Vitamin D 3 contribution to the regulation of oxiDatiVe metabolism in the liVer of Diabetic mice

this work is devoted to the study of the features of oxidative metabolism of hepatocytes in diabetic mice and those under the vitamin D3 action. We found out that a 2.5-fold decrease of 25OhD3 content in the serum was caused by chronic hyperglycemia in diabetes. Intensification of the reactive oxygen species (ROS) and nitrogen monoxide (NO) production, protein oxidative modifications (detected by the contents of carbonyl groups and 3-nitrotyrosine), accumulation of diene conjugates and tBa-reactive products of lipid peroxidation, and the decreased level of free Sh-groups of low molecular weight compounds in the liver were accompanied by development of vitamin D3 deficient state. It was shown that there was a decrease in the key antioxidant enzymes activity (catalase, SOD), while the activity of prooxidant enzymes NaD(P)h:quinone oxidoreductase, xanthine oxidase and NAD(P)H oxidase was increased. The identified oxidative metabolism lesions caused the elevation of the hepatocytes necrotic death that was tested for the ability of their nuclei to accumulate propidium iodide. Prolonged vitamin D3 administration (during 2 months) at a dose of 20 IU to diabetic mice helps to reduce the ROS formation and biomacromolecules oxidative damage, normalizes the antioxidant system state in the liver and increases survival of hepatocytes. the results suggest that vitamin D3 is a key player in the regulation of the oxidative metabolism in diabetes.

this work is devoted to the study of the features of oxidative metabolism of hepatocytes in diabetic mice and those under the vitamin D 3 action.We found out that a 2.5-fold decrease of 25OhD 3 content in the serum was caused by chronic hyperglycemia in diabetes.Intensification of the reactive oxygen species (ROS) and nitrogen monoxide (NO) production, protein oxidative modifications (detected by the contents of carbonyl groups and 3-nitrotyrosine), accumulation of diene conjugates and tBa-reactive products of lipid peroxidation, and the decreased level of free Sh-groups of low molecular weight compounds in the liver were accompanied by development of vitamin D 3 deficient state.It was shown that there was a decrease in the key antioxidant enzymes activity (catalase, SOD), while the activity of prooxidant enzymes NaD(P)h:quinone oxidoreductase, xanthine oxidase and NAD(P)H oxidase was increased.The identified oxidative metabolism lesions caused the elevation of the hepatocytes necrotic death that was tested for the ability of their nuclei to accumulate propidium iodide.Prolonged vitamin D 3 administration (during 2 months) at a dose of 20 IU to diabetic mice helps to reduce the ROS formation and biomacromolecules oxidative damage, normalizes the antioxidant system state in the liver and increases survival of hepatocytes.the results suggest that vitamin D 3 is a key player in the regulation of the oxidative metabolism in diabetes.k e y w o r d s: vitamin D 3 , 25OhD 3 , oxidative stress, antioxidant protection, experimental type 1 diabetes.I nsufficiency of insulin production by β-cells of the pancreatic Langerhans islets caused by their destruction, which develops under the influence of environmental factors (viral infections, stress, certain nutritional components) and in the presence of genetic background (genetic mutations, autoimmune processes) underlies pathogenetic development of insulin-dependent diabetes [1].The primary reason in the development of diabetes pathology is a hyperglycemia.When the glucose level is chronically elevated an excessive non-enzymatic glycosylation (glycation) of proteins that form cytotoxic advanced glication end products (AGEs) of protein-lipid complexes glycoxidation is observed.These AGEs can interact with appropriate receptors (RAGE) on the cell membrane and this leads to the intensification of prooxidant processes and increased synthesis of proinflammatory cytokines [2].The development of oxidative-nitrosative stress (ONS) under diabetes increases metabolism and endocrine disorders caused by pathological changes in the insulin secretion, pathoimmune reactions and depletion of antioxidant reserves [3].ONS is a universal non-specific mechanism of numerous morphological changes in the body.The antioxidant defense system of the cell gets damaged one of the first in the organism.
For a normal organism functional activity freeradical processes are maintained at a steady level that is ensured by the balance of the intracellular reactive oxygen species (ROS) formation rate and functioning of a complex tissue-specific system of radical traps and biomacromolecules peroxidation inhibitors.Permanent formation of prooxidants is balanced by their enzymatic and non-enzymatic detoxification by antioxidants [4].Activity of the main enzymes that generate reactive oxygen and nitrogen (specific -NAD(P)H oxidase and NO-synthase, unspecific -NAD(P)H:quinones-oxidoreductase and xanthine oxidase) is significantly changed in diabetes [5].
At the molecular level, the pathological effects of excess ROS content appear as compartment-dependent changes in cell redox status and oxidative modifications of biomacromolecules (protein carbonyl level, lipid peroxidation, mutations in nucleic acids).Accumulation of nitrosylated and poly(ADP) ribosylated proteins in different cell types indicates biochemical disorders in diabetes [6].A key role in the intensification of nitrosylation and poly(ADP) ribosylation belongs to the activation of poly(ADP) ribosopolymerase (PARP), an enzyme whose main physiological function is the NAD-dependent reparation of single-stranded DNA breaks (SSBs) [7].ONS plays a key role in the development of diabetic complications such as cardiovascular, microvascular, neuropathic, immune etc. [8].
Besides a well-known role of vitamin D 3 in the regulation of calcium and phosphorus metabolism and involvement of cholecalciferol in the bone remodeling process, it is also known that vitamin D 3 exhibits immunomodulatory, anti-inflammatory, anti-proliferative effects and is able to prevent transformation of tumor cells [9].Molecular mechanisms of vitamin D 3 cytoprotective properties can be implemented both through genomic regulation which largely corresponds to the steroid hormone-like action and non-genomic effects, including its regulatory effect on the protein expression, cellular metabolism, inflammation and oxidative stress [10].The present studies examine the effects of vitamin D 3 in cells as a biological antioxidant, consider its action mechanism in the context of structural similarity with cholesterol and ergosterol.Thus, it is associated with the ability of cholecalciferol to directly modify the physicochemical state of biological membranes [11].In addition, the antioxidant activity of vitamin D 3 can be provided through its hormonally active forms on the genes expression level and regulation of pro-/antioxidant enzyme activity.
The aim of this work was to determine the vitamin D 3 role in the regulation of reactive oxygen species formation, processes of biomacromolecules oxidative modification, state of enzymatic antioxidant defense system in the mice liver and hepatocytes survival in experimental diabetes.

materials and methods
Studies were conducted on male mice C56Bl/J6 weighing 21 ± 3 g.Type 1 diabetes was caused with 5-fold administration of streptozotocin (STZ, Sigma-Aldrich, USA) at a dose of 40 mg/kg body weight of the animal.This type of administering STZ is commonly used for the induction of experimental autoimmune type 1 diabetes in mice.In this study the animals after 6 weeks of diabetes with blood glucose 20.4 ± 4.3 mmol/L were used.After development of stable hyperglycemia mice were injected with vita-min D 3 (DSM, the Netherlands) for 2.5 months in an aqueous suspension form (800 IU/kg body weight, per os).Control animals were kept on a complete diet in the vivarium.For the adaptation period (one week) and during the experiment animals were kept in the vivarium at a temperature of 18-22 °С, humidity 50-60%, natural light mode "day-night" in standard plastic cages with free access to their food and water and in compliance with General ethical Principles for experiments on animals (Ukraine, 2001).Mice were decapitated, using diethyl ether for anesthesia.Selection of animals and the formation of groups were performed by the method of random numbers [12].
We evaluated vitamin D 3 availability in mice by the level of serum 25OHD 3 , which was determined by immunoenzyme technique (ELISA kit, Immunodiagnostic Systems Ltd., USA).This method is based on the competitive binding of serum 25OHD 3 and 25OHD 3 -biotin with vitamin D 3 binding protein (VDBP), immobilized on immunological 96-well plate.
Hepatocytes were isolated after liver perfusion with phosphate-saline buffer through the portal vein.The rinsed liver was cut into thin slices, which were incubated with collagenase at 37 °C within an hour, followed by washing with phosphatesaline buffer containing 0.146 M NaCl, 5.4 mM KCl, 0.8 mM MgSO 4 •7H 2 O, 2 mM CaCl 2 , 0.7 mM Na 2 HPO 4 •12H 2 O, 0.7 mM KH 2 PO 4 , 1% albumin, pH 7.4 [13].The number of cells was counted in a Goryaev cell chamber.
Fluorescent determination of the reactive oxygen and nitrogen content were performed with DCFH-DA (2′,7′-Dichlorodihydro fluorescein diacetate) [14].The amount of 1 µl of DCFH-DA solution (2.5 mM) was added to 100 ml of mice isolated hepatocytes suspension (0.5•10 6 cells) and incubated for 30 min at 37 °C in the dark.Samples were centrifuged at 300 g for 10 minutes and washed 2 times with a PBS solution (pH 7.4), and the final precipitate of hepatocytes was resuspended in 0.5 ml of PBS.Right after preparation, the samples were analyzed using flow cytometer COULTER® EPICSTM XLTM.The fluorescence of the DCF-DA oxidized form (Dichlorodihydro fluorescein diacetate) was measured at λ ex = 488 and λ em = 540 nm.
Fluorescent determination of NO content was carried out with DAF-FM DA (4-amino-5methylamino-2′,7′-difluorofluorescein diacetate) [15].DAF-FM (1.5 mM) in the amount of 1 µl was added екСпериментальні роботи to 100 µl of the suspension of isolated mice hepatocytes (0.5•10 6 cells) and incubated for 30 minutes at 37 °C in the dark and then resuspended in 0.5 ml PBS.Subsequently, the samples were analyzed using flow cytometry (COULTER® EPICSTM XLTM).The fluorescence of oxidized forms of DAF-FM DA was measured at λ ex = 488 and λ em = 540 nm.
The level of hepatocyte death (necrosis) was estimated using flow cytometry by the number of cells that could accumulate propidium iodide [16].
The level of oxidative modification of proteins (OMP) was evaluated by the content of carbonyl derivatives of proteins (ketone-2,4-dinitro-phenylhydrazone) in the presence of electron donors and metals with variable valences, especially copper or iron ions.Changes in absorbance were recorded using automatic microspectrophotometer μQuant (Biotek , USA) [17].Determination of lipid peroxidation products was carried out by reaction with thiobarbituric acid [18], and the content of low molecular weight compounds free SH-groups -by reaction with ortho-phthalaldehyde [19] in the liver homogenates.
The activity of xanthine oxidase (EC 1.17.3.2) was determined in the reaction of ethanol oxidation by hydrogen peroxide (which was formed due to substrate hypoxanthine oxidation by xanthine oxidase) into acetaldehyde, which was cleaved by NAD-dependent aldehyde dehydrogenase.Reduction of coenzyme NAD + was detected with spectrophotometry (λ = 334 nm) [24].
The activity of NAD(P)H:oxidase (EC 1.6.3.1) was determined in liver homogenates spectrophotometrically by the rate of NADPH to NADP + oxidation in parallel samples without addition and with the addition of NAD(P)H oxidase specific inhibitorapocynin (10 mM) [25].
The level of nitrozylated proteins was determined by Western blotting [26].The intensity of the signals on X-ray film was evaluated using GelPro32 [27].
Statistical analysis of the data was performed using the computer program (Microsoft Excel).Results were presented as means of 6 independent determinations ± standard error.The assessment of statistical significance of distinction averages were performed using a standard Student t-test for noncorrelated samples [12].

results and Discussion
The most objective and informative measure of vitamin D 3 availability is 25OHD 3 content in serum, which is in the range of 100-150 nmol/L (40-100 ng/mL) in human organisms.Reduction of serum 25OHD 3 level below 75 nmol/L shows the development of D 3 -hypovitaminosis [28].The results shown in Table 1 indicate that in terms of diabetes the level of serum 25OHD 3 decreases to 2.52 times as compared to control animals, reflecting a lack of vitamin D 3 availability and inhibition of its biologically active hydroxylated forms synthesis.The serum level of glucose in the animals with diabetes t a b l e 1. Content of 25OhD 3

and glucose in serum of mice in experimental diabetes and with the vitamin D 3 administration (M ± m, n = 6)
Here and in Table 1-3 *the difference compared with the control is reliable (P < 0.05); # the difference compared with the group "Diabetes" is reliable (P < 0.05)

Experimental groups
Content of 25OHD significantly increased to 20.4 ± 4.3 mmol/L compared to control -5.2 ± 1.1 mmol/L.Production of free radicals, their neutralization and free radical oxidation are normal physiological processes that ensure the regulation of cellular activity and there is a balanced equilibrium among them.However, overproduction of ROS and accumulation of lipid peroxidation products (LPO) under pathological conditions can lead to inactivation of cytosolic and membrane-bound regulatory proteins, alterations of their physicochemical properties, biological membrane permeability and functional genome integrity [29].
Pathologies that are accompanied with the tissue hypoxia development (including diabetes) are characterized by a significant imbalance between the rate of free radical species production and antioxidant enzyme activity (AOP), resulting in the accumulation of excess peroxide compounds, and oxidative stress progression [30].Firstly, we studied the intensity of bio-oxidants production (reactive oxygen and nitrogen species), the level of cellular macromolecule oxidative modifications and the efficiency of the antioxidant defense system in the liver to determine the role of ROS-mediated processes under chronic hyperglycemic condition (experimental type 1 diabetes).
In accordance with the data obtained using ROS-sensitive fluorescent probe DCFH-DA, we have found out that chronic diabetic hyperglycemia stimulates formation of reactive oxygen metabolites in isolated hepatocytes, increasing the intensity of fluorescence by 90.4% (P < 0.05) compared with control animals (Fig. 1).Similarly, a significant increase by 73.1% (P < 0.05) in fluorescence of DAF-FM DA, a probe specific for the nitric oxide (II) molecule, was shown for hepatocytes from diabetic mice (Fig. 2).It should be noted that the results of recent year studies confirm the direct effect of insulin signa ling on the regulation of nitric oxide synthesis [31].On the other hand NO exhibits some insulin-like effects such as stimulating glucose transport and oxidation [32].Due to impairment of insulin-producing system in type 1 diabetes we can suggest that activation of NO production in tissues (especially in the liver) may be a regulatory mechanism of partial compensation for the lack of insulin in diabetic organisms.
It is known that excessive production of reactive oxygen metabolites is one of the key mechanisms leading to cell dysfunction and death.Cytotoxic and mutagenic action of ROS is caused by increased free radical (peroxidative) oxidation of physiologically important macromolecules [33].The results of biochemical analysis of changes in integrated indicators of free radical oxidation of biomolecules evidence for a significant intensification of the oxidative process under conditions of chronic hyperglycemia.This applies to the content of TBA-reactive products (predominantly MDA) and dien conjugates (DC) in particular, which are the end products of lipid peroxidation (LPO) and can cause damage of biological membranes, proteins and nucleic acids.It was established that the level of TBA-reactive products and DC exceeded 2.5 and 1.9 times, respectively, in the experimental type 1 diabetic mice compared with control group of mice (Table 2).
It is important that in conditions of chronic hyperglycemia, ROS also causes protein oxidative degradation.It is believed that protein oxidative modifications play a key role in the molecular mechanisms of oxidative stress and may trigger damage to other cell biomolecules (lipids, DNA) [34,35].The degree of protein oxidative modifications was evaluated by the content of carbonyl groups (>C=O), which are formed in protein molecules mainly due to the direct oxidation of some amino acid radicals (especially Pro, Arg, Lys), as well as due to the interaction with lipid peroxidation products or reducing sugars.Since amino acid residues normally contain a small amount of carbonyl groups, even little increase of their content per unit of protein can be considered as a result of oxidative stress, which occurs because of an imbalance of ROS-generating and degradation processes.Moreover, determination of carbonyl groups in proteins has significant advantages compared to the detection of lipid peroxidation products because of the relative stability of oxidized proteins and their formation at the beginning of oxidative stress development [34].It was established that the content of carbonyl groups in the studied samples of liver is 1.65 times higher compared to the control group (Table 2).Among various types of macromolecules damage, mainly oxidative modification of cellular proteins often leads to loss of their function and then such molecules are to be selectively degraded.Despite the fact that moderate oxidative modification makes proteins more accessible for many proteases which play an important role in protein degradation, the increased level of modified proteins (particularly enzymes) under conditions of oxidative stress may harm cellular metabolism.As was mentioned above, chronic diabetic hyperglycemia can lead to ROS overproduction in various tissues, especially superoxide anion, which interacts with other active forms of oxygen and can be converted to hydroxyl radical, hydrogen peroxide and peroxynitrite (ONOO -).Reactive nitrogencontaining compounds, including peroxynitrite play a crucial role in the pathogenesis of diabetes and its complications [36].The growth of peroxynitrite content, which today is recognized as the number one oxidant in biological systems leads to the development of oxidative/nitrosative stress, accompanied by protein nitrosylation, lipid peroxidation, DNA breaks, changes in cell signaling, activation of poly(ADP-ribose)polymerase-1 (PARP-1), induction of necrosis and apoptosis.High level of nitrosylated proteins (estimated by 3-nitrotyrosine content) is one of the earliest markers indicating the beginning of chronic complications in diabetes [36].It was shown (due to immunoblotting analysis) that there is a 1.72fold increase (P < 0.05) of nitrosylated proteins in the liver lysates compared with the control group of mice (Fig. 3).It should be noted that the increase in nitrosylated protein content correlated with high NO production by hepatocytes (Fig. 2), which actually is one of the peroxynitrite precursors.This growth of NO concentration can be explained by inducible form of nitric oxide synthase (iNOS) overexpression in the liver under diabetes [37].

Index
A thiol-disulfide system is an important component of oxidative homeostasis in cells and tissues.It includes low molecular weight thiols, including glutathione, SH-and SS-groups of proteins, the level of which is regulated by glutathione cycle enzymes [38].Cyclic oxidation/reduction of amino acids thiol groups serves as a kind of trap that captures ROS, protecting these proteins from irreversible, more harmful oxidative modifications.Changes in the redox state of GSH/GSSG are able to regulate the reversible formation of mixed disulfides between protein thiol groups and glutathione.However, a decrease of free SH-groups content under different forms of stress and pathologies, formation of mixed disulfides in proteins may prevail over their recovery process and lead to an increase of the disulfide bonds number and the rigidity of the protein conformational structures.These changes modify cell membrane state, its permeability and adhesive properties, besides having negative influence on enzymes activity and cell survival [34,38].A disturbance in thioldisulfide system was observed in type 1 diabetes, and these changes were traced in a decreased level of free SH-groups of low molecular weight compounds in the liver to 41.1% (P < 0.05) compared with controls (Table 2).This shows a significant suppression of antioxidant protection adaptive mechanisms.Reduction of free SH-groups number in SH/SS system under conditions of oxidative stress promotes the accumulation of lipid peroxidation products and oxidative protein damage and can be an additional factor for further enhancement of the oxidative stress.Significant reactivity of ROS and macromolecules peroxidation products necessitates continuous activity of antioxidant protection (AOP) specific mechanisms, which are important components of antioxidant enzymes.The latter along with nonenzymatic antioxidants perform a cytoprotective function reliably limiting the pro-oxidant process at all stages, starting from the stage of reactive oxygen species formation.Weakening of any AOP element may contribute to the development of oxidative stress [33,39].Pro-oxidant processes activation within diabetes may result from the disruption of coordinated system mechanisms of enzymatic and non-enzymatic control over ROS and free radical oxidation products content.Furthermore alterations in enzyme activity in diabetes may be due to the post-translational modifications especially by glycosylation.
Intensification of protein oxidative modification processes, lipid peroxidation and descension of thiols antioxidant capacity in diabetes correlates with changes in the AOP enzymes activity -superoxide dismutase (SOD), catalase and glutathione peroxidase (GPO).These enzymes complement each other and play a key role in oxidation/reduction processes regulation in the liver.The most powerful natural antioxidant is SOD enzyme, which catalyzes the reaction of superoxide anion radical dismutation and converts them into less reactive molecule -H 2 O 2 .Therefore, changes in the SOD activity characteri ze the depth of tissue destruction and metabolic changes caused by oxidative stress.The studies of SOD activity (Table 3) indicate that enhancement of free radical processes in diabetes is accompanied with a significant decrease in liver enzyme activity by 34.5% (P < 0.05) compared to control.Changes in SOD activity may be caused by modifying influence of reactive oxygen species, whose content increases significantly under chronic hyperglycemia.The presence of metals with variable valences associated with the imidazole group of histidine residues in the active Cu, Zn-SOD, makes this enzyme particularly sensitive to high concentrations of both reactive oxygen species (superoxide anion radical, hydroxyl radical, hydrogen peroxide) and a number of lipid peroxidation intermediates (hydroperoxides of un- saturated fatty acids, aldehydes, ketones, etc.) [40].

Enzyme
Cu, Zn-SOD may also interact with H 2 O 2 and act as a pro-oxidant agent, initiating the formation of superoxide anion radicals and hydroxyl radicals.The next step of the antioxidant protection system includes catalase and glutathione peroxidase which eliminate hydrogen peroxide and lipid hydroperoxides.Catalase which decomposes hydrogen peroxide is a hem-containing enzyme localized mainly in peroxisomes.Simultaneously, catalase may be a source of active oxygen metabolites production because approximately 0.5% of O 2 is formed by the decomposition of H 2 O 2 .Our study showed that the catalase activity in the liver is decreased by 27.2% (P < 0.05) in diabetes compared with the control group (Table 3).This result can be explained by degradation of plasma and peroxisome membranes where this enzyme is mainly localized.Glutathione antioxidant system includes glutathione peroxidase, glutathione reductase, glutathione-S-transferase, glucose-6-phosphate dehydrogenase and cofactorsglutathione and NADPH.Protective functions of this system are determined by specific partial contribution of each individual component during oxidative stress.Glutathione forms a reserve of cellular mobile SH groups, provides recovery and isomerization of disulfide bonds in proteins, and is involved in the eicosanoids and xenobiotics metabolism, reparative processes and adaptation to the stress factors in particular.Selenium-dependent glutathione peroxidase catalyzes reduction of hydrogen peroxide and organic hydroperoxides (linoleic, linolenic, arachidonic acid, cholesterol, corticosteroids) to their hydroxylated forms using glutathione [41,42].The activity of glutathione peroxidase (GPO) -an enzyme that uses GSH, increased by 69.5% (P < 0.05) in diabetes compared with controls (Table 3).This significant increase suggests that GPO is the major enzyme which can provide antioxidant protection under oxidative stress conditions that develops in experimental diabetes in the liver cells.
It should be noted that the investigated antioxidant protection enzymes -SOD, catalase and glutathione peroxidase form not only a complete enzymatic antioxidant system of cells, but also directly protect each other from the inhibitory effects of reactive oxygen species.In particular, SOD removes their negative effects on the catalase active center while eliminating superoxide anion radicals.Howe ver, the GPO and catalase protect SOD against oxidative modification by decomposing H 2 O 2 .There-fore, the combined effect of antioxidant enzymes under oxidative stress conditions provides not only the greatest manifestation of their biological activity, but also protection against reactive oxygen species.Conversely, impairment of this cooperativity by reducing of at least one antioxidant enzyme activity due to oxidative modification alters the functioning of the whole antioxidant defense system.Therefore, the obtained results about the changes in the activity of catalase and glutathione peroxidase may also be explained by the increased activity of SOD, which catalyzes the reaction of superoxide anion conversion to hydrogen peroxide -the only substrate of catalase [43].It is known that GPO is more effective at low H 2 O 2 concentrations, and also neutralizes various organic peroxides [44].
Hyperglycemia-induced changes in the structural organization and functional status of hepatocytes may be associated with disturbances in the biotransformation systems of xenobiotics and endogenous compounds, including the processes of their enzymatic oxidation.NAD(P)H:quinone oxidoreductase (NQO) related mechanisms of xenobiotics biotransformation are not fully clarified.It is known that the development of xenobiotic-induced oxidative stress impairs the function of mitochondria oxidative phosphorylation system.Hydrophilic quinones can partially counteract negative effect in these conditions.NQO in the presence of NADH (which is over-accumulated in these conditions) reduces hydrophilic quinones to hydroquinones.Then these molecules are oxidized in mitochondria respiratory chain complex III, subsequently forming a shunt that provides a bypass of complex I [45].In the case of prolonged hyperglycemia a significant increase (2.2 times) in the liver NQO activity (Tab le 3) was found.According to literature data, the expression of this enzyme increases dramatically as a response to the oxidative stress and xenobiotics influence in diabetes [46].These results evidence that hyperglycemiainduced reduction of quinones to dyhydroquinones under the effect of NQO can be used for electron transport in the respiratory chain to restore the cell function.Although NQO as inducible antioxidant enzyme prevents the ROS formation, the formed dyhydroquinones themselves may be an additional source of ROS, as shown by N. Watanabe et al [47].Thus, the observed intensification of free radical processes in diabetes may partly be connec ted with rising of this enzyme activity.
Functional disorders in enzymatic systems of metabolites biotransformation are linked with the ac-D.O. LABUDZyNSKyI, O. V. ZAITSEVA, N. V. LATySHKO et al. cumulation of the toxic catabolism products, which can greatly enhance the process of oxidative stress and disrupt intracellular processes.From this viewpoint, it is important to study the liver xanthine oxidase (XO) activity that is involved in the oxidation of hypoxanthine and xanthine to uric acid.It is important that purine oxidation reaction is coupled with the oxygen reduction to singlet oxygen.Thus, XO is a potent ROS producer, especially in various diseases [48].We have shown that there is an increase in the liver xanthine oxidase activity in diabetic animals to 1.43 times (P < 0.05) compared with control (Table 3).It can be assumed that the increased activity of XO caused by pathological hyperglycemic conditions will result in the accumulation of purine degradation products.This can be considered as an additional factor enhancing oxidative stress and reduction of reparative processes in the liver.
Functional activity of ROS generation elements and antioxidant system are largely dependent on the cellular NADPH pool.Interdependent redox transformation of important antioxidants, in particular, like glutathione and ascorbic acid (the latter recovers tocopherol radicals and various polyphenols) occurs due to NADPH.The observed decrease in the hepato cytes antioxidant capacity could be a result of intensified competitive NADPH use in the NAD(P)H oxidase catalyzed reaction.This multi-oxidase complex provides ROS production mainly in phagocytic blood cells and resident macrophages in peripheral tissues.Moreover, it also plays a key role in the inflammatory process within Kupffer cells in the liver [49].The main product of NAD(P)H oxidase is a superoxide anion radical, which has bactericidal action and is an essential component of nonspecific immune defense in the organism.In addition, NAD(P) H oxidase is expressed in the hepatocytes and other liver cells [50].We have found out that the activity of this enzyme in liver homogenate of diabetic mice increase 1.64 times (P < 0.05) compared with control (Table 3).This data is consistent with the generalized inflammation state that is characteristic of diabetic liver tissue.
Oxidative modifications of biomacromolecules and cellular structures largely determine the cytotoxic effects of free radical oxygen and nitrogen species under conditions of their excessive production.It was important to investigate the relation between prooxidant processes, functional activity and survival of hepatocytes in diabetes.The oxidative stress induced by chronic hyperglycemia can cause mitochondrial dysfunction with subsequent initiation of hepatocytes damage and death.Our research has shown that intensification of free radical processes correlated with an increase in the proportion of dead (necrotic) cells in the isolated hepatocytes culture to 9.3% in diabetes compared with 5.8% in control (P < 0.05), Fig. 4. The destruction of parenchymal liver cells potentially can trigger the activation of other cell populations that are able to initiate inflammatory and/or adaptive immune responses and inhibit liver regeneration.
Diabetic disorders that were unveiled in the research were observed on the background of vitamin D 3 deficiency in the organism of experimental animals, and it was evidenced by more than 2.5-fold 25OHD 3 reduction in the serum of mice compared with control (Table 1).One of the reasons for the cholecalciferol decreased level under chronic hyperglycemia can be an observed reduction in vitamin D 3 25-hydroxylase expression in diabetes [26].These results indicate changes in vitamin D 3 metabolism and we can suggest that the increase in prooxidant status of the liver may be associated with vitamin D 3 deficiency in chronic hyperglycemia.
Vitamin D 3 administration in physiological doses (10 IU of D 3 for 45 days) was accompanied by 2.2-fold growth of 25OHD 3 content compared to its level in the animals with experimental diabetes (Table 1).Investigation of biochemical markers of free radical processes, lipid peroxidation products content and protein oxidative modifications in the liver under vitamin D 3 therapeutic administration, showed a decrease in the intensity of DCF-sensitive ROS and NO formation by 25.1 and 20.6%, respectively (Fig. 1 and 2); TBA-reactive compounds, diene conjugate and protein carbonyl groups content by 33.3, 24.8 and 30.8%, respectively (Table 2); nitrotrozylated proteins by 14.5% (Fig. 3); and increase of the content of thiol compounds by 32.7% (Table 2) compared to animals with diabetes (P < 0.05).Inhibition of lipid and protein peroxide modifications and increase of thiols antioxidant capacity under cholecalciferol administration correlated with partial or complete normalization of detoxification enzymes which can enhance prooxidative processes.With the vitamin D 3 treatment, NQO, xanthine oxidase and NADPH-oxidase activities were reduced by 39, 11.5 and 23.7%, respectively, compared with diabetes group (P < 0.05), Table 3.The activity of antioxidant enzymes SOD and catalase increased by 19.1 and 14.4%, respectively, while GPO activity level decreased by 32% compared to diabetic mice (P < 0.05).It was found out that vitamin D 3 administration significantly increased the hepatocytes survival, reducing the percentage of dead cells, which may be due to inhibition of free radical compounds formation and biomolecules oxidative modification.These results demonstrate that vitamin D 3 prevents the oxidative stress development in liver cells caused by chronic hyperglycemia.First of all, antioxidant effects of vitamin D 3 are mediated by hormonally active metabolite of cholecalciferol -1.25(OH) 2 D 3 .This multifaceted regulation of oxidative metabolism with vitamin D 3 is caused by complex interactions between several nuclear coactivators or co-repressors that mediate the regulation of gene transcription at the level of their interaction with receptors of hormonally active form of cholecalciferol (VDR).When binding receptors, vitamin D 3 enhances signaling and effectively controls the rate of free radicals formation in mice liver cells [9,51].As a natural ligand of VDR, vitamin D 3 influences the development of proinflammatory and prooxidant processes through the gene expression regulation of numerous important cytokines, and therefore its deficiency increases the risk of many multifactorial polygenic diseases [52].
However, there are data in literature that indicate a significant antioxidant effect of cholecalciferol in mature erythrocytes without nucleus.These findings confirm that vitamin D 3 has the antioxidant activity [10].It is believed that cholecalciferol may act as a direct membrane antioxidant, stabilizing and protecting them from lipid peroxidation through interaction with their hydrophobic areas [11].Data on the antioxidant activity of cholecalciferol correlate with the results obtained in several experimental works, which indicated that vitamin D 3 is able to slow down the lipid peroxidation process and enhance the SOD activity in the liver and other organs of mice [51].The ability of cholecalciferol to protect prostate cells from oxidative stress-induced death through cell damage inhibition associated with increased formation of ROS was also demonstrated [53].According to the literature, calcitriol (hormonally active form of vitamin D 3 ) may enhance the pathway of reactive oxygen and nitrogen species elimination, increasing the intracellular pool of reduced glutathione, partially through upstream regulation of glutamate-cysteine ligase (GCL) and glutathione reductase (GR) genes expression [54].This correlates with the data established in this research that there is increased content of low molecular weight thiols (mostly glutathione) in the liver under the cholecalciferol influence.In addition, vitamin D 3 anti-inflammatory effects can be mediated through inhibition of NF-kB-dependent activation of numerous pro-inflammatory cytokines gene transcription induced by hydroxyl radicals [55].
Thus, the results of experimental studies demon strate that chronic hyperglycemia conditions are accompanied by the oxidative-nitrosative stress development, increased content of metabolites of oxygen and nitrogen, products of biomacromolecules oxidative modification and decreased activity of antioxidant protection.All these diabetes-induced injuries correlated with lack of adequate vitamin D 3 supply, which is confirmed by a significant decrease in the content of 25OHD 3 in serum.Cholecalciferol administration to experimental animals greatly inhibits the formation of reactive oxygen and nitrogen in hepatocytes, normalizes metabolic and antitoxic function of liver cells and provides them with better survival.It was established that vitamin D 3 level supply is an essential factor in the normalization of structural and functional abnormalities in hepato-cytes caused by prolonged hyperglycemia, which is consistent with the results of the leading laboratories and demonstrates a significant therapeutic potential of vitamin D 3 .

Fig. 1 .B
Fig. 1. the rate of ROS formation in hepatocytes of diabetic mice and with vitamin D 3 administration.(a) Fluorescence cytofluorograms of DCF-DA (count -the number of events; FL1 LOG -fluorescence intensity) and (B) mean fluorescence intensity.*P < 0.05 compared with control, #P < 0.05 compared with diabetes group (M ± m, n = 6)

Fig. 2 .
Fig. 2. the rate of nitric oxide (II) in hepatocytes of diabetic mice and with vitamin D 3 administration.(a) Fluorescence cytofluorograms of DAF-FM DA (count -the number of events; FL1 LOG -fluorescence intensity) and (B) mean fluorescence intensity.* P < 0.05 compared with control, # P < 0.05 compared with diabetes group (M ± m, n = 6)

Fig. 3 .
Fig. 3.Western blot analyses of nitrosylated proteins in the liver of diabetic mice and with vitamin D 3 administration (A).Graphical representations are expressed as means ± SD (B).*P < 0.05 compared with control, # P < 0.05 compared with diabetes group (M ± m, n= 6)