Bioinfluence with Infrared Thermal and Electromagnetic Fields as a Therapeutic Approach of Hamsters with Experimental Graffi Myeloid Tumor

The aim of the present experiments was to study the effect of bioinfluence Infrared thermal field (ITF) and electromagnetic fields (e.m. fields) on the progression of experimental tumor in hamsters.Spectral analysis methods called Non-equilibrium spectrum (NES) and Differential non-equilibrium spectrum (DNES) for 1% solutions of blood serum from hamsters with cancer and healthy hamsters were investigated. Drossinakis had an effect on hamsters with cancer. Results are obtained with the DNES method compared to the control serum samples of hamsters with cancer and healthy hamsters. The spectrum was obtained in the range (-0.0937 eV; 13.23 μm; 755 cm) (-0.1387 eV; 8.95; μm; 1117 cm). The average energy (∆EH...O) of hydrogen Н...O-bonds among individual molecules H2O after treatment of Drossinakis was measured by NESand DNES-methods. The result ∆E=-0.0071±0.0011 eV is according control serum samples of hamsters with cancer and ∆E=-0.0035±0.0011 eV is for healthy ones. These results suggest the restructuring of ∆EH...O values among H2O molecules with a statistically reliable increase of local extremums in DNES-spectra. Local extremum was obtained at (-0.1212) eV, which is an indicator of anti-inflammatory effect. Another local extremum was obtained at (-0.1387) eV, which is an indicator of inhibition of development of tumor cells at the molecular level. The effects of Drossinakis influence– anti-inflammatory, antioxidant, and cancer development inhibition are shown. This article deals with the review of the basic biophysical-biochemical and biological processes underlying the influence of Drossinakis. The authors are studying their physical-chemical properties and biophysical and biological effects on the organism of hamsters. Another research by Toshkova has shown that the life span of cancer hamsters influenced by Drossinakis has been increased.

In the current study was followed the effect of influence with Infrared thermal field (ITF) and electromagnetic fields (e.m. fields) of Christos Drossinakis over the experimental myeloid tumor of Graffi, implanted in hamsters.The study was conducted by a team under the authority of Reneta Toshkova.

Purpose of the research:
To establish the therapeutic effect of infrared thermal field and e.m. fields in hamsters with experimental tumor.

NES and DNES Spectral Analysis
The device for DNES spectral analysis based on an optical principle was designed by A. Antonov.For this, a hermetic camera for evaporation of water drops under stable temperature (+22-24 0 С) conditions was used.The water drops were placed on a water-proof transparent pad, which consisted of thin maylar folio and a glass plate.The light was monochromatic with filter for yellow color with wavelength at λ = 580±7 nm.The device measures the angle of evaporation of water drops from 72.3 0 to 0 0 .The DNES-spectrum was measured in the range of -0.08--0.1387eV or λ = 8.9-13.8µm using a specially designed computer program.The main estimation criterion in these studies was the average energy (∆EH...O) of hydrogen O...H-bonds between H2O molecules in water samples and hamster serum blood.

Routes of administration of bioinfluence (ITF and e.m. fields)
Two types of bioinfluence experiments were conducted.The first one involved the application of bioinfluence to well-formed subcutaneous tumors with a diameter of about 1 cm.The second one involved bioinfluence action starting simultaneously with the tumor's transplantation.Each of the hamsters was injected with 5.10 4 tumor cells /in 1.0 mL PBS-buffered saline.
Figure 1 shows the manner of bioinfluence by Drossinakis with Infrared Thermal Field and e.m. waves on hamsters with experimental tumor.Fig. 1.Bioinfluence with Infrared Thermal Field and e.m. waves on hamsters with experimental tumor In the first trial the treatment with infrared thermal field and e.m. fields started on the 10 th day after the injecting of tumor cells in the hamsters, were conducted in 5 consecutive days.The hamsters were separated into two groups -gr.1 with bioinfluence (ITF and e.m. waves) and gr.2-control group -without a bioinfluence.In the second trial, the treatment with ITF and e.m. fields were conducted in 5 consecutive days starting simultaneously with injecting the tumor cells in the hamsters.As above the hamsters were separated into two groups -gr. 3 with bioinfluence (ITF and e.m. fields), and gr.4-control group -without a bioinfluence.

Examined tumor growth parameter
Following parameters have been examined in the course of experiments (Figure 2. A, B, C, D).Transplantability percent.It is evaluated as the ratio between the number of hamsters that have developed tumor and the total number of transplanted/injected hamsters with tumor cells.This parameter was calculated only for the second trial, where the bioinfluence has startеd on the day of tumor cells injecting.Tumor size (mm).It was defined as the average diameter based on the measurement of two mutually perpendicular diameters of tumor (A-width and B-length) with caliper on regular intervals of time after the transplantation of tumor cells.Lethality/mortality rate was evaluated as the number of died animals to the total number of hamsters in the group in percent.Average survival rate (ASR) was calculated in days for the respective group Survival rate -chart of survival rate in the groups 3.3.Results from the First trial are depicted in Fig. 2.
The size of the developed tumors was measured and compared to that of the control animals.As shown in Fig. 2, A the size of tumor in hamsters with ITF and e.m. fields (gr.1) was insignificantly smaller between 12-24 th day in comparison with that of control group (gr.2).Around the 30th day the tumors of the two groups are aligned in size.
Lethality rate (expressed in %) was demonstrated in fig.2, В.In both groups significant difference was observed.For hamsters with bioinfluence (gr. 1) low mortality rate compared to the control group (gr.2) was established.Until the 35th day, mortality was 0, at day 40-40%, at day 45 was 80%, and at day 50 it was 100%.At the same time in the control group (gr.2)-the lethality was 50% on the 30 th day and reached 100% on the 38 th day.
The chart in fig.2, D shows the extended survival rate for hamsters with therapy (gr. 1) in comparison with control group-without therapy (gr.2).
Photographs of hamsters from gr. 1 (with ITF and e.m. fields) and gr. 2 (control group) in Fig. 3 illustrate the above results.They are taken on the 17 th and the 32 nd days after tumor transplantation in gr.1 (Fig. 3, A, A1) with bioinfluence therapy, and control gr.2 (Fig. 3,B, B1) without therapy.
The conclusion we can draw from the obtained results is that bioinfluence therapy (in this scheme of application and duration) in hamsters with developed tumor doesn't stop the tumor growth (Fig. 3, A), but delayed its progression, decreased lethality and prolonged average survival time.Results are shown in fig.5, A. It can be seen from graph that the transplantability is lower in hamsters with ITF and e.m. waves (gr. 3) up to day 28, in comparison with the control group-without bioinfluence (gr.4).The reported values for gr. 3 are: on the 14 th day 33% of the hamsters developed tumors, on the 18 th day -66%, and on 28 th day 100%.Unlike them, in the control group of hamsters without bioinfluence (gr.4) 50% of hamsters developed tumor on the 14 th day and 100% on the 18 th day.
Tumor size was monitored until day 36 (Fig. 5, B).Tumor size in the control group (gr.4) was about 2 times higher than that measured in hamsters with applied ITF (gr-3) throughout the study period.
The lethality (%) in experimental (gr.3) and control (gr.4) hamsters has been reported following the 23 rd day from the beginning of experiment and was presented on fig. 4. C. High mortality rate in hamsters from gr. 4 -on the 24 th day -25%, on the 35 th day -50%, and on 38 th day -100% death was observed.For hamsters with ITF and e.m. waves on the 42 nd day -20%, on the 45 th day -40%, on 47 th day -60%, and on 56 th day -100% was calculated.
The average survival time (AST) of hamsters with ITF (gr. 3) has been established compared to the control group untreated animals (gr.4) (Fig. 5, D,E).From the chart (Fig. 5, D) was clearly visible that the hamsters from gr. 3 survive 49.0±6.63days, and from gr. 4 -33.25±6.29 days, which was around 16 days in favor of hamsters with ITF.The hamsters from gr. 3 with applied 5 days of ITF survive for over 45 days, and 40% of hamsters survive 56 days (Fig. 5, E).
To support the obtained results we have presented photographs of hamsters from gr. 3 (with ITF and e.m. fields) and gr. 4 (control group) -which present the picture on the 7 th and the 23 rd day after the transplantation of tumor cells in gr. 3 (Fig. 6, C, C1) with bioinfluence, and control gr. 4 (Fig. 6, D, D1) at the same days of investigation.

Hematological research
Cytological differences in the erythrocyte /RBCs-/ morphology and differentiation were noticed in the blood smears of control /untreated-/, vs bio-influenced hamsters with implanted myeloid tumours of Graffi as shown in Fig. 7. A, B, C.
The observed differences probably indicate positive effects of the near infrared bio-influence on the erythropoiesis of Graffi tumour-bearing hamsters, that may lead to improvement of the anemia-syndromeobligatory developed in this and/or in other experimental models of myeloid malignancies.However, more experiments are required for a definite inference.The average energy (EH...O) of hydrogen Н…O-bonds among individual H2O molecules in 1% solution of Sample 1 of blood serum of hamsters with cancer after influence of Drossinakis is measured at E=-0.1285 eV.The result for the Control sample 2 of 1% solution blood serum from hamsters with cancer is E=-0.1214eV.The results obtained with the NES method are recalculated with the DNES method as a difference of the NES (Sample 1) minus the NES (Control Sample 2) equalled the DNES spectrum of 1% solution of blood serum from hamsters.∆f(E) = f (sample 1) -f (control sample 2) Thus, the result for 1% solution of blood serum from hamster recalculated with the DNES method is ∆E=-0.0071±0.0011eV.
The result shows the increasing of the values of the energy of hydrogen bonds in 1% solution of blood serum of hamsters with cancer after influence of Drossinakis regarding control sample blood serum of hamsters with cancer.The results are effects of structuring of new hydrogen bonds at (-0.1212 eV) with common antiinflammatory effect and new hydrogen bonds at (-0.1387 eV) with anti cancer effects.This shows restructuring of water molecules in configurations of clusters, which influence usefully on human health on molecular and cellular level.
The average energy (EH...O) of hydrogen Н…O-bonds among individual H2O molecules in 1% solution of Sample 1 of blood serum of hamsters with cancer after influence of Drossinakis is measured at E=-0.1285 eV.The result for the Control sample 3 in 1% solution of blood serum of healthy hamsters is E=-0.1250eV.The results obtained with the NES method are recalculated with the DNES method as a difference of the NES (Sample 1) minus the NES (Control Sample 3) equalled the DNES spectrum of 1% solution of blood serum from hamsters.∆f(E) = f (sample 1) -f (control sample 3) Thus, the result for 1% solution of blood serum from hamster recalculated with the DNES method is ∆E=-0.0035±0.0011eV.The result shows the increasing of the values of the energy of hydrogen bonds in 1% solution of blood serum of hamsters after influence of Drossinakis regarding control sample blood serum of healthy hamsters.

Local extremums of influence of Drossinakis on hamsters
The local extremums of water samples are detected at E = -0.1112eV, E = -0.1212eV and E = -0.1387eV.The value measured at E = -0.1212eV (λ = 10.23 µm; 978 cm -1 ) is characteristic for anti-inflammatory effect (Ignatov, Gluhchev, Karadzhov et al., 2014) The value measured at E = -0.1112eV (λ = 11.15µm; 897 cm -1 ) is characteristic for the presence of Ca 2+ ions in water (Antonov, 1993).The value measured at E = -0.1387eV (λ = 8.95 µm; 1117 cm -1 ) is characteristic for inhibiting the growth of tumor cells (Ignatov, Mosin, 2012).Experiments conducted by Antonov with cancer cells of mice in water demonstrated a reduction of this local extremums to a negative value in DNES spectra for the function of distribution of energies ∆f(E) (Antonov, 1993).The results of influence of Drossinakis is effect of structuring of new hydrogen bonds at (-0.1212 eV) with common anti-inflammatory effect and new hydrogen bonds at (-0.1387 eV) with anti tumor effects.This shows restructuring of water molecules in configurations of clusters, which influence usefully on human health on molecular and cellular level.

Mathematical models (Ignatov, Mosin, 2013) of blood serum of hamsters.
The research was performed with the following types of 1% solutions of blood serums Sample 1 -1% solution of blood serum of hamsters with cancer after influence of Drossinakis Sample 2 -1% solution of blood serum of hamsters with cancer as control sample Sample 3 -1% solution of blood serum of healthy hamsters The research with the NES method of water drops is received with 1% solution of blood serum of hamsters with cancer after bio influence of Drossinakis.The mathematical models of 1% solutions of blood serum of hamsters with cancer after bio influence of Drossinakis give the valuable information for the possible number of hydrogen bonds as percent of H2O molecules with different values of distribution of energies according two control samples (Table 1; Figure 7).Sample 2 is 1% solution of blood serum of hamsters with cancer as control sample.Sample 3 -1% solution of blood serum of healthy hamsters These distributions are basically connected with the restructuring of H2O molecules having the same energies.E= -0.1112 eV is the local extremum for effect on the nervous system E=-0.1212eV is the local extremum for anti-inflammatory effect E= -0.1387 eV is the local extremum for inhibition of development of tumor cells of molecular level * The result (-Evalue) is the result of hydrogen bonds energy for one parameter of (-E) ** The result (-Evalue) is the total result of hydrogen bonds energy Figure 8 shows the distribution (%, (-Evalue)/(-Etotal value) of H2O molecules in and 1% of water solution of serums from hamsters, as follows: (red line) -after bioinfluence of Drossinakis on hamsters with cancer (blue line) -hamsters with cancer (green line) -healthy hamsters In particular statistical re-structuring of H2O molecules in water samples according to the energies was observed.It could be assumed that the experimental data proves the restructuring of H2O molecules on molecular level which could lead to anti-inflammatory effects with influence on the immunology system.However, for the value E = -0.1387eV or λ = 8.95 µm there is biggest local extremum (28.8 (%, (-Evalue)/(-Etotal value)) corresponding to the re-structuring of hydrogen bonds among H2O molecules for inhibition of development of tumor cells of molecular level.The distribution (%, (-Evalue)/(-Etotal value) of water molecules in Sample 1 according Control sample 2 (10.5 (%, (-Evalue)/(-Etotal value)) is different.
The experimental data for the effect of Drossinakis may prove that H2O molecules are restructured on molecular level, and the biophysical effects are: E=-0.1212 eV is the local extremum for anti-inflammatory effect E= -0.1387 eV is the local extremum for inhibition of development of tumor cells of molecular level

Conclusions
In conclusion, the results achieved from the two types of tests with 5 days course of ITF and e.m. waves treatment of hamsters with experimental subcutaneous tumor are positive.Prolonged survival rate and decreased mortality (in the first and second trial), as well as lowered transplantability and slowed tumor growth (in the second trial) were observed.The present results are the base for conducting further tests that aim to establish the optimum regimen of bioinfluence with regards to frequency and duration of the therapeutic procedures, also valid with other experimental models.
The basic conclusion is that Drossinakis is able to increase the average energy of hydrogen bonds among water molecules in the blood of hamsters with cancer after treatment compared to the average energy of hydrogen bonds among water molecules in the blood of non-treated hamsters with cancer and healthy hamsters as control groups.
The mathematical model of blood serum solution of hamsters with cancer after the Drossinakis' influence gives significant information about the possible number of hydrogen bonds as a percent of H2O molecules with different distribution of energy relative to the same number in the two control groups.
As a result of different energies of hydrogen bonds, the surface tension of the blood serum solution of cancer hamsters is increased after the treatment relative to the control samples.This effect is connected with the preservation and increase in the energy of the biochemical processes between water molecules and biomolecules.
The achieved results of hamsters from experimental bio-influence of Christos Drossinakis reveal their biological efficiency and can be subject of future studies.Extending the life of the hamsters is an indicator of improving immune status.The obtained results correspond to recent data from the medical scientific literature for the positive effect of the near infrared irradiation on the structure and function of erythrocyte membrane in normal and pathological conditions.The mitochondrial polarity in cancer cells was found to be lower than that of normal cells.Drossinakis is increasing the mitochondrial polarity.

Fig. 3 .
Fig.3.Pictures of hamsters from gr. 1 (with ITF) (top line) and gr.2 (control group without bioinfluence) (bottom line) on the 17 th and 32 nd day respectively.3.4.Results from Second trial are depicted in Fig. 5. Similar results are obtained in the second trial.Small differences concern the appearance of tumors in hamsters which was established through daily inspection and palpation (touching) of the site, where the tumor cells have been injected following the 5 th day after injection.Initially, at the spot of injection some firmness has been palpated that gradually was increasing, and when it reached around 10 mm in size it can be measured with caliper Fig. 4.

Fig. 6 .
Fig. 6.Pictures of hamsters from gr. 3 (with ITF) (top line) and gr. 4 (control group without bioinfluence) (bottom line) respective to the days of investigation.The presented photographs of hamsters from experimental groups on the 7th and the 23 rd day from the beginning of trial, show delay in appearance of tumors and lesser size tumors in hamsters with applied ITF and e.m. waves therapy (Fig. 6.C, D), which was shown numerically and graphically in the calculated biometrical parameters of tumor -transplantability and tumor size on Fig. 5.A and Fig. 5. B. Based on the results obtained in the second type experiments, we can draw the following conclusion: The application of ITF and e.m. waves in 5 consecutive days at the same time with injecting of tumor in trial hamsters revealed lowered percent of appearance of tumor, decreased percent of mortality, slowed tumor growth, increased survival rate and prolonged average survival time in group (gr.3) in comparison with the same indicators for the control group -untreated hamsters with tumor (gr.4).
A) Control healthy (x20); B) Control tumor (x 20); C) Tumor with therapy (x40)3.6.Results with spectral analysis with methods NES and DNES of 1% solutions of blood serums of hamsters NES and DNES of blood serums after influence of Christos DrossinakisThe research was performed with the following types of 1% solutions of blood serums Sample 1 -1% solution of blood serum of hamsters with cancer after influence of Drossinakis Sample 2 -1% solution of blood serum of hamsters with cancer as control sample Sample 3 -1% solution of blood serum of healthy hamsters

Fig. 8 .
Fig. 8. Mathematical model (Ignatov, Mosin, 2013) of 1% water solutions of 1% solution of serums from hamsters.Notes: E=-0.1212 eV is the local extremum for anti-inflammatory effect E= -0.1387 eV is the local extremum for inhibition of development of tumor cells of molecular level