis about the data of the percentage of dead cells shows the following: The highest was the percentage of dead cells (63.5%) after three impulses in Chicken fibroblasts. Also the percentage of dead cells (38.9%) was higher after three impulses. The lowest was the percentage of dead cells in MDBK cells after three impulses (16.1%). Surprisingly, in the case of MRC-5 cells, again the percentage of dead cells after three impulses is 70.1%. And the lowest was the percentage of

The comparison of the data about the percentage of Caspase-3 positive cells shows the following: (a) The pattern of Caspase-3 positive cells after 24 h and three impulses was the highest in MRC-5 cells with 55.9%, followed by Chicken fibroblasts with 44.7%, Vero cells with 30.5%, MDBK cells with 11.7%, and finally in HeLa cells with 11.4%. (b) The increase of percentage of Caspase-3 positive cells

percentage of dead cells.

*Electromagnetic Field Radiation in Matter*

**on different cells growing in monolayer**

When both events occur, cell death can arise.

dead cells in the case of HeLa where it was 14.5%.

**132**

**4. Discussion**

1.3% after three impulses. Therefore, the percentage of Caspase-3 positive cells is independent from the number of impulses.

The percentage of Caspase-3 positive cells after 72 h in the lymphoblast cells FB1 was 2.1% after one impulse and fell down to 1.7% after three impulses. In the case of Lymphoblast cells K562, the percentage was from 1.2% after one impulse to 0.9% after three impulses.

### **Acknowledgements**

This research was supported by Ivan Čermak, Crodux plin D.o.o., 10000 Zagreb, Croatia, and it was performed via the Project: "Oncolytic Newcastle disease virus in the veterinary medicine' of the Croatian Institute for Experimental and Translational Medicine, Koledinečka 03, 10040 Zagreb, Croatia. Authors are indebted to Tomaž Velnar for English suggestions.

The picture of such a square wave is very similarly with this produced on the bioinduction device described by Pretnar et al. [14] with the number of harmonics

*Square-Wave Electric Impulses of 10 ms and 100 V/cm of Field Force, Produced…*

*DOI: http://dx.doi.org/10.5772/intechopen.90506*

of 23.

**135**

#### **Conflict of interests**

All authors declared no conflicts of interest.

### **Appendix**

The PGen-1 device generates the characteristic square-wave impulse. This can be mathematically described as Gibbs phenomenon, which is the result of an instantaneous change to a system with a finite number of samples. A pure sine wave has only a single fundamental frequency and no higher-order harmonics. A square wave has an infinite number of frequency components. A sine wave can be transformed into a square wave by adding harmonics to its fundamental. As such, a number of harmonics will produce a perfect square wave with a number of harmonics of 25. Such use of harmonics to build a square wave is demonstrated in the figure below:

*Square-Wave Electric Impulses of 10 ms and 100 V/cm of Field Force, Produced… DOI: http://dx.doi.org/10.5772/intechopen.90506*

The picture of such a square wave is very similarly with this produced on the bioinduction device described by Pretnar et al. [14] with the number of harmonics of 23.

1.3% after three impulses. Therefore, the percentage of Caspase-3 positive cells is

This research was supported by Ivan Čermak, Crodux plin D.o.o., 10000 Zagreb, Croatia, and it was performed via the Project: "Oncolytic Newcastle disease virus in the veterinary medicine' of the Croatian Institute for Experimental and Translational Medicine, Koledinečka 03, 10040 Zagreb, Croatia. Authors are

The PGen-1 device generates the characteristic square-wave impulse. This can

transformed into a square wave by adding harmonics to its fundamental. As such, a number of harmonics will produce a perfect square wave with a number of harmonics of 25. Such use of harmonics to build a square wave is demonstrated in the

be mathematically described as Gibbs phenomenon, which is the result of an instantaneous change to a system with a finite number of samples. A pure sine wave has only a single fundamental frequency and no higher-order harmonics. A square wave has an infinite number of frequency components. A sine wave can be

The percentage of Caspase-3 positive cells after 72 h in the lymphoblast cells FB1 was 2.1% after one impulse and fell down to 1.7% after three impulses. In the case of Lymphoblast cells K562, the percentage was from 1.2% after one impulse to 0.9%

independent from the number of impulses.

*Electromagnetic Field Radiation in Matter*

indebted to Tomaž Velnar for English suggestions.

All authors declared no conflicts of interest.

after three impulses.

**Acknowledgements**

**Conflict of interests**

**Appendix**

figure below:

**134**

*Electromagnetic Field Radiation in Matter*

#### **Author details**

Bratko Filipič<sup>1</sup> \*, Lidija Gradišnik<sup>2</sup> , Kristine Kovacs<sup>3</sup> , Ferenc Somogyvari<sup>4</sup> , Hrvoje Mazija1 and Toth Sandor<sup>5</sup>

1 CIETO (Croatian Institute for Experimental and Translational Oncology), Zagreb, Croatia

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8440406

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electromagnetic fields interactions with biological systems. FASEB Journal. 1993; **7**:272-281. DOI: 10.1096/fasebj.7.2.

*DOI: http://dx.doi.org/10.5772/intechopen.90506*

*Square-Wave Electric Impulses of 10 ms and 100 V/cm of Field Force, Produced…*

biology. Bioelectrochemistry and Bioenergetics. 1999;**48**(2):355-360. DOI: 10.1016/s0302-4598(99)00012-4

DOI: 10.1002/glia.20951.w

10.3109/15368379909012912

electromagnetic fields on cells: Physiological and therapeutical

000093061

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15368379009119803

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**137**

Costato M. Low-frequency

s13534-017-0042-3

2 Medical Faculty of Maribor, Institute of Biomedical Sciences, Maribor, Slovenia

3 Sylvester Comprehensive Cancer Center, Coral Glades, University of Miami, USA

4 Department of Medical Microbiology and Immunobiology, University of Szeged, Szeged, Hungary

5 TEWA Consultant, Szeged, Hungary

\*Address all correspondence to: bratko.filipic@gmail.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Square-Wave Electric Impulses of 10 ms and 100 V/cm of Field Force, Produced… DOI: http://dx.doi.org/10.5772/intechopen.90506*

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[3] Bolognani L, Del Monte V, Francia F, Costato M. Low-frequency electromagnetic pulsed field stimulation of yeast. Electromagnetic Biology and Medicine. 2009;**11**(1):1-10. DOI: 10.3109/15368379209012847

[4] Im C-H, Jun CS, Sekihara K. Recent advances in biomagnetism and its applications. Biomedical Engineering Letters. 2017;**7**:183. DOI: 10.1007/ s13534-017-0042-3

[5] Cui Y, Liu X, Yang T, Mei YA, Hu C. Exposure to extremely low-frequency electromagnetic fields inhibits T-type calcium channels via AA/LTE4 signaling pathway. Cell Calcium. 2014;**55**(1): 48-58. DOI: 10.1016/j.ceca.2013.11.002

[6] Araújo QFO, Alice Z, Coelho ZAM, Isabel CP, Margarit CPI, Vaz-Junior AC, et al. Electrical stimulation of *Saccharomyces cerevisiae* cultures. Brazilian Journal of Microbiology. 2004; **35**(1–2):97-103. DOI: 10.1590/ S1517-83822004000100016

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[9] Huang J, Ye Z, Hu X, Lu L, Luo Z. Electrical stimulation induces calciumdependent release of NGF from cultured Schwann cells. Glia. 2010;**58**(5):622-631. DOI: 10.1002/glia.20951.w

[10] Velizarov S. Electric and magnetic fields in microbial biotechnology: Possibilities, limitations, and perspectives. Electromagnetic Biology and Medicine. 1999;**18**(2):185-212. DOI: 10.3109/15368379909012912

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[13] Katsuki S, Li Y, Miyakawa D, Yamada YR, Onishi N, Lim S. Response of mammalian cells to non-thermal intense narrowband pulsed electric fields. In: 11th European Conference of Antennas and Propagation (EUCAP). 2017. DOI: 10.23919/EuCAP.2017.7928345

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[15] Filipič B, Kovacs K, Somogyvari F, Ihan A, Ocosovsky I, Koren S, et al. The effects of medium-strength electric

**Author details**

Szeged, Hungary

\*, Lidija Gradišnik<sup>2</sup>

Hrvoje Mazija1 and Toth Sandor<sup>5</sup>

*Electromagnetic Field Radiation in Matter*

5 TEWA Consultant, Szeged, Hungary

provided the original work is properly cited.

\*Address all correspondence to: bratko.filipic@gmail.com

, Kristine Kovacs<sup>3</sup>

1 CIETO (Croatian Institute for Experimental and Translational Oncology), Zagreb,

2 Medical Faculty of Maribor, Institute of Biomedical Sciences, Maribor, Slovenia

3 Sylvester Comprehensive Cancer Center, Coral Glades, University of Miami, USA

4 Department of Medical Microbiology and Immunobiology, University of Szeged,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

, Ferenc Somogyvari<sup>4</sup>

,

Bratko Filipič<sup>1</sup>

Croatia

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## *Edited by Walter Gustavo Fano, Adrian Razzitte and Patricia Larocca*

This book is dedicated to the interaction of electromagnetic wave radiation in matter, such as the wave propagation in a plasmonic and conductive state, that are dispersive media. The different measurement methods of electrical properties of soils have been studied using several applications. The experimental results of the thermoelectric properties of a chalcogenide system and the electrical conductivity of molten salts and ionic conduction in electrolyte solutions are discussed. The application of an electric field impulse and its influence on the immune responses of animals by increasing different elements of the immune response is discussed. The electromagnetic radiation transmission through skin samples of pigs of different ages have been measured in order to understand the process of absorption and conversion. The methods and results are covered in the book.

Published in London, UK © 2020 IntechOpen © sakkmesterke / iStock

Electromagnetic Field Radiation in Matter

Electromagnetic Field

Radiation in Matter

*Edited by Walter Gustavo Fano,* 

*Adrian Razzitte and Patricia Larocca*