[Effect of essential lipophilic nutrients on free radical processes in liver mitochondrial fraction of the tumor-bearing rats].

Oxidative stress is a universal mechanism of cellular damage of hepatocytes, leading to a decrease in the detoxification function of the liver, which is especially important during oncogenesis. An early correction of these mechanisms by lipophilic essential nutrients could increase the effectiveness of antitumor treatment and prevent the development and progress of cancer. Aim to study the effect of separate and combined use of ω-3 PUFA and vitamin D3 on the intensity of free radical processes, mitochondrial swelling and cytochrome c content in the liver mitochondrial fraction of the tumor-bearing rats during the intensive growth of the tumor has been studied. Material and methods. Studies were carried out on white outbred female rats weighing 130-150 g, which were divided into 5 groups (each n=12). Guerin's carcinoma was used as a model of malignant neoplasm. Carcinoma transplantation was carried out by subcutaneous injection of 0.5 ml of a 30% suspension of cancer cells into saline in the upper thigh region of the right limb. ω-3 PUFAs (120 mg/kg of body weight, per os) and vitamin D3 (600 IU/kg of body weight, per os) were pre-administered for 28 days before the transplantation of Guerin's carcinoma and after transplantation for the entire period of tumor growth in the body (14 days). Liver mitochondrial fraction was isolated by differential centrifugation. The intensity of lipid peroxidation was judged by using spectrophotometry by the content of primary, secondary, and tertiary products in isopropanol extracts. The rate of formation of the superoxide radical was recorded in a test with nitro-blue tetrazolium, the swelling of mitochondria was assessed by a decrease in the optical density of isolated mitochondria, the content of cytochrome c in the mitochondrial and cytosolic fractions was determined by multi-wavelength visible light spectroscopy. Results and discussion. An increase in the content of primary (diene and triene conjugates), secondary (ketodienes; conjugated trienes; TBA-active products) and terminal (Schiff bases) lipid peroxidation products with a simultaneous increase in the generation of superoxide anion-radical was found in the liver mitochondrial fraction of the tumorbearing rats. With the administration of ω-3 PUFA and vitamin D3, both separately and especially when used together, a decrease in the intensity of free radical processes in liver mitochondrial fraction of tumor-bearing rats has been observed. At the same time, mitochondrial swelling decreased, this prevented the release of cytochrome c from mitochondria into cytosol. Conclusion. The administration of the complex ω-3 PUFA and vitamin D3 reduces the processes of lipid peroxidation in the mitochondrial fraction of the liver of tumor-bearing rats while simultaneously restoring the functional ability of mitochondria.

[Lipid peroxidation in cardiac mitochondrial fraction of rats exposed to different supplementation with polyunsaturated fatty acids].

The effect of diet supplementation with polyunsaturated fatty acids (PUFAs) used at different ratios of w-6/w-3 was studied on the content of primary (diene conjugates, DC; triene conjugates, TC), secondary (ketodienes, CD; coupled trienes, CT; TBA-active products) and terminal (Schiff bases) lipid peroxidation products (LPO) and generation of superoxide anion-radical in rat heart mitochondrial fraction. It was shown that diet supplementation with high doses of w-6 or w-3 PUFAs increased the content of primary, secondary and terminal LPO in rat heart mitochondrial fraction. Llipid peroxidation was accompanied by the intensification of superoxide anion-radical generation in rat heart mitochondrial fraction. During diet consumption with the PUFAs leading factor affecting the intensity of lipoperoxidation in rat heart mitochondria is fatty acid composition, rather than the level of their saturation.

Hepatotoxicity of bisphenol A under conditions of differential supplementation with retinoids.

Classical xenoestrogenic in vivo effects of bisphenol A (2,2-bis(4-hydroxyphenyl)propane, BPA) are well-described in the literature, however the molecular mechanisms of BPA-induced hepatotoxicity are not fully characterized. The work is aimed to assess biochemical markers of BPA induced hepatotoxicity under conditions of differential supplementation with retinoids. We demonstrate that the absence of hepatic retinyl esters as the main form of vitamin A storage provides for a resistance to BPA induced liver damage. Retinoid supplementation increases the hepatotoxic effects of bisphenol A, evidenced in higher indexes of oxidative damage of lipids, proteins and non-protein thiol groups as well as increase of serum alanine aminotransferase activity and myeloperoxidase activity in liver parenchyma. The absence of hepatotoxicity signs when hepatic retinoid stores are depleted and their presence during normal or excessive retinoid supplementation suggest that hepatic retinoid availability is one of the factors determining the hepatotoxicity of bisphenol A.

Monooxygenase system in Guerin's carcinoma of rats under conditions of ω-3 polyunsaturated fatty acids administration.

The aim of the study was to determine the variations of function in components of monooxygenase system (MOS) of rat Guerin's carcinoma under ω-3 polyunsaturated fatty acids (PUFAs) administration. The activity of Guerin's carcinoma microsomal NADH-cytochrome b5 reductase, the content and the rate of cytochrome b5 oxidation-reduction, the content and the rate of cytochrome Р450 oxidation-reduction have been investigated in rats with tumor under conditions of ω-3 PUFAs administration. ω-3 PUFAs supplementation before and after transplantation of Guerin's carcinoma resulted in the increase of NADH-cytochrome b5 reductase activity and decrease of cytochrome b5 level in the Guerin's carcinoma microsomal fraction in the logarithmic phases of carcinogenesis as compared to the tumor-bearing rats. Increased activity of NADH-cytochrome b5 reductase facilitates higher electron flow in redox-chain of MOS. Under decreased cytochrome b5 levels the electrons are transferred to oxygen, which leads to heightened generation of superoxide (O2•-) in comparison to control. It was shown, that the decrease of cytochrome P450 level in the Guerin's carcinoma microsomal fraction in the logarithmic phases of oncogenesis under ω-3 PUFAs administration may be associated with its transition into an inactive form ­ cytochrome P420. This decrease in cytochrome P450 coincides with increased generation of superoxide by MOS oxygenase chain.

HEPATOPROTECTIVE ACTIVITY OF EXOGENOUS RNA.

Hepatoprotective activity of Nuclex, a pharmaceutical composed of low-molecular yeast RNA, was investigated during acute and chronic thioacetamide-induced hepatotoxicity. It is demonstrated, that Nuclex administration at a dose of 200 mg/kg during acute and chronic liver injury produces hepatoprotective effect, which is associated with decrease in liver parenchyma lesions and in its inflammatory infiltration. Nuclex application attenuates thioacetamide-induced free radical damage of hepatic biopolymers, expressed in the reduction of TBA-reactive products, carbonyl derivatives, and recovery of protein thiol groups and reduced glutathione levels.

[The effect of diet ratio of polyunsaturated fatty acids of omega-3 and omega-6 families on activity of aminotransferases and gamma-glutamyltransferase in rat blood serum].

The effect of diet fat compositions with various ratio of omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) on alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyltransferase (GGT) activities in blood serum of 45 white mongrel rats weighing 90-110 g (9 animals in group) has been investigated. Fat components in the semi-synthetic diet, compiled on the basis of AIN-93 diet, and sources of omega-6 and omega-3 PUFA were presented by sunflower oil, soybean oil and fish oil. It has been shown that four-week inclusion of linoleic acid (LA) and alpha-linolenic acid (alpha-LNA) in a ratio of 7:1 into the diet (soybean oil) as well as use of only omega-6 PUFA (sunflower oil) has lead to an increase in the activity of ALT and GGT in rat blood serum compared to control animals treated with the complex of linolenic, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid through the mixture of sunflower oil and fish oil (9:1) with the ratio of omega-6 and omega-3 PUFA 7:1. Along with this, the AST:ALT ratio (de Ritis ratio) was lower (p < 0.05) as compared with the control group of rat, amounting respectively 0.92 +/- 0.08 and 0.79 +/- 0.12 vs 1.26 +/- 0.10. The use of high doses of omega-3 fatty acids (600 mg EPA and 400 mg DHA per kg of animal weight per day coming through fish oil) did not affect the activity of ALT and GGT, but increased AST serum activity (0.47 +/- 0.04 micromoles/min per mg protein) and the de Ritis ratio (2.53 +/- 0.23). The diet deprived with fat increased enzyme activity of ALT, AST and GGT in rat blood serum.

[The state of the mitochondrial energy-supplying system of blood leukocytes in the dynamics of Guerin's carcinoma growth under the low-level irradiation conditions].

Mitochondrial NADH-dehydrogenase, succinate dehydrogenase and cytochrome oxidase activities of peripheral blood leukocytes of rats with the grafted Guerin's carcinoma were studied in the dynamics of oncogenesis under the conditions of the preliminary low-level irradiation. Tumor growth was accompanied by a decrease in NADH-dehydrogenase activity, an increase of succinate dehydrogenase activity. Cytochrome oxidase activity of leucocytes remained at the control level up to the terminal stages of tumor growth. Preliminary low-level irradiation of the tumor bearing animals caused a tendency to the decrease of enzymatic activities studied. This tendency was observed from the initial stages of oncogenesis.

Effects of low-dose radiation on glutamate dehydrogenase activity in tissues of rats with transplanted Guerin's carcinoma.

Glutamate dehydrogenase activity in mitochondrial fraction of the liver and muscle tissue of tumor-bearing rats was studied in the dynamics of Guerin's carcinoma growth and after preliminary low-dose irradiation. At the initial stages of Guerin's carcinoma growth, maximum activity of glutamate dehydrogenase was observed in the liver, while in the muscle tissue, catabolic transformations of amino acids was activated at the logarithmic phase of carcinogenesis and tended to inhibition at the terminal stages. Low-dose irradiation was followed by activation glutamate dehydrogenase in mitochondrial fraction of the liver and inhibition of this enzyme in mitochondrial fraction of the muscle tissue.

[Functional activity of NADH-dependent reductase system in the liver microsomal fraction and Guerin's carcinoma in rats exposed to preliminary irradiation].

The activity of liver and Guerin's carcinoma microsomal NADH-dependent reductase system has been investigated in tumor-bearing rats exposed to preliminary irradiation. Preliminary irradiation of rats (before transplantation of Guerin's carcinoma) resulted in the decrease of NADH-cytochrome b5 reductase activity and contents of cytochrome b5 in the Guerin's carcinoma microsomal fraction in the logarithmic phases of oncogenesis compared with the non-irradiated tumor-bearing rats. The effect of irradiation preceding transplantation of the tumor to rats results in the increase of enzymatic activities of liver microsomal NADH-cytochrome b5 reductase in the latent and logarithmic phases of tumor growth as compared with non-irradiated tumor-bearing rats. At the same time the contents of cytochrome b5 decreases. During longer periods after irradiation the investigated parameters approached to those in the group of non-irradiated tumor-bearing animals.

[The state of the cytochrome part of respiratory chain in tumor carrier rats' liver in the conditions of preliminary low-level irradiation].

The effect of low-level irradiation on the structural and functional organization of the cytochrome part of the respiratory chain in tumor carrier rats' liver is studied. The preliminary low-level irradiation leading to the mitochondrial cytochrome a, b and c content reduction at the latent stage of Guerin's carcinoma is shown. At the same time, the maximal reduction of the content of all liver cytochromes is observed at the terminal stages of oncogenesis. The content of cytochome c undergoes the most significant changes in the liver mitochondrial fracture. The possible mechanism of mitochondrial haem-containing cytochromes content reduction may be associated with the disorder of their formation caused by the heam synthesis inhibition found in our study. Simultaneously, the cytochrome oxydase (key enzyme of the cytochrome part) activity inhibition is observed to be caused by preliminary low-level irradiation at the latent growth stage of Guerin's carcinoma. The determined differences between irradiated and non-irradiated tumor carrier groups allow us to come to the conclusion that low-level irradiation has an impact only at the initial stages of the aftereffect. At the following stages, the state of the cytochrome part of the respiratory chain is defined by growth conditions of tumor.

[Activity of enzymatic detoxification systems in the mice liver under conditions of different retinoid provision].

The activity of cellular components of liver detoxification system was studied under the conditions of the absence of vitamin A stores. It is shown that a decrease of p-hydroxylase and N-demethylase activity of cytochrome P-450 simultaneously with a decrease of glutathione-S-transferase activity takes place in the liver microsomal fraction of vitamin A-deficient animals. At the same time the absence of retinoid stores in knock-out animals influences the decrease of only p-hydroxylase activity of cytochrome P-450 system. The increase in glutathione-S-transferase activity is observed in the liver postmicrosomal fraction in mice, kept on vitamin A-deficient diet, while its parametres in knock-out group animals were not statistically different compared to the control.

[Cytochrome p450 system components and nitric oxide synthase activity in mouse liver under conditions of retinoid stores absence].

p-Hydroxylase and N-demethylase activities of cytochrome P450 system, NO-synthase activity and the intensity of nitric oxide and superoxide anion production in mitochondrial, postmicrosomal and microsomal cellular fractions were studied in mouse liver under conditions of retinoid stores absence. It is determined, that under conditions of retinoid stores absence the activation of NO-synthase is occurring with decreasedp-hydroxylase activity of cytochrome P450 system. The results of the generation intensity analysis showed the level of NO and O(2-) in liver mitochondrial fraction of knock-out mice, and changes in NADPH-dependent O(2-) production in microsomal fraction of mouse liver cells.

[Generation of superoxide anion-radical in the liver monooxygenase system of preliminary radiation-exposed tumor-bearing rats].

Generation of superoxide anion-radical (O2*-) in reductase and oxygenase electron-transport chains in the liver monooxygenase system was investigated in tumor-bearing rats exposed to preliminary irradiation. Preliminary irradiation of rats (before transplantation of Guerin's carcinoma) resulted in the increased generation of superoxide anion-radical by components of the liver monooxygenase system in the logarithmic phase of oncogenesis. It is shown that the increased NADPH-cytochrome P-450 reductase activity is accompanied with the intensification of superoxide anion-radical generation in liver microsomal fraction of preliminary radiation-exposed rats. At the same time the cytochrome P-450 hydroxylase activity in the microsomal fraction of tumor-bearing rats subjected to preliminary irradiation was decreased.

[The change in the structural and functional organization of the Guerin's carcinoma cytochrome part of respiratory chain in tumor carriers in the conditions of preliminary low-level irradiation].

The effect of low-level irradiation of tumor-bearing rats on the structural and functional organization of the cytochrome part of respiratory chain of mitochondria isolated from Guerin's carcinoma has been investigated. The maximal reduction in the mitochondrial cytochromes a, b and c content was observed at the terminal stage of Guerin's carcinoma. A low-level irradiation during initial stages of oncogenesis produced opposite changes in the mitochondrial cytochrome content. The possible mechanism of mitochondrial haem-containing cytochromes content reduction may be attributed to impairment in their formation caused by inhibition of the key enzyme of haem synthesis, 5-aminolevulinate synthase. The determined changes of the mitochondrial cytochromes quantitative content were accompanied by decreased activity of cytochrome oxidase. The preliminary low-level irradiation of the tumor-bearing animals produced further reduction in the cytochrome oxidase activity observed in all experimental periods.

[Biochemical features of cancer cachexia under conditions of different provision with vitamin A].

On the model of tumor growth of Guerin's carcinoma and experimental modeling of vitamin A provision the biochemical characteristics of tumor cachexia in animals with malignant tumors and different vitamin A provision status were studied. It is determined that tumor growth in the body, deprived of vitamin A, is characterized by negative nitrogen balance, decrease of glucose, free fatty acids and ketone bodies level in the blood serum, which indicates the increase of cancer cachexia.

[Effect of low doses of x-ray irradiation on the liver detoxication system in rats with transplanted Guerin's carcinoma].

The activity of detoxication enzymes in liver microsomal fraction of preliminary radiation-exposed rats was investigated. It was shown that preliminary organism exposure to radiation reduced cytochrome P450 and glutathione-S-transferase activity in liver microsomal fraction in the latent and logarithmic phases of oncogenesis compared with the unirradiated rats with tumor. Low level of cytochrome P450 activity can be caused by transition of microsomal cytochrome P450 in P420 inactive form. The preliminary radiation does not influence the enzyme activity of liver cytochrome P450 and glutathione-S-transferase on terminal stages of Guerin's carcinoma growth.

[Peculiarities of fractional composition of serum glycoproteins in Guerin's carcinoma development under condition of different vitamin A provision].

The most substantial change in the ratio between glycoprotein fractions distributing in the blood serum of rat under conditions of different vitamin A provision (0.50-0.70 micromol/L) are observed; they are characteristic of gamma-globulin, haptoglobin and alpha 1-acid glycoprotein; the intensity of colouring of their bars decreases on serum electrophoregrammes. At the same time, the decline of intensity of bar coloration of ceruloplasmin fraction takes place under the conditions of middle vitamin A deficiency (0.35-0.50 micromol/L) while that of transferrin takes place at marginal A-deficient state (< 0.35 micromol/L). In latent and logarithmic periods of Guerin's carcinoma growth, regardless of conditions of different vitamin A provision, the intensity of bars colouring, on electrophoregrammes, which correspond to immunoglobulin, haptoglobin, ceruloplasmin alpha2-Macroglobulin, alpha 1-acid glycoprotein and alpha1-antytrypsyn, increases. In a stationary period of tumour growth a decrease of coloration intensity of gamma- and alpha2-globulin fractions is observed.

[Xanthine oxidase activity in transplantable Guerin's carcinoma in rats].

Influence of low-dosage pre-irradiation on xanthine oxidase levels in Guerin's carcinoma was studied with particular emphasis on free oxygen generation and free sulphohydrils in a fraction of proteins characterized by xanthine oxidase activity. Enhanced growth of tumor correlated with that of xanthine oxidase activity involving higher levels of enzymatic O-form due to thiol groups oxidation. Under similar conditions, xanthine oxidase and free oxygen generation levels were higher due to SH-group oxidation than in intact animals. Terminal stages involved dropping xanthine oxidase levels and enzymatic protein degradation.

[Histone specific proteinase and DNase activity of spleen lymphocyte nuclei during oncogenesis and under the effect of hydrobromide-5-(5',6' benzocoumaroyl-3')-methylaminouracil].

The features of histone proteolysis, their carbonylation level and endonucleolysis intensity were studied in the spleen lymphocytes of rats with transplanted Guerin's carcinoma and under the conditions of hydrobromide-5-(5',6' benzocoumaroyl-3')-methylaminouracil (BCU) action. The intensification of oxidizing histone destruction and histone-specific protease activity during oncogenesis and under conditions of BCU action was shown. DNase I and DNase II enzymatic activities of spleen lymphocytes of rats with tumour were decreased on different stages of tumour development, however, they were increased under conditions of chemical compound administration.

[Xanthine oxidase activity in the rat liver tissue in the process of oncogenesis].

The work is dedicated to the investigation of xanthine oxidase activity in liver homogenate of rats with tumour at different stages of Guerin carcinoma oncogenesis on the preliminary low-dose irradiation background. Special attention is paid to studying enzyme oxidase form activity, the level of its reactive oxygen species generation and content of thiol groups in protein fraction, which possesses xanthine oxidase activity. The period of Guerin carcinoma active growth is characterized by the increase of total xanthine oxidase activity in the liver. It was found that oxidase activity and the level of O2* generation increase in the preliminary irradiated organism in the period of Guerin carcinoma active growth, as a result of enzyme SH-groups oxidation. At the terminal stages of oncogenesis the decrease of xanthine oxidase activity is accompanied by degradation of the molecule protein part.