Societatea Stiintifica Romana de Oxigen - Ozono - Terapie ( S.S.R.O.O.T.)

Ozonoterapia in oncologie

OZONE THERAPY IN THE ONCOLOGY

(S.P.Aljochina, T.G.Tscherbatyuk “Ozone therapy: clinical and experimental aspects” – Nizhny Novgorod, 2003)

Ozone Therapy of Malignant Tumors

 

  1. Altman (1995) summarizing the gathered experimental and clinical experience of world practice of ozone therapy in his book “Oxygen healing therapies” wrote that ozone has been used in the oncology for about ten years.

The works dedicated to investigations into the action of ozone in malignant tumors were made by the scientists from Germany, Cuba, the USA, Italy, Switzerland and have been recently continued in Russia.

The foreign researches based their attempts at using ozone in the oncology on two discoveries.

The first discovery was made by O. Warburg (1966, Germany) that the key reason for development of tumor is oxygen deficiency on the cell level.

The second discovery made by J. Varro (1974, Germany) showed intolerance of peroxides by tumor cells. In this connection there was made a supposition that ozone and hydrogen peroxide can affect metabolism of cancer cells. However the works of J. Varro as well as the results of earlier investigations made by the Russian scientists A.I. Zhuravleva, B.N. Tarusova (1962) that the content of peroxides in tumors is decreased as compared with normal tissues were not put to use in clinical oncology.

In 1980 F. Sweet et al. furnished proof about a tumor-inhibitory effect of ozone in vitro. The investigations into the influence of certain ozone concentrations on the development of cultures of tumor cells received from malignant tumors of lung, mammary gland and uterus have established the suppression of neoplasma growth in 90% of cases. At the same time, it has been pointed out to a slight ability of tumor cells to compensate the oxidative stress induced by ozone in comparison with normal cells (Sweet, 1980). M. Arnan, L. DeVries (1983) investigated the influence of ozone on carcinoma inoculated to mice. The results of that experiment showed that the animals exposed to ozone lived by 30-40 days longer that the animals of control group.  H. Karlic et al. (1987) established the selective suppression of tumor cell growth of ovarian carcinoma, ovarian adenocarcenoma and endometrial carcinoma. The similar results were demonstrated by K. Zanker and Krozek (1989).

In the conditions of normal cell respiration reactive-inert molecular oxygen converts to active metastable form only through enzyme-substrate complexes of electronic-transport chain of mitochondrions. In cancer cell this enzymatic chain is inactivated and destroyed. The use of ozone results in the formation of active oxygen metabolites that can react with hydrogen substrate and put into action the disturbed end oxidation of tumor cell thereby activating the respiration through the substituted mechanism as the protein-containing enzymes cannot be substituted. Thus, ozone provides an anti-tumor effect (N. Altman, 1995).

As discussed earlier, the growth of induced and inoculated tumors is associated with aggravation of oxygen supply, tissue hypoxia, and the pharmaceutical agents for therapy and prevention of hypoxic conditions in different diseases are mostly focused on the improvement of oxygen transportation to the tissues. In this connection the antihypoxic effect of ozone allows to make a supposition that ozone can be also used as electron-acceptor compound – radiosensibilizator of hypoxic tumor cells. So, in cultures of skin cancer cells through ozone in vitro it came to sensibilization to radiation. Zanker at al. proved a synergetic effect of ozone and chemotherapeutic preparation 5-fluorine-urazila on cell cultures of large intestine cancer and mammary gland cancer.

At the same time, the use of ozone in oncology is restricted as in the most of cases it has been used empirically without rational basis and appropriate methods of control. It is known that the increased concentration of ozone in air contributes to pulmonary toxicity (V. Bocci, 1997). This problem was subject of several works, one of which showed an insignificant statistically unreliable increase in pulmonary tumors in mice of strain A/J that allowed to make a conclusion that ozone is not a carcinogen for this strain (Witschi et al., 1999). At the same time, there are some investigations pointing out to inefficiency of ozone introduced intravenously in case of Erlich’s carcinoma and sarcoma-180 or establishing an increase in pulmonary metastasis in mice with fibrosarcoma NR-FS (Kobayashi et al., 1987). The availability of such contradictory data can be explained by the fact that ozone therapy is dose-depending and requires careful selection of conditions when ozone therapy is reasonable for use.

However, analyzing the mechanisms of the known anti-tumor physical-chemical methods we have formed an opinion that they all are focused on changing oxygen, free-radical homeostasis. From our point of view, ozone therapy (in particular, in the form of ozonated physiological saline) is the most physiological, effective and accessible method allowing influencing free-radical processes.

A very interesting investigation was conducted by the scientists of the Tcherkasskiy Engineering Institute G.S.Stolyarenko, V.N.Vyazovik and the oncological health center M.T.Shaposhnikova, A.V.Gromiko. In the experiments in vitro they found such conditions (selection of ozone solvents, saturation time of solution by oxidant) when it was possible to observe lysis, cell pyknosis, numerous destruction of nuclear substance of tumor cells. The authors pointed out that the observed processes are similar to radiation as in the zone of tumors in liquid phase under exposure to radiation it also comes to synthesis of oxygen-containing oxidants or active oxygen forms. But in case of ozone-radical chemodestruction there is no burn, necrosis of normal cells (along the line of radiation) and apparently no influence due to effect of high-frequency fluctuations. The specialists called this process “mild radiation” (G.S. Stolyarenko, 2001).

Exactly because of this we believe that ozone must occupy its own deserving niche in the complex treatment of malignant tumors.

 

Results of Experimental Investigations

 

Our own investigations dedicated to experimental substantiation of using ozone in the complex treatment of malignant tumors have being conducted in the Central Scientific Research Laboratory of the Nizhny Novgorod Medical Academy since 1994.

We performed a many-stage work on studying the possible use of ozone in neoplasia “in vitro”. The work was made in the Biochemistry Department of the Central Scientific Research Laboratory of the Nizhny Novgorod Medical Academy.

The first stage covered the experiment conducted on the modeled systems of sarcoma-45 (not-metastasizing strain) and Pliss’ lymphosarcoma (metastasizing strain).

For local intensification of oxidative reactions in tumor tissue we used ozone in the form of ozonated physiological saline by methods of intra- and paratumoral administration.

It is important to add that the use of ozonated physiological saline was started just after the transplanted tumor has reached a size of 1 cm in diameter, on the 14th day after inoculation when self-rejection of tumor tissue is already impossible. We believe that this is the important condition to investigate the effect of ozonated physiological saline. From our point of view, the experiment presented at the 2nd International Symposium on Ozone Applications (Cuba, 1997) was conducted incorrectly. The authors made a report that rectal application of ozone is able not only to stop the growth of tumor cell colonies, but also leads to complete resorption of tumor tissues of strain: RL-67, L-1210, LP-388, S-37 (Y. Rodriguez, 1997). However the use of ozone was started after first 24 hours from tumor transplantation when there were no proofs about its transplantability in the organism of animal-recipient. Even if we would ignore this remark and consider that the transplanted cells were vital, the anti-tumor effect of ozone as reported by the authors is the effect in vitro, according to our opinion.

The results of our investigation have shown that the use of ozonated physiological saline providing the increased free-radical environment of tumor initiates peroxidation processes in tumor tissue. Independently from tumor terms that was confirmed by the increased intensity of chemiluminescence shining as well as the increased level of molecular products of lipid peroxidation: diene conjugates, triene conjugates, Schiff’s bases in homogenates of tumor tissue.

Along with initiation of lipid peroxidation in sarcoma cells it comes to a significant decrease in the activity of antioxidant enzymes: superoxiddismutase and catalase.

Probably: the destruction of the antioxidant defense of malignant tumor induced by the local use of ozonated physiological saline facilitates that the tumor is not able to hold back the intensified free-radical oxidation that finally destroys tumor cells.

As a result of the intra- and paratumoral use of ozonated physiological saline (20-day tumor) it comes to a reliable decrease in glycolysis products – pyruvate and lactate in comparison with control. So, in homogenate of sarcoma-45 after the use of ozonated physiological saline the level of pyruvate was 0,041 + 0,001 mcmol/g of tissue that is by 61% (p<0,05) less than in control tumor tissue (0,104 + 0,001 mcmol/g of tissue). And the level of lactate in tumor tissue exposed to ozonated physiological saline decreased by 41% in comparison with control (p<0,05) (from 1,555 + 0,007 mcmol/g of tissue in control group to 0,924 + 0,002 mcmol/g of tissue in experimental group).

The second stage of sarcoma development was characterized by the following features. Ozonated physiological saline introduced directly into the tumor led to a more significant decrease in the level of pyruvate as compared with control – by 83% (p<0,05) (from 0,122 + 0,001 mcmol/g of tissue in control group 2 to 0,021 + 0,001 in experimental group 2). At the same time the level of lactate decreased by 46% (p<0,05).

The biochemical investigations of tumor homogenates on the 40th day after inoculation of sarcoma showed the following changes. In tumor tissue of animals exposed to ozonated physiological saline the level of pyruvate decreased by 83% (p<0,05) as compared with control (0,022 + 0,005 mcmol/g of tissue and 0,131 + 0,001 mcmol/g of tissue, respectively).

The level of lactate in tumor tissue homogenate of animals exposed to ozonated physiological saline decreased by 47% (p<0,05) as compared with animals of control group (0,993 + 0,008 mcmol/g of tissue and 1,877 + 0,005 mcmol/g of tissue, respectively).

The investigation of histological preparations of sarcoma-45 of experimental animals in latent and final stages of oncogenesis has pointed out to the following destructive changes of tumor tissue:

–          a significant (45% and 55,2%) increase in specific volume of necrotized tissue;

–          a decrease in volume density of cells of preserved tissue;

–          well-manifested disturbances of microcirculation.

It is important that the direction of necrotizing in tumor knot is determined by the localization of using ozonated physiological saline. So, in case of subcutaneous injections the area of necrosis is located from subcutaneous tissue along the peripheral zone of tumor knot. When ozonated physiological saline is introduced directly into the tumor, there is necrotization of the central and transitional zones of tumor.

The results of investigation into the ultrastructure of Pliss’ lymphosarcoma cells have shown (see table below) that ozonated physiological saline has a better-manifested effect than the oxygenized one.

 

Table:

 

Manifestation of morphological changes of tumor tissue exposed to oxygenized and ozonated physiological saline.

 

 

Features of differentiation Whole blood Hematoma Edema Dystrophy Necrosis
1 control group + + + + +
2 group with oxygenized physiological saline ++ ++ ++ ++ ++
3 group with ozonated physiological saline +++ ++ +++ +++ ++++

 

The use of oxygenized physiological saline leads to an insignificant increase in necrotic areas established by means of light microscopy of half-thin sections. On some places there were small areas of destruction including large cells with a big hyperchromatic nucleus apparently as a result of therapeutic pathomorphosis. The electronic microscopy did not detect any significant shifts in the ultrastructure of tumor cells.

The effect of ozonated physiological saline with ozone concentration in gas mixture of 3000 mcg/L leads to well-manifested disturbances of lymphosarcoma. The tumor knot with well-manifested invasive growth. There are appearances of whole blood, edema, dystrophic changes, numerous areas of hematomas in stroma. Thus, there are well-manifested disturbances of microcirculation that results in damage of vascular penetrance, integrity of vascular walls. There is an increase in the quantity of necrotic appearances in cell cytoplasma up to destruction of plasmalemma and exit of organelles into the surrounding space.

The well-manifested irreversible disturbances of tumor cell metabolism apparently lead to weakening of the systemic action of malignant tumor on the organism.

That was primarily manifested as restoration of prooxidant-antioxidant balance of the organism- tumor carrier.

The effect of ozonated physiological saline on tumor in the organism of animals results in the suppression of free-radical oxidation that is manifested by decreasing the level of both radical products evaluated according to chemiluminescence activity of blood plasma and molecular products of lipid peroxidation: diene conjugates, triene conjugates, Schiff’s bases. The inhibition of lipid peroxidation is connected with intensification of the organism’s antioxidant capacity. The results of the investigation have shown an increase in the activity of enzymatic components of the organism’s antioxidant defense: superoxiddismutase, glutathion peroxidase and glutathion reductase.

In blood of tumor animals exposed to ozonated physiological saline it comes to an increase in the level of reduced glutathion and a-tocopherol as well as normalization of ceruloplasmin in blood serum (see table below).

 

Table:

 

The level of non-enzymatic antioxidants in the process of oncogenesis after injections of ozonated physiological saline (M+m).

 

Groups of animals sChL, rel. units Ceruloplasmin, mcmol/l a-tocopherol, mcmol/l
Intact 42 + 7 0,36 + 0,04 1,05 + 0,004
Control-1 68,1 + 0,4* 0,45 + 0,12* 0,761 + 0,002*
Control-2 75,1 + 1,7* 0,489 + 0,009* 0,641 + 0,003*
Control-3 92,6 + 10,2* 0,524 + 0,007* 0,51 + 0,004*
Experimental-1 50,2 + 1,9** 0,426 + 0,023** 0,79 + 0,001**
Experimental-2 57,4 + 2,1** 0,439 + 0,014** 0,81 + 0,002**
Experimental-3 47,1 + 0,6** 0,397 + 0,011** 0,93 + 0,001**

 

* – p<0,005 reliable differences with regard to the initial condition (intact animals)

** – p<0,05 reliable differences with regard to the control

 

The destructive effect of ozonated physiological saline on tumor tissue results in the normalization of ion exchange in blood of tumor animals: an increase in the concentration of iron ions and a decrease in the concentration of copper ions. At the same time, it comes to an increase in the level of hemoglobin in blood and OL in blood plasma.

Considering the fact that in some cases of anemia there is not true iron deficiency, but its blockade in the macrophages, it is possible to suppose that the normalization of the concentration of iron and hemoglobin in blood of animals exposed to ozonated physiological saline is induced by iron release from the phagocytes.

It has been established that ozonated physiological saline introduced into the tumor knot leads to gradual normalization of carbohydrate metabolism of the organism-tumor carrier. It has been established an increase in the blood level of glucose testifying to the organism’s exit from the hypoglycemic condition, on the one hand, and a decrease in the level of pyruvate and lactate characteristic of glycolysis inhibition, on the other hand.

It has been established that in spite of development of intensive necrotic tumor processes induced by ozonated physiological saline introduced into the tumor there is a decrease in the level of total intoxication in blood of animals with transplanted tumors

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