International O3 Congress - Oxygen Medicine:

2nd International Symposium in O3 Ozone Applications

O3 Ozone in Biology

Ozone in Medicine - May 24th to 26th 1997

2nd International Symposium in O3 Ozone Applications

O3 Ozone in Biology

Ozone in Medicine - May 24th to 26th 1997

• Ozone in Biology: Ozone Therapy, Basic Immunological Response:

IMMUNE SYSTEM CHANGES IN INFLAMMATORY PROCESS DURING OZONE THERAPY APPLICATIONS.

The aim of this study was to asses the effect of ozone therapy on lymphocyte subpopulations CD3, CD4+, CD8+, leukocyte function CD45, HLA-DR molecule expression in the peripheral blood and the serum levels of IgE in patients with inflammatory diseases. Initial high values in the expression of HLA-DR molecule were significant decreased after endovenous ozone therapy sessions. A tendency to achieve normal values was observed in CD3, CD4+, CD8+ and CD45 parameters.

These results suggest that ozone treatment can be a beneficial alternative therapy in inflammatory process.

IMPROVED LYMPHOCYTE PROLIFERATION RESPONSE IN AIDS AFTER MAJOR AUTO-HEMOTHERAPY WITH O3 OZONE

F. Shallenberger.
USA.

Four subjects with advanced AIDS were treated with a course of ozone therapy administered in the form known as major auto-hemotherapy. This technique involved the withdrawal of 200 mL of heparinized whole blood into a vacuum bottle to which 200 mL of an ozone-oxygen gas mixture at a concentration of 80 gamma (80 g of ozone to 1 mL of oxygen) was added.

The blood was admixed with the ozone gas mixture for 30 second and then promptly reinfused into the patient. The entire procedure took about 40 minutes. Each patient was administered one treatment per day for from 5 to 10 consecutive days.

In order to test the effect that the treatment had on the functional characteristics of CMI, a mitogen response panel was performed both as a baseline and after the treatment period was over. This treatment resulted in an overall increase of the lymphocyte proliferation responses as follows:

1. Response to Con-A increased 41 %.

2. Response to PHA increased 22 %.

3. Response to PWM increased 101 %.

This small study suggest that when properly administered, Major Auto-Hemotherapy (MAH) modulates the inmune response in a direction that can be clinically useful. This outcome can be predicted based upon Bocci's studies on cytokine modulation by MAH.

SOME USEFUL EFFECTS OF O3 OZONIZED OXYGEN IN ANIMAL STUDIES.

S. Schulz, A. Banhofer.
Philipps Universität Marburg, Germany.

Ozone has been the focus of considerable research efforts for several decades. The inhalated ozone dominated the animal studies and some few human studies to look for the potential risks to human health represented by chronic low-level exposure to ozone.

There is fundamental information on the range of biochemical, functional and morphological responses to these low level of ozone exposures to the lungs. In some animal models with induced disease compared to humans we studied the effects of extrapulmonar application and inhalation with ultra-short and high concentrations of ozone.

ON THE INFLUENCE OF O3 OZONATED OXYGEN ON EXPERIMENTAL OSTEOMYELITIS.

S. Schulz, H. Steinhart, R. Mutters.
Philipps Univesität Marburg, Germany.

For hyperbaric oxygen therapy a minimal blood supply is needed, but in chronic osteomyelitis there are usually necrotic infected areas which are not nutrified and therefore not assessable for hyperbaric oxygen therapy.

Ozone is known to be an oxidizing medium with strong bactericide effect. The standard therapy for the treatment of chronic osteomyelitis are antibiotics and surgical procedures. Hyperbaric oxygen therapy seems to be a further useful instrument in refractory cases of chronic osteomyelitis. We investigated the influence of locally applied ozonated oxygen on the progress of chronic osteomyelitis in an experimental model (femur of the rabbit).

Forty rabbits were prepared at the proximal side of the femur and needle was inserted into the intremedullary cavity. Osteomyelitis was induced with Staphyloccus aureus(3x106 CFU/mL) and sodium morrhuate. The needle was left intramedullary. After a latency period of four weeks the animals were randomly divided in control and therapy groups. The infected femur was treated three times a day with 20 mL of ozonated oxygen (ozone concentration 107 µg/mL) over periods of two or four weeks.

Clinical, radiographic and microbiological results were documented. We were able to induce a chronic osteomyelitis in all animals. Ten rabbits were excluded during the investigation, because of loss of 15% of the original weight. Microbiological evaluation showed no sterile femur neither in control group nor in therapy group. In eight femurs from animals of the therapy groups (out of 17) the culture revealed possible polymicrobial infections with concomitant gramnegative species.

Comparison of radiographic results revealed less serious osteomyelitis related bone damages in the therapy groups (p: 0.01). Ozonated oxygen therapy was not able to heal chronic osteomyelitis, but radiographic and clinical evaluations showed significant less severe damages in the therapy groups.

In cases of serious and refractory osteomyelitis in the head and neck region the locally applied therapy with ozonated oxygen may be useful in combination with the standard therapies (antibiotics, surgery, hyperbaric oxygen).

PASSIVE CUTANEOUS ANAPHYLAXIS IN RATS USING SERUM OF MICE PREVIOUSLY TREATED WITH O3 OZONE BY MEANS OF RECTAL INSUFFLATION.

Ozone therapy applications are a very controversial topic. Many papers show this potent oxidant agent as a pollutant related with the incidence of different illnesses (e.g., bronchial asthma, chronic obstructive pulmonary disease, interstitial pulmonary fibrosis). Other papers report therapeutical benefits when the gas is applied by a not harmful way and with a suitable dose.

Based on the passive cutaneous anaphylaxis reaction, a preliminary study about the ozone effect on the anaphylactic reaction is performed. 90 NMRI mice were used, divided into 3 groups of 30 animals each. One milliliter of a mixture of ozone/oxygen, at a concentration of 50 mg/L was applied daily, up to 20 treatments, to the first group, using rectal insufflation. Group 2 received oxygen in the same way and time as group 1, and group 3 received no treatment (positive control group). All animals were sensitized with an ovalbumin solution at the day 5 and 19.

Twenty four hours after finishing the treatments, animals were sacrificed, total blood was collected and the correspondent serum was stored at -70 oC. The serum (0.1 mL) of the different groups was injected at both sides back of Sprague Dawley male rats (that were not submitted to any kind of medication or drug). After 24 h an ovalbumin solution with Evans Blue dye was injected intravenously and the reaction measured one hour later.

Our results indicated statistical significant differences on the anaphylactic reaction based on the rats skin lesion diameters, among ozone (1.2 mm), oxygen (8.6 mm) and positive control groups (12.4 mm).

It is concluded that ozone, applied by rectal insufflation in mice, influenced on the reaginic antibody synthesis reaction.

• Ozone in Biology: Experimental and Scientific Principles:

BIOCHEMICAL MECHANISMS PRESENT IN MEDICAL O3 OZONE APPLICATIONS.

Taking into account that medical ozone applications gather strength, day by day, it is necessary to study the pharmacological actions, as well as the biochemical mechanisms that are involved all around the ozone therapy.

This is the most important objective to fulfill in order to eliminate the strong prejudice for its use. It is presented all the knowledge that exist, international and national ones, in the ozone mechanism of action by means of the different therapeutic properties of this dual molecule.

Ozone, being a powerful oxidant, can offer beneficial actions, when it is applied by a therapeutic way and at controlled doses, producing, for example, an activation of enzymes involved in peroxide or oxygen scavenging (glutathione peroxidase, catalase, superoxide dismutase).

This phenomenom have been demonstrated in different pathologies: Retinitis Pigmentosa, ischemic cardiopathy, arterial insufficiencies, asthma, glaucoma, senile dementia, vascular strokes, etc.). This enzyme activation can induce an acceleration of glycolysis in erythrocytes, with a stimulation of deoxygenating substances (2,3 diphosphoglycerate) with a release of oxygen to tissues.

Ozone therapy can activate the citric acid cycle, with a direct influence on the mitochondrial transport system and also, increase blood fluidity, arterial pO2 and red blood cell pliability. The influence that this therapy exerts in different biochemical parameters, fundamentally in the lipidic profile, demonstrating that ozone can act as a metabolic modulator, as well as in the release of certain eicosanoids is discussed.

Also, the influence that ozone exerts in the immunological system, as an ideal cytokine inducer, as well as the different hypothesis about its germicidal power is been analyzed here. The use of ozonized oils, against bacteriae, virus and fungi is presented, too.

O3 OZONE OXIDATIVE PRE-CONDITIONING AND ITS INFLUENCE ON CALCIUM HOMEOSTASIS.

National Center for Scientific Research.
Ozone Research Center.

It has been demonstrated that oxygen-ozone therapy could be effective in the treatment of different diseases. Nevertheless, its pharmacological action mechanism has not been yet explained.

We start from the hypothesis that controlled ozone administration could be able to promote an oxidative pre-conditioning preventing the hepatocellular damage mediated by free radicals.The oxidative challenge was carried out using carbon tetrachloride (CCl4) as an inductor of free radicals (1 mL/kg CCl4 by intraperitoneal administration of a solution of 10% CCl4 in vegetable oil). 30 adult female Sprague Dawley rats (220-250 g) were used for this study.

The rats were divided in 6 experimental groups: 1, a negative control group treated only with vegetable oil by intraperitoneal route; 2, a positive control group using 1 mL/kg of 10 % CCl4 solution; 3, ozone group, were the rats were submitted, daily, to 15 ozone treatments (dose=1 mg O3/kg of weight), by rectal insufflation and after the last ozone treatment a challenge with CCl4; 4, oxygen group, the same as group 3 but using oxygen instead of ozone; 5, ozone control group, the same as group 3, but without oxidative challenge with CCl4 and group 6, oxygen control group, the same as group 4, but without oxidative challenge with CCl4.

The ozone protective effect against cellular damage, induced by CCl4, was determined through biochemical trials: parameters related with hepatical function (transaminase and cholinesterase), mediators of oxidative stress (Superoxide Dismutase, Catalase, Phospholipase A, Calcium dependent ATPase, reduced Glutathione, Glucose 6 Phosphate Dehydrogenase, Lipid Peroxidation), calcium (free and associated) and histopathological studies.

Between group 1 (negative control) and group 3 (ozone) no significant differences were obtained in the different parameters studied. Group 2 (positive control) and group 4 (oxygen) demonstrated significant differences respect to group 1 and 3, with severe hepatic damages (hepatocellular necrosis, ballonic degeneration, lipidosis, mesenchimal reaction and glucogenic depletion).

The results obtained showed that ozone treatment in the experimental conditions described above, was able to protect the liver against the damage originated by free radicals, regulating the lytic activity of the phospholipase

A dependent of calcium in function of the free levels of this cation, demonstrating a protective effect of calcium dependent ATPase and maintaining calcium homeostasis, hepatocellular integrity and antioxidant endogenous systems, which are responsible for offsetting the damage associated with an oxidative stress.

These results are the first ones in demonstrating ozone pharmacological efficiency against cellular damage originated by free radicals in terms of oxidative preconditioning.

BIOCHEMICAL, MORPHOLOGICAL AND FUNCTIONAL RENAL STUDY IN KIDNEYS OF RATS PRETREATED WITH O3 OZONE AND SUBMITTED TO A HOT ISCHEMIA.

institute of Basic and Preclinical Sciences
Ozone Research Center
National Center for Scientific Research
Center for Research and Biological Evaluation (Pharmacy and food Institute)

It is known that the time of hot and cold ischemia is determinant in the kidney viability and in the transplantation success. After the reimplantation, in a significant proportion of kidneys, an acute tubular necrosis is present and the recipient must required the dialysis.

In literature are referred several papers that study how to increase the quality of the solutions of kidney preservation, as well as, to decrease the oxidative stress produced during the reperfusion of the transplanted organs.

Taking into account that ozone, being a strong oxidizer, can stimulate the cellular antioxidant enzymes, inhibiting the oxidative stress, we decided to study the kidney morphology, the renal function and different mediators of oxidative stress (superoxide dismutase, catalase, phospholipase A, calcium dependent ATPase, reduced glutathione and lipid peroxidation), when rectal ozone is applied before a hot ischemia. White Wistar rats, 250 ± 9 g weight, were divided in four groups.

The first group (control) were anesthetized, using sodium pentobarbital at doses of 30 mg/kg of weight, receiving 50 I.U. of heparin by intraperitoneal injection and after that, a medial abdominal incision was performed for the exposure of the kidneys.

The second group (ischemia) were processed in the same way, but after the kidney exposition they were submitted to a bilateral renal ischemia by arterial clamping during 30 minutes with subsequent reperfusion before the biochemical, morphological and functional renal study.

The third group (ozone-ischemia) received the same procedure as group 2, but the animals were previously treated with daily rectal ozone during 15 days, at doses of 0.5 mg/kg of weight.

The fourth group (oxygen-ischemia) with similar procedure to group 3, but using rectal oxygen instead of ozone.

According to the study of the plasmatic renal flow and the glomerular filtration rate by means of plasmatic clearance of p-amino-hypurate and inulin, respectively, the results demonstrated a significant decrease of the flow and renal filtration in group 2 (ischemia) and 4 (oxygen-ischemia) with respect to group 1 (control) and 3 (ozone-ischemia).

Between group 1 and 3 any statistical differences were obtained, the normal values remained in both groups. In the same way the morphological alterations found (cortical-medullar hemorrhage, mitochondria tumefaction of the tubular epithelium, tubular cells necrosis and convoluted tubules dilatation) were present in the 100 % of the animals in group 2 and 4 and only in 28 % of group 3.

According to the biochemical parameters measured, the ozone protective effect on ischemia-reperfusion damage might be attributed to upregulation of the expression of antioxidant systems.

This ozone preconditioning is able to protect the cells against the free radical injury produced during the ischemia-reperfusion process. This finding suggests novel therapeutic approaches to tissue damage in kidney transplantation.

O3 OZONE IN PROPHYLAXIS OF HYPOXIC CHANGES OF METABOLISM AND MORPHOFUNCTIONAL STATE OF ORGANS IN POST ISCHEMIC PERIOD.

I. Mukhina, L. Snopova, N. Andreyeva, N. Zhemarina, E. Tuvaleva, M. Balandina.
The Medical Academy of Nizhni Novgorod, Russia.

To prevent hypoxic and reperfusion lesion of organs the possibility to use ozonated solutions and ozonated blood in post ischemic period was studied. The trial was based on well-known metabolic effect of ozone low concentrations.

The experiments were carried on non-linear white rats on the model of total cerebral and cardiac ischemia followed by reperfusion in vivo and by isolated cardiac perfusion according to Langerdorff-Fallen.

The use of ozonated blood in 40 minutes was shown to cause specific changes in carbonic metabolism of tissues Sensomotoric part of brain cortex in the control group revealed the prevalence of anaerobic processes of glucose utilization, lactate with reduction of oxidoreductase activity.

The experimental group showed the increase in activity of lactate diminishes with aerobic reorganization of glucose. Redistribution of enzymatic activity was observed between cerebellum glial and nervous cells.

On being ozonated myocardium revealed increase in activity of succinic dehydrogenase with reduced PDG, elevate pyruvate level and lessening of lipid exchange substrates (triglyceride, free fatty acids).

This gives ground to suppose that it is rather free fatty acids that get oxidized in the heart than glucose. The level of adenylic nucletides (including ATP) was found to be elevated both in the brain and in the heart.

It contributed to a quicker normalization of neurologic status in experimental animals, stabilization of EKG and of arterial pressure. Transcapillary exchange in tissues has been improved and diffusion radius diminished. There has been revealed activation of compensatory hepatocides reactions to maintain cellular membrane structures in the liver.

Thus, the received tendency of changes in metabolism, structure and function of the examined organs makes it possible to recommend ozone as a preventive or corrective method in recirculatory pathologies in post ischemic period.

EXPERIMENTAL GROUNDS FOR MEDICAL O3 OZONE USE IN PANIC ATTACKS PREVENTION.

S. Kotov, A. Gustov, C. Kontorschikova.
The Medical Academy of Nizhni Novgorod, Russia.

An animal model was used to study anti-anxiety and vegetotropic characteristics of ozonated physiological solutions (OPS) in parenteral administration. OPS safety and efficiency was studied with the aim to block the development of anxiety in rats. A state of anxiety was induced by intraperitoneal infusions of sodium lactate (600 mg/kg) in 1 mL of ozonated 5 % glucose solution, ozone concentration being 1000 µg/L of ozone/oxygen mixture. The evaluated indexes were divided into 3 groups:

1. Anxiophobic state in rats (the aggregate of behavior reactions according to 9 etiologically adequate tests, based on inducing emotional conditions).

2. Integral indexes of vegetative homeostasis-deep-body temperature, systolic and respiration rate.

3. Biochemical products content in blood and brain of experimental animals (catalase, superoxide dismutase, diene conjugates, Shiff bases).

The results of the experiment showed that 1 mL of OPS infusion made the anxiophobic state less pronounced. OPS anti-anxiety effect was much more significant when used with prophylactic purpose.

Besides, OPS produced a marked vegetotropic effect leveling the shifts of vegetative indexes that were caused by sodium lactate infusion (decrease of deep-body temperature and reduction of systolic rate). Normalization of rats emotional state and vegetative indexes went along with cerebral changes, characterizing the stage of urgent adaptation to stress factor -LP inhibition.

Blood changes registered 2 hours after the start of the experiment revealed activation of free radicals reactions with compensatory (feed-back principle) enforcement of antiradical defense system inspite of discrepancy of biochemical changes in the brain and blood of experimental animals, LP reduction was observed in rats infused with OPS.

The registered tendency was more pronounced in prophylactic use of ozone. The results of the experiment open wide prospects for ozone therapy in prevention and treatment of panic conditions.

THE PHARMAKOCINETIC PROFILE OF OXYGEN-OZONE O3 THERAPY.

H. G. Eberhardt.
Germany.

The administration of oxygen-ozone mixtures is indicated for the treatment of both chronic and acute disease. The "dual" effect of therapeutic intervention with oxygen-ozone mixtures, which has both a local and a systemic impact, is relevant in this context.

The groundwork for the theoretical demonstration of the pharmacodynamic efficiency of ozone-oxygen therapy, a therapy which is aimed specifically at the inmune system, was laid by Rodney Berg. Berg was able to prove in animal studies that the living intestinal contents (i.e. the intestinal flora) are by definition an integral part of the "inmunological cascade".

According to Berg, structural modifications of the bacterial population caused, for example, by the invasion of system-incompatible microbes have negative consequences for the functional integrity of the cellular and humoral defense organs.

Conversely, the complete colonization of the intestinal boundary organ with system-compatible, "autochthonous" strains of coli bacteria creates a sound foundation for an effective inmunological defense.

The autochthonous coli flora exhibit a strong affinity toward oxygen a finding which is of great therapeutic significance. Since this microbe is able to use molecular oxygen as a terminal electron acceptor in energy metabolism, both local and systemic administration of ozone-oxygen mixtures result in a competitive advantage for E. coli during the recolonisation of the intestines with bacteria.

A high population density of this microbe leads, in turn, to an anaerobic milieu in the intestinal lumen, thereby creating the hospitable "climate" which these strictly anaerobic bacteria require to establish stable populations.

These findings constitute objective substantiation of the contribution to be made by oxygen-ozone therapy to restoring the inmunological competence of the human organism.

• Ozone in Biology: Experimental Findings in Ozone Therapy:

CONTROL BIOCHEMICAL PARAMETERS IN O3 OZONE THERAPY.

PROPOSED MECHANISM FOR O3 OZONE THERAPHY IN BRONCHIAL ASTHMA.

Bronchial Asthma is a disease that has gained interest from many countries due mainly to its increasing death rate and its high hospital costs.

In literature, ozone has been recognized as a potent oxidizing agent, capable of producing adverse effects when it is inhaled and it has also been associated with asthmatic attacks. Conversely, it is also known that when ozone is applied to humans in appropriate doses and by a therapeutic way, it produces assorted biological actions with beneficial results without causing adverse reactions or genotoxic damage.

In this case, it is possible to use in a great variety of pathological processes. In some papers ozone therapy is referred as a treatment for bronchial asthma, though the physiological mechanisms are not precisely outlined. It is considered that the airway inflammation is a result of activity from inflammatory stimuli interacting with a numerous quantity of primary effects cells of the respiratory tract. Within this cellular group macrophages, mastocytes, neutrophils, eosinophils, platelets and limphocytes T (CD4) are emphasized.

This cellular group, once activated, can release inflammation mediators that include: histamine, proteolytic enzymes, lipid mediators, cytokines, and reactive oxygen species (ROS), among others. All these mediators can exert different biological effects on the respiratory epithelium and in addition can contribute to the recruitment of new cellular elements with the consequence of the inflammatory process amplification.

In our judgment, the possible therapeutic actions of ozone on bronchial asthma can be based on:

- Its immunomodulating action.

- Its stimulating action on the antioxidant systems.

- Its action on the synthesis and/or release of certain autacoides.

This work intends to explain the ozone mechanism, that avoids airways and that can be effective as a therapeutic method in the treatment of bronchial asthma.

ANTI-TUMOR ACTIVITY OF O3 OZONE. EXPERIMENTAL RESEARCH.

Ozone, alone and combined with other therapeutical practice, has proved to be effective on patients with cancer. It diminishes the negative effects of other therapies, such as chemotherapy and radiotherapy.

Taking into account these properties, the aim of this paper is to carry out a preliminary study about the influence that ozone can exert on tumor growth and also to determine whether it has antimetastatic effect.

To achieve our goal, different treatment models were applied. Laboratory mice BDF1 and MNRI were injected with ozone rectally and intratumorally at doses of 6, 12 and 49 µg. These animals, divided in groups according to the doses they would received, were inoculated with 1x106 and 2x106murine tumor cells TAE, RL-67, S-37, L1210 and L-P388 via intramuscular, intraperitoneal and endovenous treatment at frequencies of 5, 6, 8, 9 and 20 ozone administrations.

Results showed that ozone therapy didn't produced any evidence of tumor growth hastening and also any significant therapeutic effect, with the treatment models applied.

According to the antimetastatic effect, ozone was capable to inhibit significantly the colonization of tumor cells (when these cells were inoculated by endovenous way), but any influence was achieved in the development of metastasis (in this case the ozone treatment didn't covered the period of their growing).

THE INFLUENCE OF O3 OZONIZED OXYGEN ON LUNG TUMOR DEVELOPMENT (MULTIPLICITY AFTER DIFFERENT FORMS OF APPLICATION ON MICE (NMRI).

S. Schulz, M. Wagner.
Philipps Univesität Marburg, Germany.

A pilot-study with urethane (ethyl carbamate) was carried out within the teaching aims (goal: anaesthesia) for medical students to demonstrate the unwanted long-term side-effects (carcino-genity) of this anaesthetic drug. This was achieved in laboratory animal surgery sessions, and the hazard and potential risk for the personnel (Green, 1979, Pöllman & Schulz 1987) was stressed.

Secondly, the possible post-anesthetic influence of ozone/oxygen on mouse tumor development was also demonstrated. Furthermore, we compared the pre-and post-anesthetic influence of ozonized oxygen to extrapulmonar applications on the modulation of lung tumor development.

In one part of the study we could demonstrate a decrease of 77 % of the number of lung tumors after a single ultra-short inhalation with highest doses of ozone, and a 32 % decrease after repeated periods of inhalation with lowered ozone concentrations over a period of 44-55 weeks in this 2 different exposure groups. The results of the extrapulmonar applications are discussed.

O3 OZONE APPLICATION IN NEOPLASIA TREATMENT OF EXPERIMENTAL ANIMALS.

T. Scherbatyuk, C. Kontorschikova.
Medical Academy, Nizhni Novgorod, Russia.

The aim of investigation was to study the ozone influence on some metabolic indexes of experimental animals affected by sarcoma-45. The experimental was done on 140 white male rats. The sarcoma was transplanted subcutaneously in the thing of the animals. 60 rats without ozonetherapy constituted the control group. Ozone therapy was carried out by introduction of ozonized saline solution (beginning 15th day of tumor cells implantation through 30 days) directly into the tumor. By the end of experiment samples of blood and tumor tissue were taken to study ozone effect on glycometabolism; lipid peroxidation (LP) system and phagcytosis activity of polymorphonuclear leukocytes (PAPL). There were determined the amount of glucose, lactate, pyruvate; molecular products of LP: diene and triene conjugates (DC, TC), Shiffbases; ceruloplasmin (Cp), reduced glutation (GSH), superoxide dismutase (SOD), catalase and PAPL.

The received data revealed a valid decrease of lactate, pyruvate in the tumor, wile glucose amount in whole blood was higher compared with the intact. The latter is evidence that ozone abrogated glycopenia.

It should be noted that ozone reliably decrease of SOD, catalase in tumor tissue.

There was a slight rise in DC and TC readings. Blood analysis has shown that ozone valid increase of enzyme activity, Cp, GSH, PAPL but insignificantly change of LP products. According to the data it is possible to suggest that ozone may alter the metabolism in tumor and give carcinolytic effect.

O3 OZONE CORRECTIVE EFFECT ON LIVER METABOLISM INDEXES IN EXPERIMENTAL ANIMALS WITH NEOPLASIA.

C. Kontorschikova, T. Goncharova.
Medical Academy, Nizhni Novgorod, Russia.

The results of the experiments to study ozone effect on phospholipid (PL) composition, lipid peroxidation activity (LP), and energetic state of liver cells in cancerogenesis dynamics are presented. Sarcoma-45 strain was reinoculated to adult white male rats. In two weeks of tumor growth the animals were infused ozonated saline.

The first groups of rats received intraperitoneal infusions (1.5 mL) of ozonated saline for a week. The animals of the second group were intraperitoneally infused during the first week and directly into the tumor during the second week. Every species received 120 µg of ozone.

Control group did not receive any treatment. With the tumor development liver examination revealed valid increase of primary (diene conjugates-DC) and final molecular LP products (Shiff bases-SB) and sphyngomyeline accumulation. It might have caused structural and functional state of cellular membranes and lowered down the energo-genetic processes in liver cells that was revealed in valid decrease of adenylic nucleotides.

Ozone infusions caused a valid decrease of SB level by the end of the first week and brought DC content to normal one by the end of the second week.

PL spectrum of the first group revealed prevalence of lysophosphatidyl choline and sphyngomueline in reduced phosphatidyl choline and phosphatidyl ethanolamine, making activation of oxidative processes evident in membrane.

PL spectrum of the second group came completely to the initial level due to activation of antioxidant systems and phophollipases.

By the end of the second week cellular energetic state was to be normalized. The latter is essential to restore basic liver functions that defend living organism against malignant tumors.

• Ozone in Biology: Posters:

INFLUENCE OF THE O3 OZONATION ON THE DOGS ERYTHROCYTE'S METABOLISM.

Y. V. Veretelnikov, V. D. Conway.
Russian Federation Khanty-Mansi Autonomous District Tyumen.

In the real work had been studied the influence of the difference ozone's concentrations in the isotonic solution of NaCl on the energetical exchanges figures and peroxide oxidation of the lipids in the erythrocytes and in the plasma. It was determined the maintenance of the glutathion, glucose, piruvat, lactat, and activity of glucose-6-phosphate dehydrogenase with an aim of the specifying of the action's ozone mechanism at the blood.

On the basis of the analyses of the biochemical facts is established the factors, which are capable of the irreversible injury of the blood cells, what must be taken into account before ozone's concentrations selection, which is possessing by the therapeutic effect.

THE O3 OZONE INFLUENCE ON DYNAMIC OSCILLATION OF BIOCHEMICAL ERYTHROCYTE INDEXES.

I. Ivanova, C. Kontorschikova.
The Medical Academy of Nizhni Novgorod, Russia.

The influence of ozone on dynamic oscillation of biochemical erythrocyte indexes has been studied. Upon exposure of intact erythrocytes to the ozone/oxygen mixture (in therapeutical dosage of 50 µg/L) both erythrocyte response and adaptation to oxidative stress depended on dynamic status of all system parameters at that time.

The experiments were done on dog erythrocytes in vitro during 60 minutes interval. It has been established that up to 60 minutes after obtaining blood the levels of lipid peroxidation products (LPP) - diene conjugates (DC), triene conjugates (TC), malone dialdehyde (MD) and fluorescent bases (FB) - oscillated with 30 minutes period: DC, TC and FB shared coincident phase that was opposite to the MD and superoxidedismutase oscillation. The free radical process in erythrocyte membrane influences, first of all, the phospholipid contents of membrane regarding both types and amounts of phospholipids.

The lisoform changes in our experiment had the same oscillation regime as the primary and secondary LPP. Phosphatidilcholine (PC) along with phosphatidilethanolamine (PE), on the contrary, oscillated in opposite phase to the LPP. Phosphatidilserine and sphyngomyeline had identical oscillation patterns characterized by opposite phase to PC and PE.

Analysis of glycolisis-mediated ATP formation revealed dynamic changes with 30 minutes period in glucose, ATP, ADP and AMP concentrations that corresponded to the changes of LPP albeit had been variously phase-shifted comparing the latter.

EFFECTS OF EXTRACORPOREAL TREATMENT OF BLOOD BY O3 OZONE AND OXYGEN MIXTURE ON CONDITION OF AEROHEMATIC BARRIER AND ULTRASTRUCTURE OF MYOCARDIUM IN THE EXPERIMENT.

Y. Yakovleva, V. Rogachevsky, O. Rogachevskaya.
The Medical Academy of Nizhni Novgorod, Russia.

Experiments on dogs (p-21) showed that efferent ozone therapy improves pulmonic microcirculation and functional condition of lungs under respiratory-distress syndrome and during posthemorrhagic period (improves aggregate condition of blood, external respiration and metabolism of lungs, reduces edema of aerohematic barrier).

Extracorporeal treatment of blood by ozone and oxygen mixture with ozone concentration of 48 µg/L and exposition of 30 minutes on intact animals (p-7) revealed under electronic and microscopic analysis of pulmonic capillaries free erythrocites with clear-cut membranes or sometimes their microaggregates, isolated neutrophils, some exhibiting degranulation condition in some cases with adhesion to endothelium and symptoms of swelling. The area of interstice featured a significant number of obese cells. Basal membrane and alveolar epithelium mostly remained in usual condition.

Myocarduim showed slight perivascular edema. Cardiomyocites exhibited in most observations slight diffusive lumens of sarcoplasm, swelling and heterogeneity of mitochondria with partial cryst distruction and lumens of matrix. Chromatin was diffused over the nucleus and kernel was clearly detected. Arteriovenous extracorporeal perfusion (control check series) revealed more clear cut changes in minor blood circulation circle vessels - increased content of neutrophils, leukocytic and erythrocytic microaggregates.

In neutrophils adhesion to endothelium areas sites of its lysis were detected as well as slight focal edema of interstitial zone. Myocardium revealed a large number of plasmatic capillaries and hightened pinocytosis of endothelium. Perivascular and internucleus edema exhibited more clearly before extracorporeal treatment of blood.

Cardiomyocytes showed symptoms of hydration - diffusive lumens of sacroplasm and sublimately especially expansion of sarcoplasmatic reticulum cisternae, slight lumen of nucleoplasm. Heterogeneity of mytochondria was accompanied by crysts fragmentation and lumen of matrix was higher than was observed after extracorporeal treatment of blood.

Thus extracorporeal treatment of blood provides for better microcirculation, improvement of aerohematic barrier ultrastructure and cardiomyocytes after extracorporeal perfusion when carrying out diagnostic reviews in early period of disease and blood loss.

O3 OZONE TREATMENT IN MASTITIS, METRITIS AND RETENTION OF FETAL MEMBRANES IN THE COW.

P. Scrollavezza, M. Abblondi1, B. Pogliacomi1, D. Guareschi1, R. Dallaglio2, R. Poldi, G. Pezzoli.
Study University of Parma, Italy.
1DVM, Parma, Italy.
2DM, Parma, Italy.

Considered germicidal, metabolic and inmunomodulant properties of ozone and the standardization of the methodical therapeutics in human medicine, we wanted to study the effects of ozone on common bovine pathologies, particularly mastitis, metritis and the retention of fetal membranes.

Since 1993, we have been treating streptococcal, staphylococcal, coliform, pseudomones, corynebacterium pyogenes and mycotic mastitis with local ozone insufflation associated or not with ozonized autohemotherapy.

The ozone treatment can cure all kinds of mastitis and sterilize the milk that can be shortly after used in the derived processing, butter and cheese. Furthermore, we observed a decrease in the number of the milk leukocytes cells and an increase of milk production in cows treated with ozonized autohemotherapy from 5 to 30 %.

All the kind of metritis and retention of fetal membranes cause by bacteria, virus, fungi and allergic reactions were treated locally with ozone water flushing or with ozonized ovules, associated or not with ozonized autohemothrearapy.

The recovery from metritis and retention of fetal membranes was better and faster than the normal allopathic pharmacological treatment. Authors reports the results of ozone treatment and the administration route in different situations.

HISTHOLOGICAL AND BIOCHEMICAL EFFECTS OF O3 OLEOZON® (OZONIZED OIL) IN A MOUSE TAIL TEST.

Center for Research and Biological Evaluation. Pharmacy and Food Institute
1National Center for Scientific Research.

Psoriasis is a common pathology distinguished by an increase in the keratinocites growth. There are abnormal differentiations and alterations on the antioxidant defense system.

On the other hand ozone/oxygen therapy causes an "oxidative preconditioning" with gives protection against free radical damage.

A protocol with "Mouse tail test" has been developed using 4 groups: OLEOZON® 1500 mg/kg b.w. Sunflower oil 3 mL/kg b.w. and 2 negative control groups both without medication, but one of them with manipulation simulating the stress of the test. At the end of the experiment (30 daily applications) the tail was removed and sections were processed histhopatologically and biochemically.

The main result indicated a significant increase (p<0.05) in the orthokeratosis percentage in the Oleozon and Sunflower Oil treatment groups with respect to negative controls, but this effect is not greater than that produced by the traditional drug.

The biochemical finding aims at an important role of calcium (decrease it's intracellular level p<0.05 in Oleozon and Sunflower Oil group with respect to negative controls) and SOD (increase it's enzymatic activity p<0.05 in OLEOZON®group with respect to the other groups, demostrating a protection action of Oleozon) in the action mechanism.

DERMAL ACUTE TOXICOLOGY OF OLEOZON® (O3 OZONIZED OIL) IN RATS.

Center for Research and Biological Evaluation. Pharmacy and Food Institute.
Pharmacal Biological Laboratory.
National Center for Scientific Research.

"OLEOZON®" (Ozonized Sunflower Oil) is a topical medicine with recognized therapeutical efficiency as a germicide. This drug has been evaluated on an acute dermic toxicity assay on rats skin and as a requirement to be registered as a medicine for human use. Groups of animals were formed by 8, each of them according to sex and type of treatment. The

y were observed for 14 days. During the treatment it was taken into consideration different parameters to know the presence of retarded toxicity signs. Also, studies were carried out in anathomopathology, hemathology and biochemistry; the last one was directed to know the local or systemic alterations on the oxidative reaction system.

The results proved that the doses of 2000 mg/kg body weight, by dermic way was not lethal. After the application of "OLEOZON®" toxic signs (erithema and edema) not appeared with severity. The systemic effect are not detected. The biochemical and anatomopathological studies confirmed these results.

STRUCTURAL AND ULTRA-STRUCTURAL MORPHOLOGICAL STUDY OF DIFFERENT ORGANS OF MICE TREATED BY RECTAL O3 OZONE.

Ozone Research Center.
Medical and Surgical Research Center.
National Center for Scientific Research.

Ozone is recognized in frequent publications as an ubiquitous air pollutant with powerful oxidizing capacity. Higher concentrations of inhaled ozone are sufficient to cause adverse effects to humans and animals producing histological lung damages, just as cellular and epithelial damages, pleural adenomas, hyperplasia, metaplasia and toxicological effects as DNA damage.

Also, are present, airway inflammation with measurable transient changes in lung function and bronchoconstriction. Increasing neutrophil infiltration and release of inflammatory mediators (as leukotriens), cytokines (as interleukin 8) and chemoattraction were observed. On the other hand, some controversial results have been obtained using inhaled ozone.

For example, inhaled ozone in low doses, produced in NMRI-mice after urethane treatment, a protection against lung tumor multiplicity.

Also, an adaptation syndrome has been shown in the prevention of mortality with lethal ozone doses after pre-exposures to low level with intermittent ozone exposures.

In clinical practice, it has been demonstrated the therapeutic effects of ozone therapy, when it is applied avoiding the airways and at appropriate doses.

The aim of this paper is to study the pulmonary and rectal histology (using optical and electronic microscopy) of mice previously treated with rectal ozone (20 sessions, in doses equivalent to therapeutic doses used in humans), comparing with a control group without ozone.

The histopathology study, at pulmonary level, demonstrate focal atelectasia, focal hemorrhage, as well as edema and focal congestion with a homogeneous distribution in both group of animals.

This alterations were related to the technique procedure employed for the obtainment of the fragments. Any of the histological damages described in the literature, when inhaled ozone is used, were detected. At rectal level, any morphological changes were observed.

THE INFLUENCE OF DIFFERENT O3 OZONE DOSES IN THE OXIDATIVE PRECONDITIONING.

Center for Research and Biological Evaluation. Pharmacy and Food Institute.
National Center for Scientific Research.
Ozone Research Center.

It has been demonstrated that controlled ozone administration could be able to promote an oxidative preconditioning, preventing the hepatocellular damage mediated by free radicals. The aim of this study is to evaluate the influence of different ozone doses on the oxidative preconditioning. The oxidative challenge was carried out using carbon tetrachloride (CCl4) as an inductor of free radicals (1 mL/kg CCl4 by intraperitoneal way of a solution of 10 % CCl4 in vegetable oil). 50 adult female Sprague Dawley rats (220-250 g) were used for this study.

The rats were divided in 5 experimental groups: 1, a negative control group treated only with vegetable oil by intraperitoneal route; 2, a positive control group using 1 mL/kg of 10 % CCl4 solution; 3, 4 and 5 ozone groups, were the rats were submitted, daily, to 15 ozone treatments at doses of 0.5, 1 and 2 mg O3/kg of weight, respectively, by rectal insufflation and after the last ozone treatment a challenge with CCl4.

The ozone protective effect against cellular damage, induced by CCl4, was determined through different mediators of oxidative stress (Superoxide Dismutase, Catalase, Phospholipase A and Lipid Peroxidation) and histopathological studies.

The results demonstrated that the mechanism of protection against de cellular damage, mediated by CCl4, were dependent of the different ozone doses used in this experimental model.

The behavior of the biochemical parameters measured were dependent of the ozone doses, demonstrating their functional relationship. An ozone dependent oxide-reduction factor, which well correlates with the biochemical parameters, was found.

Theoretically, an ozone minimum effective dose, equals to 1.52 mg/kg, was estimated. The histopathological results, according to lipidosis and glucogenic depletion are in correspondence with the behavior of the biochemical parameters measured and the ozone dependent oxide-reduction factor.

THE ACTION OF O3 OZONIZED OIL ON THE HEALING PROCESS OF SKIN WOUNDS AND NORMAL SKIN IN LAB ANIMALS.

Faculty of Medicine. Holguín.

The simple comprised sixty adult male isogenic mice, line balc-c weighting between 25 and 30 g. A 1 cm perforation, with loss of tissues, was performed in the central area of the auricular chamber of each animal. The animals were divided into two groups of 30 animals each.

Control group: sunflower oil was applied in the wound daily, for 7 days.

Ozone group: ozonized oil was applied daily in the wound for 7 days .

All cures were performed twice a day. Three animals of each group were killed within the next 24 hours, 2, 3, 5, 7, 10, 14, 21, 28 and 32 days. Measurements of the diameter, superficial area and wound perimeter were taken in each animal, once they were killed, using stereoscopic microscopes and the morphometric system, as well as computing COMSDI-PLUS.

The samples were processed later with paraffin technique. Serial longitudinal cuts of the pound of the right auricular chamber were performed with an approximate thickness of 7 or 8 micrometers. The coloring techniques used were hematoxilline and eosin. A qualitative study of the hystological samples was made assessing the development of the phases of the healing process.

The results showed that the measurements taken in the wounds had lower values in the ozone group. The used of ozonized oil for the cure of local skin wounds does not modify the general sequence of the phenomenon that takes place during the healing process.

HISTOPATHOLOGICAL EVALUATION OF THE INNER EAR OF GUINEA PIGS AFTER OTOTOXIC DRUG DAMAGE FOLLOWED BY O3 OZONE TREATMENT.

National Center for Scientific Research.
Cira García Central Clinic.
Ozone Research Center.

The histopathological changes in the inner ear of guinea pigs after ototoxic damage with streptomycin followed treatment with ozone or oxygen was examined. In this study celoidin and paraffin methods were employed.

The group damage with 300 mg/kg streptomycin and treated with ozone showed complete loss of the cells of the organ of Corti, of the cells of the limbus spirals and atrophy of the stria vascular.

On the other hand the groups damaged with 100 and 200 mg/kg streptomycin and treated with ozone showed a less extent of damage in comparison with the former group.

In fact, these two groups showed no morphological differences with similar groups but treated with oxygen. These findings may provide a morphological basis for the hypothesis that inner ear damage could be in part reversible under specific treatments with ozone.

THE ERYTHROCYTE GST AND ITS RELATION WITH THE ENDOVENOUS O3 OZONE THERAPY.

Ozone Research Center.

Ozone therapy is an alternative therapy of recent application which has turned out to be efficacious for the treatment of several pathological processes, it needs the knowledge of biochemical markers which can be used in the safe control in its application and follow up.

Since it is known that Glutathione S - transferase (GST) is a family of isoenzymes involved in the metabolic initiation of almost all the products of ozonization of biomolecules and that ozone exerts a stimulating effect on the antioxidant enzymes of the body, the possibility of erythrocyte GST as a biomarker in ozone therapy was studied. In vitro studies were carried out with ozone concentrations between 0.4 and 98.4 mg of ozone gas per milliliter of blood. Direct effect of ozone gas increased the enzyme activity at concentrations under 2 mg/L and at higher one was inhibited.

However, derivated products from ozonized blood increased the erythrocyte GST activity in a concentration dependent manner until 71 mg/L of ozone concentration in blood. In vivo studies were carried out with humans of both sexes and ages between 15 and 50 years. GST activity was measured at 10 and 40 mg/L ozone concentrations after 15 autohemotherapy sessions. GST activity was compared with erythrocyte glutathione peroxidase activity (GPx), which is a known antioxidant biomarker measured in the same blood sample.

Patient subgroup with basal activity values below population reference range showed a remarkable increase of GST and GPx with both ozone concentrations used.

On the other hand, patient subgroup with basal activity values upper the population reference range presented a decrease in the activity of both enzymes reaching approximately the normal values. Although GST showed the same behavior of GPx, the GST activity was more sensitive than GPx activity during ozone therapy.

The results of this paper evidenced that erythrocyte GST can be used as biomarker of endovenous ozone therapy.

EFFECTIVENESS OF O3 OZONIZED CACAO BUTTER FOR THE TREATMENT OF VAGINAL CANDIDOSIS.

Ozone Research Center.
Center for Research and Biological Evaluation. Pharmacy and Food Institute.
Pedro Kourí Institute.
National Center for Scientific Research.

Candida vaginitis continues to be a prevalent disease with a world-wide distribution.

Some ozonized substances as: sunflower oil and cacao butter have shown antimicrobial effects against Candida albicans, Candida tropicalis and other fungi.

Lately several experimental models of candidosis in mice and rats have been developed. The purpose of this study was to determine the effectiveness of ozonized cacao butter to treat vaginal candidosis.

Candida vaginitis in Sprague-Dawley rats was developed with an inoculation of 106. Candida albicans 3153 in 0.1 mL of PBS. Six groups of infected animals were studied: three different doses of ozonized cacao butter ovules and three control groups; one was treated with ketoconazol ovules, other with untreated cacao butter ovules and last one without treatment.

The experimental model used in this paper allowed to develop vaginal candidosis in 80 % of inoculated rats; it made possible to study the effectiveness of ozonized cacao butter for the treatment of Candida vaginitis.

IN VITRO ACTIVITY OF O3 OLEOZON® AGAINST BACTERIAL AGENTS OF SKIN INFECTION.

Ozone Research Center.
1National Center for Scientific Research.

Pyogenic skin infection are produced in 90 % of cases by Staphylococcus aureus and Streptococcus pyogenes; Pseudomonas aeruginosa and Escherichia coli can participate as secondary agents. OLEOZON® has antimicrobial effects against different microorganisms.

In the present study, the antibacterial activity of OLEOZON® againstStaphylococcus aureus,lStreptococcus pyogenes, Pseudomonas aeruginosa and Escherichia coli clinical isolates andStaphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 was examined.

Susceptibility tests, Minimal Inhibitory Concentration and Minimal Bactericidal Concentration, were performed by agar dilution and macrodilution techniques based in NCCLS 1992, 1993; as well as Bioactivity tests by microcalorimetry technique were studied.

The results showed the potent antimicrobial activity of OLEOZON® against the bacterial strains analyzed in this study. It made possible to recommend other experiments in order to establish the effective therapeutics doses.

OLEOZON® (O3 OZONIZED SUNFLOWER OIL) GENOTOXIC EVALUATION USING MICRONUCLEUS ASSAYS IN BONE MARROW AND PERIPHERAL BLOOD OF MICE.

National Center for Production Animals of Laboratory.
Ozone Research Center.

It was accomplished the study of the OLEOZON® genotoxic activity through the micronucleus assay in bone marrow and in peripheral blood, using Cenp: NMRI mice of both genders. these assays provides an indirect measure of the induction of structural or numerical chromosome aberrations.

The evaluation was carried out through analysis and comparison of micronucleus frequencies in peripheral blood reticulocytes and polychromatic erytrocytes in bone marrow of an experimental group of 10 mice treated with ozonized sunflower oil and a negative and positive control group of 10 mice treated with sunflower oil and cyclophosphamide (40 mg/kg of corporal weight), respectively.

They were accomplished 5 consecutive administrations by oral gavage, with intervals of 24 hours of a limit dose (2 g/kg/day), based on no evidence of toxicity in subchronical studies by oral gavage with this product.

No evidences of toxic effects in the erytrocyic population studied were obtained as well as no cittotoxity and clastogenicity inducement (chromosome aberrations inducement). were achieved.

SUBCHRONIC DERMAL TOXICITY ASSAY OF SUNFLOWER OZONIZED OIL (OLEOZON®) IN CENP: SPRD (SPRAGUE DAWLEY) RATS.

National Center for Production Animals of Laboratory.
Ozone Research Center.

OLEOZON® is one of the most important product used in Ozone therapeutical applications particularly as external antimicrobial agent. For safety testing of OLEOZON®. A subchronic dermal toxicity studied was carry out in Cenp:SPRD (Sprague Dawley) rats was developed at CETEX. To fulfill the requirement for sanitary registration.

The test substance was applied on the dorsal shaved skin of the rats daily during 13 weeks, 6 days a week, to 6 groups (10 of each sex per group) of healthy young adult animals at three dose levels. Groups were arranged as follows: Group 1: Vehicle control (just sunflower oil) Group 2: lowest level doses (10 mg/kg b.w) Group 3: intermediate level doses (100 mg/kg b.w) Group 4: highest level doses (200 mg/kg b.w) Group 5: satellite group schedule for follow up observations Group 6: sentinel group for integrals observations.

Clinical observations, haematological and blood chemistry tests, and histophatological examinations including electron microscopy observations were performed. It was observed a proper growth in all groups as well as an adequate food and water consumption. There were changes in some haematological and blood chemistry parameters, some of them statistically different in specific statistical tests.

This differences may be associated to physiological variations due to strain, age and sex and they do not seem associated to OLEOZON® treatment. The variations rank within the established limits of the 2 SD related to the mean value.

Histophatological changes of low intensity were found in the groups of rats treated and untreated. Other finding and results are fully discuss in this report.

Source: Ozone Research Center