Introduction: MRET Activated Water is produced with the help of patented in the USA Molecular Resonance Effect Technology (MRET). MRET water activator is the stationary source of subtle, low-frequency, resonant electromagnetic field of composite structure. The origin of the low-frequency composite electromagnetic field is the intensive electrical activity of nano-rings formed by linear molecular groups of MRET polymer compound (volumetric fractal geometry matrix) when polymeric body is exposed to the external electromagnetic fields of specific frequency and wavelength [Vysotskii, Smirnov 2005].
This particular article relates to a detailed observation of the effect of MRET activated water with the modified molecular structure, physical and electrodynamic characteristics on the tumor resistance in vivo in animal models. The research was conducted under supervision of Prof. Vladimir I. Vysotskii, S. Olishevsky, Ph.D. and Y. Yanish, Ph.D. on 500 mice at Kiyv Institute of Experimental Pathology, Oncology and Radiobiology, Ukrainian Academy of Science. The best results were observed in the groups of mice on MRET water activated for 30 minutes (optimal regime of activation). The significant anti-tumor effect of MRET Activated Water on mice was close to the action of the chemotherapy agents and allowed to avoid the side effects that typically follow chemotherapy treatment in oncology.
The research regarding the physical parameters of water conducted earlier at Moscow State University, Russia confirmed that MRET treatment of distilled water leads to substantial modification of basic physical-molecular properties of distilled water. The anomalous viscosity of MRET water (subject to very low tangent pressure) and electrodynamic characteristics of MRET water (subject to applied electromagnetic field of low frequency range) confirmed the high level of long-range dynamic structuring of water molecules in polarized-oriented multilayer formations in activated water produced with the help of MRET activation process [Smirnov 2007-2]. The fundamental biophysical theories revealed the scientific paradigm regarding polarized-oriented multilayer structuring of cell water in biological systems [Ling 2003, 2001, Drost-Hansen 1971, 1991]. The interaction of water dipoles with pervasive matrix of fully-extended proteins constitutes the basis for the cellular transduction mechanism. Based on this scientific approach the similarity of molecular formations of cell water and MRET activated water can contribute to their compatibility, easy bio-availability and assimilation of MRET activated water, as well as to the enhancement of cellular functions in biological systems. The introduction of MRET water to biological systems can contribute to the enhancement of the cellular transduction mechanism and the proper function of cells in biological systems.
The previous studies confirmed the ability of MRET water to enhance morphology of blood cells, immune response and to inhibit the growth of mutated cells (Fig 1) [Smirnov 2006-1, 2006-2, 2003 and 2002, Vysotskii 2006-1].
Fig 1: The effect of MRET water (30 minutes and 60 minutes of activation) on the growth and development of kaluss tissue (mutated cells of botanical origin) [Vysotskii 2006-1].
The antibacterial activity of MRET water (the inhibition of growth and reproduction of pathogens) was confirmed by testing conducted at C.A.I. Environmental Laboratory, Carlsbad, USA. It revealed the significant reduction of the amount of total coliforms following the process of MRET activation. The reduction of coliform population by 86% was observed in the rainwater activated for 30 minutes compare to non-activated rainwater. [Smirnov 2002].
The high antibacterial properties of MRET water were also confirmed by the study of the effect of MRET water activated for different periods of time (30 and 60 minutes respectively) on metabolic activity and growth of conditionally pathogenic microbial culture Escherichia coli K-12. The investigation [Vysotskii 2006-2] revealed the significant effect of MRET-activated nutrient medium in aerobic environment on the process of growth and reproduction of E.coli microorganisms, their division, the size of colonies and the modification of forms of culture cells. It was observed that at low initial concentration of cells of investigated culture Escherichia coli K-12 the nutrient medium MRET-activated during 30 minutes and 60 minutes inhibited the growth of culture 27 and 303 times respectively during the 25 hours of experiment. It confirms the sterilization effect of MRET water and allows admitting that the process of MRET activation can be applied in food industry and for water purification. Taking in account that a small population of pathogenic bacteria, such as E.coli, is usually presented in complex microbial associations in the large intestine (colon), the test on metabolic activity of E.coli bacteria in anaerobic environment was conducted. The investigation showed that the metabolic activity of E.coli bacteria in anaerobic environment practically did not change. In order to simulate the environmental conditions similar to the conditions in the small intestine of humans and animals the test on metabolic activity of complex microbial associations was conducted in anaerobic environment. It was found that MRET activated water substantially increased metabolic activity of microbial associations during the first several hours of experiment. Thus, this investigation showed that the ingestion of MRET water was beneficial for the process of digestion and could enhance the metabolism of the body. [Smirnov 2008].
Recently the study of the effect of MRET water on Staphylococcus aureus was conducted in animal model on 400 mice and in vitro at the Division of Microbiology and Immunology, Biological Department of Kyiv National Shevchenko University, Ukraine. The research in animal model revealed the fact that the consumption of MRET water stimulated the phagocytic activity and the immune response. The phagocytic system is one of the main factors of natural non-specific cellular resistance to infections and inflammations. It is the first line of protection of an organism against the penetration and reproduction of pathogenic microorganisms. The protective role of phagocytes is based on their capacity to identify, engulf and neutralize the alien agents penetrating into internal environment of a macro-organism. Particularly, the consumption of MRET water reduced the death rate from 30% (control group of mice on non-activated water) to 0% (two groups of mice on MRET water) during the first 9 days of experiment after intra-peritoneal inoculation of Staphylococcus culture. The significant bacteriostatic effect of 70 – 100% (depending on initial concentrations of pathogens) was observed in vitro for MRET-activated nutrient medium in this investigation.
Taking in consideration the beneficial effect of MRET water on the homeostasis of the body and its ability to the inhibition of growth of mutated cells in vitro, as well as the high antibacterial activity of MRET water confirmed by the previous studies, the goal of this investigation was to study the effects of different fractions of MRET water on mutated cells in tumors and on the cells of immune system in vivo. As a result of this investigation the significant positive effect of MRET activated water for prevention and enhanced tumor resistance in oncology was observed in the investigation conducted on 500 mice (laboratory models of Ehrlich's ascites tumor and Sarcoma ascites form). The best results were observed in the groups of mice on MRET water activated for 30 minutes (optimal regime of activation). The substantial anti-tumor efficacy was confirmed by very high level of reduction of Total Number of Viable Tumor Cells by 76% for animals in “preventive treatment” group which ingested MRET water for 2 weeks prior to the inoculation of cancer cells and during the 3 weeks of experiment after the inoculation. The application of MRET water in “therapeutic treatment” regime – only during 3 weeks of experiment after the inoculation of cancer cells – was less effective. The reduction of Total Number of Viable Tumor Cells by 55% was observed for the optimal 30 minutes activated water. The survival of the investigated animals was daily monitored in order to study the effect of different activated water fractions on dynamic and survival indices of tumor-bearing mice. Water activated for 30 minutes produced the most significant effect on survival of mice with transplanted tumors. The life span of mice which received optimal activated water in “preventive treatment” regime increased by 61%. The increase of life span by 43% was observed in “therapeutic treatment” regime for the optimal 30 minutes activated water. [Smirnov 2007-1, Vysotskii 2006-1].
Method and Materials: In order to find out how different fractions of MRET Activated water affect the tumor resistance of organism the following experimental approaches and techniques were used in this research:
study of possible anti-tumor efficacy of preventive administration of different fractions of MRET activated water; for this purpose mice received MRET water during 2 weeks before tumor cell inoculation and for 3 weeks after inoculation (“preventive treatment” regime);
study of possible anti-tumor efficacy of therapeutic administration of different fractions of MRET activated water; for this purpose mice received MRET water for 3 weeks after tumor cell inoculation (“therapeutic treatment” regime);
investigation of functional cytotoxic activity of NK-cells (a type of lymphocytes called Natural Killer cells) extracted from the spleens of mice (without tumors) which received MRET water; for this purpose lymphocytes were further incubated with tumor target cells.
Five different fractions of MRET activated water were prepared to elucidate effectiveness of anti-tumor effects of MRET activated water depending on time of its activation. Four water fractions were obtained after water activation for 15, 30, 45 and 60 minutes respectively. Furthermore, before the beginning of investigations a large volume of water was activated for 30 minutes and stored at 4°C during 45 days. This fraction of activated water was called "old activated water". The inbred adult male BALB/c mice, 11 weeks old, with 23 – 24 g corporal weight were used in the study. These white mice are very susceptible to various oncology diseases. In both experiments (ascitic Ehrlich carcinoma and Sarcoma ascites form) the 5 experimental groups of mice received activated water in “preventive treatment” regime. The other 5 groups were treated with activated water in “therapeutic treatment” regime. One of the 11 groups served as a control and mice from this group received non-activated distilled water. Each group consisted of 20 mice. The cytotoxic activity of NK-cells was studied on 11 groups of mice with 5 mice in each group. Natural Killer cells or NK-cells are a type of cytotoxic lymphocytes (toxic to cells) that constitute a major component of the innate immune system (non-specific immune response). NK-cells play a major role in the rejection of tumors and cells infected by viruses. The cells kill by releasing small cytoplasmic granules of proteins called perforin and granzyme that cause the target cell to dye.
The ascitic Ehrlich carcinoma tumor cells were inoculated to all groups of mice for the first experiment. The first stage of the investigation was finished on the 8th day after tumor cell inoculation, when 10 mice from each group were sacrificed and ascitic fluids containing tumor cells were obtained from peritoneal cavities. The comparison of ascitic fluid volumes and the number of viable tumor cells from “preventive treatment”, ”therapeutic treatment”, and ”control” groups of mice allowed to determine the effects of applications of different fractions of activated water on growth and size of tumors in tumor-bearing mice. At the second stage of experiment the life span of tumor-bearing mice was studied.
In order to understand the possible mechanism of anti-tumor effects of MRET activated water the study of modification in cytotoxic activity of lymphocytes was conducted on another 11 groups of mice without tumors treated with different fractions of activated water. The lymphocytes contain natural killer cells (NK-cells) which have the natural function of spontaneous cytotoxicity (without prior immunization), very important for proper immune system reactions. Due to such capacity NK-cells are considered to play a critical role in immune surveillance and cancer therapy. NK-cells that infiltrate tumors may protect against tumor spread. They also produce cytokines which can contribute to the elimination of infection. Due to such capacities NK-cells and the methods of enhancement of their functional activity based on the substances of natural origin have great potentials for immune-stimulation.
The purpose of this investigation was to evaluate optimal regimes of MRET water activation and the regime of application of activated water in order to maximize the stimulation of NK-cells cytotoxic activity. In the first stage of the research mice of experimental groups received activated water during different periods of time. Mice of “preventive treatment” groups received different fractions of MRET activated water for 21 days, and mice from “therapeutic treatment” groups received it for 14 days. After the treatment with activated water mononuclear lymphocyte fractions containing NK-cells were isolated from spleens of mice of experimental groups. In the second stage cytotoxic activity of NK-cells incubated with tumor target cells obtained from peritoneal cavities of mice transplanted with ascitic Ehrlich carcinoma was studied. The incubation was conducted for 18 hours at 37°C in humidified atmosphere with 5% CO2 and then the micro plates were gently centrifuged during 5 minutes.
Results: The experimental results confirm that consumption of all types of MRET activated water leads to the significant inhibition of tumor growth observed in mice with inoculated tumors. The best results were observed in the groups of mice on MRET water activated for 30 minutes (optimal regime of activation). The substantial anti-tumor efficacy was confirmed by very high level of reduction of Total Number of Viable Tumor Cells which is comprised of two processes: diminishing volume of ascitic fluid in peritoneal cavity of tumor-bearing mice (by 50% for animals in “preventive treatment” group) and decrease of the number of viable tumor cells per unit of tumor tissue (by 52% in the same group). The resulting decrease of the Total Number of Viable Tumor Cells was 76%. The viable tumor cells were determined by Trypan blue exclusion test: the uncolored cells were considered as viable. The test results show the dual mechanism of MRET water effect on tumors: the prevention and reduction of volume of tumors together with the inhibition of viable tumor cells. Approximately the same level of efficacy was observed in other groups (water activated for 15 and 45 minutes) with “preventive treatment” regime of application of activated water. The application of MRET water in “therapeutic treatment” regime was less effective. The reduction of Total Number of Viable Tumor Cells by 55% was observed for the optimal 30 minutes activated water. It is important to note that the long-term preservation of activated water for 45 days decreased its anti-tumor efficacy but left it on the significantly high level compare to other fractions and non-activated water. Thus, this investigation confirms that MRET activated water is efficient anti-tumor agent. The results of experimental measurements of Average Total Number of Viable Tumor Cells of ascitic Ehrlich carcinoma are presented on Fig 2.
Fig 2: The effect of preventive (1–5) and therapeutic (6–10) application of MRET activated water on average total number of viable cells <C> in an ascetic tumor, obtained from mice inoculated intraperitoneally with tumor cells of Ehrlich carcinoma.
The survival of the investigated animals was daily monitored in order to study the effect of different activated water fractions on dynamic and survival indices of tumor-bearing mice. The data of the dependence of changes of life span of tumor-bearing mice for both application regimes and all types of activated water are presented on Fig 3. It shows that the consumption of MRET Activated water substantially increased survival of tumor-bearing animals. The increase of life span was observed in all groups of mice except “therapeutic treatment” group on water activated for 60 minutes. Water activated for 30 minutes (optimal regime of activation) produced the most significant effect on survival of mice with inoculated tumors. The life span of mice which received optimal activated water in “preventive treatment” regime increased by 61%. Significant increase in life span (about 45%) was also observed when mice were treated with MRET activated water (activation time 15 minutes, and 45 minutes) in “preventive treatment” regime. The increase of life span by 43% was observed in “therapeutic treatment” regime for the optimal 30 minutes activated water.
Fig 3: The change of the percentage increase of life span of tumor-bearing mice with ascitic Ehrlich carcinoma which received different types of MRET activated water in “preventive treatment” and “therapeutic treatment” regimes. The digits under the charts correspond with the duration of water activation in minutes.
The similar investigation conducted on mice with inoculated ascitic sarcoma revealed similar tendencies in the efficacy of MRET water as anti-tumor agent with the best results on optimal 30 minutes activated water.
The effect of application of different fractions of MRET activated water on the development of cytotoxic activity of mononuclear lymphocytes extracted from the spleen of mice (NK-cells) is shown on Fig 4. The increase of cytotoxic index in both regimes (21 days and 14 days of application of activated water for mice without tumors) by 26% and 10% respectively was observed only in the groups of mice on MRET water activated for 30 minutes. The cytotoxic index also increased in the group of mice under “old” 30 minutes activated water in “preventive” regime (21 days of MRET water application). No significant changes in cytotoxic index were observed for other water fractions.
Fig 4: The effect of MRET activated water on cytotoxic activity of NK-cells. MRET water was applied for mice without tumors in two regimes (for 21 and 14 days), called “preventive” (1–5) and “therapeutic” (6–10).
The comparative picture of tumor-bearing mice on Non-activated water and on MRET activated water (optimal activation time 30 minutes) is presented on Fig 5.
Fig 5: The appearance of mice from “control” (A) and “preventive treatment” groups (optimal activation time 30 minutes) (Â) on the 18th day after ascitic Ehrlich carcinoma cell inoculation.
Conclusions: The results of investigation of the application of water activated by non-ionizing Molecular Resonance Effect Technology (MRET) process for prevention treatment and enhancement of tumor resistance of animal organisms to two types of oncology diseases in vivo on 500 mice are presented. The research conducted on physical parameters of water confirmed that MRET activation process contributed to substantial modification of the basic physical-molecular properties of distilled water (substantial reduction of viscosity as a function of applied tangent pressure, as well as substantial decrease of electrical conductivity and dielectric permittivity as functions of the frequencies of applied electromagnetic field). The significant positive effect of MRET activated water on tumor resistance of biological organisms was observed in the process of this investigation in all groups of mice on different fractions of activated water. The best results were observed in the groups of mice on MRET water activated for 30 minutes (optimal regime of activation). The 30 minutes activated MRET water inhibited the tumor growth by 52-76% and increased the life span of tumor bearing animals by 43-61%. The results were better in “preventive treatment” regime compare to “therapeutic treatment” regime. Additionally, this investigation confirmed that the long-term preservation of activated water at low temperature (around 0°C) for 45 days decreased its anti-tumor efficacy but left it on the significantly high level compare to other fractions. The discussion and detailed description of the mechanism of “long-term memory effect” of MRET activated water was earlier presented and published in [Vysotskii, Smirnov 2005]. The test results show the dual mechanism of MRET water effect on tumors: the prevention and inhibition of tumor growth together with the reduction of quantity of viable tumor cells. The significant anti-tumor effect of MRET activated distilled water on mice was close to the action of the chemotherapy agents and allowed to avoid the side effects that typically follow chemotherapy treatment in oncology.
In the process of investigation of cytotoxic activity of NK-cells the significant increase of lymphocyte cytotoxicity levels was observed when donor mice were treated with MRET water activated for 30 minutes. The results also showed that the extension of the application of MRET water from 14 days to 21 days significantly increased the value of cytotoxicity index. It is possible to admit that the extension of time of application of MRET water will lead to higher levels of enhancement in NK-cells activity. Thus, the application of MRET activated water can be quite promising approach for non-drug stimulation of NK-cells immunization vaccines.
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