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Chapter Four

Results and Discussion

4-1 Introduction
Chemical carcinogens, radiation, UV light or free radicals cause direct damages to DNA (base alterations, cleavage of DNA strands)[72].A small numbers of genes that we call proto-oncogenes, whose functions are essential for normal cell growth , appear to be important targets for mutations that can occur spontaneously in the process of normal cell division when DNA of the cell duplicated. These critical changes can occur as a result of DNA damage from environmental agents which increase the probability of mutations [73]. There is good evidence, for example, that agents such as XRay (ionizing radiation) and variety of chemicals which increase the frequency of DNA damage and mutations increase the risk of acquiring cancer [74].There are very few studies investigated the biological effect of magnetic field on DNA. Some of these studies reported in vitro that Chines hamster lung cells exposedto 15T DC magnetic field cause a chromosomal aberration for metaphase cells [75].

4-2 Lymphocyte Transformation


The analysis of the mean value for lymphocyte transformation cells of 60 blood sample for three experiment exposed to (0.1T- 0.5T) magnetic field intensity is shown in Fig.(4-1)(Table 4-1).The percentages of lymphocyte transformation were significantly increased compared to normal healthy blood sample (control).Same results found for the percentage of lymphocyte transformation correlated with time of exposure (30min-180min),Figs.(4-2, 4-3).Researcher in this field found that 1mT magnetic field intensity show a slight increase in cell proliferation p=0.03[32].At magnetic field intensity 0.2T, percentage of lymphocyte transformed cell show high significant increase than other magnetic field intensity, Henderson [21],suggested that there is a correlation between the amplitude of plasma membrane fluctuations and the response of cell to MF. Fluctuated protein molecules that oscillate with frequency simulate natural earth frequency that cause resonance and give
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energy rise to enhance growth factor for cell.

Mean Number of Lymphocyte Transformed

85 80 75 70 65 60 55 50 45 40 35 30 25 20 0 0.1 0.2 0.3 0.4 0.5 0.6

Cell%

Tim e=30m in. Tim e=60m in. Tim e=120m in Tim e=180m in.

M agnetic Field (Tesla)

Figure (4-1 ): The effect of different intensities m agnetic fields on lym phocyte transform ation of three norm healthy volunteers al .

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85 80 75 70 65 60 55 50 45 40 35 30 25 20 0 30 60 90 120 Time of Exposure(min.)

Results and Discussion

Mean Number of Lymphocyte Transformed Cell%

B=0.1T B=0.2T B=0.3T B=0.4T B=0.5T

150

180

210

Figure (4-2):The effect of different time of exposure on lymphocyte transformation of three normal healthy volunteers .

Table (4-1) Number of Lymphocyte transformation cell % exposed to different magnetic field intensity at different time of exposure

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No. of Experiment 1 2 3 Mean .Stdv* 1 2 3 Mean .Stdv* 1 2 3 Mean .Stdv* 1 2 3 Mean Stdv*

Number of Lymphocyte Transformed cell% Control 36 28 33 32 4.0 36 28 33 32 4.0 36 28 33 32 4.0 36 28 33 32 4.0 0.1T 58 49 52 53 4.5 59 56 59 58 1.7 62 59 63 61 2.0 77 71 73 74 2.4 0.2T .Time=30min 63 56 58 59 3.6 Time=60min 67 60 65 64 3.6 Time=120min 69 63 67 66 3.0 Time=180min 79 79 78 79 0.4 77 70 77 75 3.2 76 68 75 73 3.5 78 62 76 72 7.1 68 62 62 64 3.4 65 60 59 61 3.2 69 64 60 64 3.6 64 61 60 62 2.0 62 59 58 60 2.0 64 62 61 63 1.5 62 51 57 55 5.5 62 52 59 57 5.1 62 54 57 58 4.0 0.3T 0.4T 0.5T

*Stdv. (standard deviation)

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Results and Discussion

a
b

a
b

Figure (4-3): Picture shows Lymphocyte transformation (a) Normal cell, (b) Transformed cell. Giemsa stain,1000X Therefore exposure of peripheral blood lymphocyte to static magnetic field may have induced DNA damage that was associated with increased lymphocyte transformation as a consequence normal repair process. From the results of our research we can concludes that magnetic field cause DNA damage, this is correlated with the duration and intensity of MF. This in agreement with Aaronson results[74]. By prolonging the duration of magnetic exposure, DNA strand breaks was observed at lower flux density[75].Such effect could normally followed by DNA repair activation process which was reflected in our study by increase of lymphoblasts at all magnetic field intensities. The effect of magnetic fields on formation of free radicals is hypothesized by many researcher that suggest that iron may play role in the effect of magnetic fields as iron is closely involved in free radicals formation (via the Fenton reaction) in cells. Relevant that magnetic field can

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cause iron dependent DNA strand breaks is that iron is present in higher concentration in the nucleas than in the cytoplasm, because of the presence of an ATpase related iron pump on the nuclear membrane[76].Another study has reported iron atoms intercatalet in DNA molecules, and DNA ferrous iron complexes could enhance hydroxyl radical formation from hydrogen peroxide compared with ferrous iron alone[77].These make DNA more vulnerable to iron-catalyzed free radical attack. In addition to DNA damage, free radicals can cause damage in other biological molecules, such as lipids and proteins, and can profoundly affect cellular homeostasis [78]. Disturbance in free radical metabolism could affect these biomolecular processes and cell functions. Magnetic field can induced DNA strand breaks caused by an iron mediated free radical process, probably via the Fenton reaction, which converts hydrogen peroxide to the more toxic hydroxyl radical. Iron induced oxidant formation is known to cause DNA strand breaks, alter calcium homeostasis in cells [76,79, 80, 81,82]. Effects of magnetic fields manifest through a two stage process. In the first step, magnetic field exposure effect iron homeostasis in certain cells, leading to an increase in free iron in the cytoplasm and nucleus, which in turn leads to an increase in hydroxyl radicals, via the catalytic reaction of the Fenton reaction, which damage DNA, lipids and proteins. Damage to lipid (lipid peroxidation) in the cellular membrane in turn leads to an increase in calcium leakage from internal storage sites in the cell. Magnetic field affects the water which is the media for exchanging information. [83] H2O H2O++ H2O H2O+ +eOH + H3O+ (4-1) (4-2)

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There are always 10-20 water molecules around each protein molecules [46]. Magnetic energy affects water molecules and can transform water into small orderly cluster[Albert szent-Gyrgyi- Nobilprize Laureati, 1978][45].This means that the four hydrogen bridge connection containing proton that spin to the direction.These cluster are not stable, very sensitive and become more alkaline and orderly organized that can pass easily through the cell membrane into the cell [46].This will lead to ion exchange through out the cell membrane which cause potential difference. The polarity of water make it a powerful solvent for ionic substance, for polar, non ionic and amines. This ionic solute dissolved in a solvent, destroy the bond between the molecules or ions of the solute and new bonding between molecules of the solvent and the ions of the solute form. Therefore electrostatic attractions within solute are separated by specific alignment of water dipoles; this will cause a hydration shell [54]. A physiological stress is an external factor on a cell in way that interferes with its normal functioning [52]. The physical properties of magnetized water may cause changes in glycoprotein which bind loosely and externally to polar group of membranous phospholipids; the water in the cell was oriented in multilayer polarized by alternating fixed charges on the surface of extended proteins, and that this multilayer extended over a considerable distance. So the cardinal site acts as receptors capable of communication with external environment [53]. When these sites are filled by specific adsorbants they can initiate a chain of induced changes in the electron distribution around each site along the extended protein and produce cooperative interactions between sites with subsequent changes in the association and dissociation of specific ligands [53].The strong effect of magnetic field is on the production of growth factor and on binding this growth factor to receptor protein. This implies a possible reduction in normal immune system response. As this was found by some researcher that

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phosphorylation of the T-cell receptor zeta subunit in Jurkat T-cell was found to inhibit by exposure of cell to EMF [84]. The damage can lead to two possible outcomes; a) cellular antioxidation processes will keep the damage in check by neutralizing free radicals, and eventually the cell will repair itself and survive. However, DNA damage and repair could lead to mutation and increase the chance of carcinogenesis. b) If the processes of free radical damage are not checked by cellular antioxidation and repair processes, the cell will die, because free radical peroxidation of lipids is a chain reaction. The outcome of oxidative damage induced by magnetic fields will therefore depend on various factors, including the oxidative status of the cell, capability of endogenous antioxidation enzyme and processes to counteract free radical buildup, availability of exogenous antioxidants, iron homeostasis (a balance of iron influx, storage and use), the parameters of exposure (e.g. intensity and duration of exposure and possibly the waveform of the magnetic field), and whether the oxidative damage is cumulative.[84]. However Onodera et al [86] found that strong static magnetic field has acute effect on immune cells during cell division while the field exposure has a minimal effect on immune cell in a nondividing phase.

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4-2-1 Lymphocyte Transformation for Cells Stimulated with Mitogen Phytohaemogglutainin(PHA)


Lymphocyte transformation stimulated with plant product mitogen (PHA)for T-cell mitogen, it caused cells to enter the cell cycle and proliferate, The analysis of this result, percentage of lymphocytes transformation are significantly decreased with time of exposure (30-180 min) at magnetic field intensity 0.2T (64 1.3, 59 0.5 , 55 1.1 ,52 1.5) compared to normal healthy cell (control) not exposed , not stimulated with PHA (34.6 6.5) and to stimulated cell with PHA but not exposed to magnetic field (66 7) as shown in Fig.(4-4)(Table4-2).

70 Mean Number of LymphocyteTransformed Cell% stimulated With PHA 60 50 40 30 20 10 0 0 30 60 90 120 150 180 Time of Expouser(min) B=0.2T C ontrol(not stimulated) Test (stimulated not exposed)

F igure(4-4):T he effec t o f magnetic field o n 0.2T lym hp o c yte trancfo rmed c ell stim ulated with P HA at d ifferent tim e o f exp o s ure

Table (4-2): Number of Lymphocyte transformed cell % stimulated with PHA exposed to 0.2 T magnetic fields at different time of exposure

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No. of Experiment 1 2 3 Mean **stdv 1 2 3 Mean **Stdv

Number of Lymphocyte transformed cell % exposed to 0.2T (Control (c 35 28 41 35 6.5 (Test PHA stimulate cells (T* 63 61 74 66 7 ***Test (T") 30min 60min 59 59 58 59 0.5 120min 55 53 55 55 1.1 180min 52 50 53 52 1.5

1 2 3 Mean Stdv**

66 64 63 64 1.3

*Test (T) = cell stimulated with PHA , **stdv.= standard deviation ***Test (T") = cell stimulated with PHA and exposed to magnetic field,

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Our results of lymphocyte transformation of PHA stimulated cultures exposed to static magnetic field show a decrease lymphoblast compared to stimulated samples these results correlated with data obtained from Leo.D`Souza et al[7],the worker found that magnetic fields produces a decrease of DNA synthesis at about 18%-24% as determined by tritiated thymidin in uptake measured by autoradiography.which indicate that proliferated lymphocytes to PHA are more vulnerable to static magnetic fields than resting cells. Which may suggest that signal transduction are a possible target through Ca+ ion intervention. Activation of normal human T- cell with plant derived lectin (PHA).As mitogen stimulates most of T-cells for proliferation so the effect of magnetic field on the response of these cells to mitogen can be recognized in Fig(4-4). Such effect can be explained as a direct consequence to transformation signal conducting to PHA. The initial findings suggest that the initial membrane surface events associated with the mitogen activity and coupled to intercellular enzymatic sequences essential in the initiation of cell division may substantially modified with magnetic field. Taken together with the evidence on field sensitive sites for calcium binding and field sensitive to hormone binding mechanism, these data point strongly to the membrane surface as the prime site for cellular interaction with certain imposed electromagnetic field [87].The possible effect on signal transduction include stimulation or inhibition of receptors or ion channels, alteration of ligand binding, alteration gene expression, induction of oncogenes, effect incross regulation of pathways[88].Therefore we can conclude that EMF from low to high level are biologically active i.e. interact with various cells or systems of the body. The most likely site of interaction of the superoxide radicals is the cell membrane, through interactions with signal transduction pathways [89]. It is reported that the intercellular calcium ion concentration is significantly raised, when the cell is exposed to the magnetic fields in mitotic period [29].

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Results and Discussion

4-3 Viable and non Viable Assay


The analysis of number of live cell and dead cell at magnetic field intensity 0.2T exposed for 30min are shown in Table (4-4).There are significantly decreases with viable cell stimulated with PHA compared to non stimulated lymphocyte exposed to magnetic field which shown a slight changes in percent of viable cell, thats mean the magnetic field exposure Significantly increased the death of PHA stimulated lymphocyte. As the interaction of the growth factors with their receptors on the plasma membrane cause somatic cells to leave G0 , traverse to G1 and enter S phase (DNA synthesis phase)[72].This mean that magnetic field exposure significantly increase the death of PHA stimulated lymphocyte by apoptosis[74]. Apoptosis is mainly triggered by DNA damage [85].These results suggest that strong static magnetic field has acute effect on immune cells during cell division, while the field has a minimal effect on cells in a nondividing phase [86]. When the magnetic field exposed to the cells in G0 / G1 phase, the respiration of the mitochondria is enhanced, this enhanced the energy activity of mitochondria [29]. This is important for understanding the mechanism of interaction since the magnetic penetrate beyond the cell membrane and potentially interact, field interact with structures inside the cell [88].So suggesting a desensitization of the growth regulatory receptor. And also showing that transformed cells respond to MF with increased apoptotic events including micronucleus formation, while non transformed cell did not.

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Table (4-4): Result from triplicate experiment of viable and non viable cell exposed to 0.2T magnetic field for 30min.

Type of cells Control (c) Stdv* Not stimulated cell exposed to MF.% (c') *Stdv PHA stimulated cellsTest (T) Stdv* PHA stimulated cells exposed to MF. Test (T") Stdv*
*Stdv = standard deviation

Viable 1425 823.6 1271 812.1 1116 682.1 867 631.6

Non viable 57 37.2 73 49.2 87 73.1 102

Percent %4 4%

9%

11% 71.1

4-4 Phagocytosis
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The analyses of the mean value for phagocytic cell of 60 samples from a normal healthy volunteers exposed to different magnetic field intensity are present in Fig.(4-5).The percentages of phagocytosis are significantly decrease with time of exposure (30min - 180min) (Table 4-3).The same results obtained for the percentage of phagocytosis correlated with magnetic field intensity (0.1T-0.4T) at different time of exposure as in Figs.(4-6,4-7). The process of phagocytic can however be hampered local environment of excessive ionic strength and deficiency of certain cations such as magnesium [90]. As the magnetic field affect water which makes it easily inter through the cell membrane, the ion exchange will be influenced. The rest potential of the cell is proportional to the ion exchange occurring on the cell membrane [85]. Phagocytic cell and polymorphonuclear (PMNs) neutrophils play a central role in defense of the host against infection [64].The process of particle engulfment by neutrophil requires energy as well as complex interaction between cytoplasm contractile protein and the plasma membrane [90]. This mechanism is affected with environment, as the water at magnetic field become alkaline, this will reduce the process of killing microorganisms inside the cell.

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70 Mean Number of Phagocytic Cell % 65 60 55 50 45 40 35 30 0 30 60 90 120 150 180 210


B=0.1T B=0.2T B=0.3T B=0.4T

Time of Exposure (min) Figure(4-5):The effect of different time of exposure on phagocytosis for three normal healthy volunteers

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70 Mean Number of Phagocytosis Cells% 60 50 40 30 20 10 0 0 0.1 0.2 0.3 0.4 0.5 Magnetic Field (T) Figure(4-6): The effect of different magnetic field intensities on phagocytosis of three normal healthy volunteers
Table (4-3) Number Phagocytic cell % exposed to different magnetic field intensity at different time of exposure
Time=30min Time=60min Time=120min Time=180min

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No. of Experiment 1 2 3 Mean .Stdv* 1 2 3 Mean .Stdv* 1 2 3 Mean .Stdv* 1 2 3 Mean Stdv*

Number of Phagocytic cell% Control 65 67 68 67 1.5 65 67 68 67 1.5 65 67 68 67 1.5 65 67 68 67 1.5 0.1T 54 56 55 55 1 52 47 52 50 2.8 50 44 49 48 3.2 48 41 44 44 3.5 0.2T 52 58 53 54 3.2 50 49 49 49 0.5 48 47 47 47 0.5 46 43 42 43 2.0 0.3T 48 58 49 52 5.5 47 45 45 47 1.5 47 46 40 44 3.7 45 40 38 41 3.6 0.4T 47 49 47 48 1.1 46 47 43 45 2.0 46 48 41 45 3.6 43 43 36 41 4.0 .Time=30min

Time=60min

Time=120min

Time=180min

*Stdv. (standard deviation)

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Figure (4-7) Picture shows phagocytic cell. Giemsa stain 1000X Magnetic field may affect the mechanism of interaction and changes in radical pair recombination, where one radical species is a metal ion (e.g. Iron in hemoglobin)[91]. Mitochondria utilize magnetic spatial energy. This magnetic energy will effect some complex photochemical reaction; this free radical generated in the triplet state and must move to singlet state before reacting. This interchange can occur as a result of local magnetic field from nearby magnetic nuclei through hyperfine interaction [72], this reaction in static magnetic field, the interconversions are affected. The magnetic field
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removes the degeneracies of the triplet state sublevel, in high magnetic fields can cause separation between triplet states greater than the hyperfine interaction, in low field strength smaller effect may occur due to increase reaction yields by a few percent due to Zeeman splitting that removes the constraints of spin angular momentum conservation of the systems allowing

singlet radicals pairs to interconvert to more triplet state.So magnetic field effect on radical processes. As an example a reaction between two species (A and B) can generate a pair of radicals in the triplet state with parallel electron spin.If one of the electrons converts to a different state, changing its spin, the radical pair can react to form product [73]. A+B [R1 + R2] [R1 + R2] product

This changes involves transfer of the electron between the three triplet state T0, T1 and T+1 . These states are normally degenerate, but in a magnetic field the energies separate. When this separation is less than the hyperfine interaction for the system, the radicals created in the triplet states can be transformed into singlet and react. When the separation is greater than the hyperfine reaction, radicals created in T1 and T+1 triplet states cannot interconvert and hence reaction cannot take place, Fig (4-8). The molecular oxygen (dioxygen) and it's electronic structure is described by the one-electron molecular orbital diagram, which indicate that the ground state,3g has two unpaired electrons in separate orbital with their angular momentum opposed but with parallel spins [92]. In contrast the first two excited states 1g and 1g have all electrons paired. Thus O2 (3g), O2 (1g), O2(1g ), O2, HO2, H2O2 and HO2 are all oxidants relative to water. Since anions are stabilized by protons or cations, equilibrium involving them, will be sensitive to PH, metal ions and solvent. Magnetic field affect free

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radicals which are molecular species containing electrons unpaired in spin [64]. This mechanism of phagocytosis is suppressed due to defect in plasma membrane NADPH oxidase which is an enzyme used as a defense against bacteria by using molecular oxygen to stimulated neutrophils for generating reactive oxygen species for respiratory burst, which act on microorganism degradation.

Energy

T+1

Hyperfine interaction

T0
S

All can interconvert

Only S and T0 convert

T1

Magnetic field Figure (4-8) Separation of energy in magnetic field [72]


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In summing up the effect of magnetic field, researcher in this field proposed that magnetic field initiate an iron dependent free radical generation process in cells, which can lead to genotoxic changes and/or cell death. From this hypothesis, one can make the following speculation regarding the biological effect of magnetic field: cells with high rates of iron intake (e.g. proliferating cells, cells infected by virus, and cells with high metabolic rates such as brain cells) would be more susceptible to the effect of magnetic fields because of hydrogen peroxide, the substrate of Fenton reaction, which is a metabolic product of mitochondria [85].

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