OZONE OXIDATIVE PRECONDITIONING AND ITS INFLUENCE ON CALCIUM HOMEOSTASIS.
O. S. León, N. Merino1, S. Menéndez2, R. Castillo, S. Sam, L. Pérez, E. Cruz, R. López, F. Jouseph, A. Fernández.
Center for Research and Biological Evaluation (Pharmacy and Food Institute of Havana University), Cuba.
1National Center for Scientific Research.
2Ozone 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 preconditioning 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 OZONE AND SUBMITTED TO A HOT ISCHEMIA.
E. Barber, M. O. Barber, S. Menéndez1, N. Merino2, O.S. León3, J. L. Calunga1.
Institute of Basic and Preclinical Sciences Victoria de Girón, Cuba.
1Ozone Research Center, Cuba.
2National Center for Scientific Research, Cuba.
3Center for Research and Biological Evaluation (Pharmacy and food Institute of Havana University), Cuba.

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 Ī 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.

 

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.