Research of Catholyte Water on Experimental Graffi Tumor on Hamsters. Kangen Device for Catholyte Water

The paper describes the results of a pilot study aimed at the investigation of the influence of catholyte (electrolyzed alkaline water) on the development of tumors. In the experiments solid Graffi tumor was transplanted subcutaneously in the back of the experimental group of Golden Syrian hamsters. Tumor appearance and growth were registered every day. Blood parameters were measured on the 10 and 25 day after transplantation and blood smears were prepared. Hamsters treated with catholyte developed tumor with some delay compared to untreated (drinking tap water) ones. Also, the tumor growth was slow and the survival rate was increased. The analysis of blood parameters and cell morphology has shown significant differences in the value of some hematometric parameters and morphological changes of some blood cells. The obtained results suggest about the beneficial influence of catholyte and the possible use of it as a supporting non-invasive therapy of cancer diseases.The studies of the author show that the highest quality of Catholyte water if from device Kangen. The research of effect on Experimental Graffi Tumor on Hamsters is with co-authors Reneta Toshkova, Elissaveta Zvetkova and Georgi Gluhchev.


Experimental tumor
The experimental Graffi solid tumor is maintained on a monthly basis in vivo in hamsters from the research team at IEMPAM-BAS (17) via subcutaneous (s.c.) transplantation of live tumor cells ((1-2.10 6 ) in the area of the back. Between days 7 and 15 in the spot of injection appear tumors, which grow progressively, and the hamsters die aproximately 30-35 days after the injection of tumor cells. In such a tumor model it is observed 100% attachment/appearance (transplantability) of umor and 100% mortality rate. Spontaneous regression, i.e. spontaneous shrinking and disappearance of the tumor is not observed.

Catholyte water
During the experiment catholyte water was produced every day using the Actvator-2 device, developed in the Institute of Information and Communication Technologies at the Bulgarian Academy of Sciences. In this way acidity pH of the water was kept between 9.0 and 9.5.

Experimental design.
All the animals were divided into 4 groups each of 6 animals, as follows. Gr.1 The hamsters from this group started drinking catholyte water 10 days before the injection with 5х10 4 Graffi tumor cells per hamster in the back area, and continued drinking it until the end of the experiment. Gr.2. This group was used as a control. The hamsters from it were s.c. injected with the same amount of tumor cells on the 10 th day of the experiment as the hamsters from Gr.1, and were receiving tap water all the time. Gr.3 consisted of healthy hamsters drinking catholyte water during the experiment. Gr.4 consisted of healthy hamsters drinking tap water all the time. The first two groups have to reveal the influence of the catholyte water on the appearance and growth of transplanted tumor compared to the tap water, as well as for evaluation of haematological parameters and peripheral blood cell morphology. The last two groups were used as control for hematological research.

Measured parameters
The following parameters of tumor development are determined: -tumor transplantability success (% of hamsters with tumor to the total number of injected ones), -tumor size (the average diameter of tumor measured with caliper in mm), -survival and average survival (calculated for the respective group in days), -lethality (% of dead animals in the group). Animals from each group were sacrificed preserving the ethical aspects of the European convention for protection of vertebrate animals, used for experimental and other scientific purposes (OJ L 222), and approved by the National Veterinary Medical Office in Bulgaria, and blood samples have been prepared at different time periods: on days 10 and 25 after Graffi tumor implantation. Hematological/hematometric parameters and indices as shown in Fig.1 were measured on the automated hematological analyzer BC-2800 Vet (Mindray, China). The WBC/LR (White blood cells to Lymphocyte ratio and NLR (Neutrophil to lymphocyte ratio) were calculated.

Statistical analysis
For the evaluation of the significance of the differences between the average values of a specific parameter t-test was used at levels of significance α = 0.10 and α = 0.05, respectively.

Research with spectral analyses with methods DNES and NES and chemical compositions of Catholyte water with different types of Catholyte water
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  = 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--0.1387 eV or  = 8.9-13.8 µm using a specially designed computer program. The main estimation criterion in these studies was the average energy (∆EH.. As can be seen from the graph on ( Figure 1A) the tumor transplantation was delayed for the hamsters taking catholyte. While all the hamsters from Gr.2 developed tumor on the 12 th day this happened only for 1/3 of hamsters from Gr.1. In the hamsters receiving catholite, tumors were detected at 100% on day 20. Similar effect is observed for the tumor size ( Figure 1B). Until the 10 th day was not established subcutaneous firmness in none of the hamsters drinking catholyte. This group showed an inhibition in tumor growth rate as compared to control throughout the study period.
The increased lethality for the hamsters ( Figure.1C) shows from Gr.2 after 35 days, compared with the hamsters from Gr.1. The control group (Gr.2) had a 100% mortality on day 41, while in the group 1 -on the 53rd day of the study.
The evaluated average survival for Gr.1 is 43.4±6.9 days, while it is 37.5±4.5 days for Gr.2. Same conclusion could be taken from (Figure.1E).
These data suggest that the catholyte water slows down tumor development and as a result increases survival rate.

Hematological parameters
Hamsters from the trial and control groups were euthanised after the application of deep anaesthesia on the 10 th and 25 th day after tumor transplanting. The obtained blood was used to report hematological parameters, for serum and preparation of blood smears.
The evaluated parameters for all groups are displayed in (Table 1). The developing experimental Graffi myloid tumor in hamsters influenced diversely the two main WBC subpopulations -neutrophil granulocytes and lymphocytes (column 4). Significantly elevated WBC, granulocyte count and granulocyte number (%) as well as significant reduction of absolute lymphocyte count and lymphocyte number (%) were observed in the Graffi myeloid tumor-bearing hamsters (Gr.2, column 4) and (Figure 3). These effects are well expressed on the day 15-th, and are profound on the 25-th day in our experimental model. The treatment of tumor bearing animals with catholyte as drinking water improved the values of same parameters during the investigation (Gr.1, column 3) and (Figure 3).
The mean values and standard deviations of some parameters are shown in Table 2 (Figure 3).
Differences in some of the hematological parameters (WBCs, Ly) and WBCs/LR, and NLR hematometric index for groups are shown in (Figure 3).
Simultaneously, a significant reduction of the lymphocyte number was evaluated in the same animals. Additionally, we obtained that catolyte water influenced (elevated) some main PLT-hematometric values in both -control and experimental animals (Table 1, PLT, MPV and and PDW). The hematometric results obtained were confirmed by our cytological studies on PLTs (thrombocytes) in the peripheral blood smears of hamsters where one could see clusters of activated thrombocytes -more pronounced in the blood of tumor-bearing animals ( Fig.4 -i).

Cytological study
Images from blood smears are shown on (Figure. 4). Atypical myeloid cells and blast-like cells from the peripheral blood of Graffi myeloid tumor-bearing hamsters, at the day 10 th from tumor inoculation were observed. One could see atypical immature granulocyte with peripherally localized ring-shaped nucleus and eosinophile granules -in the central part of cytoplasm (Figure 4 -d,

e,f). middle line
In Graffi tumor bearing hamsters, treated with electrolyzed alkaline water (catholyte) atypical activated lymphocytes and cluster of PLTs in the peripheral blood smear were noticed. Lymphocytes from these hamsters, although atypical, show characteristic signs of activation as in healthy ones (Figure 4 -g,h,i) bottom line.

Electrolyzer for Catholyte water and Electrolyzer for Kangen Water 3.4.1. Electrolyzer for Catholyte water
The main stage of electrochemical treatment of water is the electrolysis of water or aqueous solutions with low mineralization as aqueous solutions of 0,5-1,0 % sodium chloride (NaCl), which occurs in the electrolysis cell, consisting of a cathode and an anode separated by a special semipermeable membrane (diaphragm) which separates water to alkaline fraction -the catholyte and acidic fraction -the anolyte ( Figure 5). When the electric current is passing through the water, the flow of electrons from the cathode as well as the removal of electrons from the water at the anode is accompanied by series of redox reactions on the surface of the electrodes. As a result, new elements are being formed, the system of intermolecular interactions, as well as the composition of water and the water structure are changed. The products of electrode reactions are the neutralized aqueous admixtures, gaseous hydrogen and oxygen generated during the electrolytic destruction of H2O molecules, metal cations (Al 3+ , Fe 2+ , Fe 3+ ) in the case of metal anodes made of aluminum and steel, and molecular chlorine. The gaseous hydrogen is generated at the cathode while the oxygen is produced at the anode. Water also contains a certain amount of hydronium ions (Н3О + ) depolarizing at the cathode with formation of the atomic hydrogen: In an alkaline environment a disruption of Н2О molecules, accompanied by formation of atomic hydrogen and hydroxide ion (OH -) occurs: The reactive hydrogen atoms are adsorbed on the surface of the cathode, and molecular hydrogen H2, released in the gaseous form after recombination are formed: At the same time atomic oxygen is released at the anode. In an acidic environment, this process is accompanied by the destruction of H2O molecules: 2Н2О -4е -→ О2 + 4Н + , (4) In an alkaline environment the OHions moving under the electrophoresis from the cathode to the anode are a source of oxygen: 4ОН -→ О2 + 2Н2О + 4е -, (5) The normal redox potentials of these reactions compiles +1,23 V and +0,403 V, respectively, but the process takes place in certain conditions of electric overload. The cathodes are made of metals that require high electrical voltage (lead, cadmium) allowing for the generateion of reactive free radicals as Cl*, O*, OH*, HO2*, which react chemically with other radicals and ions. In bulk oxidative processes products of electrolysis of water -oxygen (O2), hydrogen peroxide (Н2О) and hydrochlorine acid (HClO) play a special role. During the electrolysis, an extremely reactive compound Н2О2 is formed. Its formation occurs due to the hydroxyl radicals (OH*) which are products of the discharge of hydroxyl ions (OH -) at the anode: 2ОН -→ 2OH * → Н2О2 + 2е -, (6) where ОН * is the hydroxyl radical. The chlorine-anion is transformed to Cl2: 2Cl -→ Cl2 + 2e -,

Device for Kangen water
The device for Kangen water is applying with tap water. Kangen devices produce catholyte water with pH 8.5; 9.0 and 9.5 with negative oxidation reduction potential (ORP) with values from (-500 to -250) mV, The figure 6 shows one type of Kangen device.