Influence of « Metisevit » on the activity of enzyme and nonenzyme link of antioxidant protection under the bull ’ s body cadmium loading

The article contains the research results of the effect of cadmium chloride on the indexes of enzyme and nonenzyme systems of antioxidant defense system in young cattle, such as the activity of catalase, superoxide dismutase, glutathione peroxidase, glutathione levels of vitamins A and E. It is established that feeding calves at a dose of toxicant 0.04 mg / kg activity of catalase, superoxide dismutase, glutathione peroxidase, glutathione levels of vitamins A and E in the blood of experimental animals decreased throughout the experiment. The lowest indicators of antioxidant in the blood of young cattle is set on the twenty –fourth day of the experiment, which is associated with increased activation of lipid peroxidation and the balance between antioxidant system and lipid peroxidation intensity. Given the cadmium load of young cattle it is used a new integrated drug with antioxidant action «Metisevit», which includes metifen, sodium selenite and vitamin E wich is founded as stimulating effects on the activity of antioxidant protection. In particular,it is established probable increase in activity of catalase, superoxide dismutase, glutathione peroxidase, glutathione levels, vitamin A and vitamin E in the blood of young cattle, which has performed cadmium stress. These changes occur through comprehensive action components of the drug «Metisevit» that leads to the normalization of metabolic processes and free radical in the body of the bull. The results of the research indicate antioxidant drug «Metisevit» in the application of its young cattle and the validity of his administration to improve the body's antioxidant status of chronic cadmium toxicosis.


Introduction
Due to the intensive development of science and technology progress over the past decades the number of chemical compounds to which human and animal contact has increased significantly.Especially dangerous is pollution with heavy metals, at a high biological activity, particularly cadmium and its salts (Honskyy et al., 2001;Melnychuk et al., 2004;Gutyj, 2012).
Oxide and reduction reactions occupy an important role in the metabolic changes and their violation play an important and sometimes decisive role in the development of pathological processes.The processes of oxidation in the liver have very important role, where their participation is carried performing its basic body functions, including disposal of endo-and exotoxins.Toxic liver cadmium damages are accompanied by significant flow disturbances oxidative reactions that are characterized by some researches as «oxidative stress» (Honskyy et al., 2001;Gutyj, 2013).
Having established that during cadmium toxicity occurs enhance lipid peroxidation (Gutyj, 2013;Gutyj, 2015), we concluded that the action of cadmium to suppress excessive free radical reactions in animals, it is necessary to use drugs with a strong antioxidant effect that can inhibit processes of peroxidate oxidation of lipids.With a large number of antioxidants in cadmium toxicosis bull, we studied the «Metisevit» prophylactic effect.This drug blocks the free radicals and prevents the development of oxidation stress in animals.
The purpose of our research was to determine the effect of the drug «Metisevit» on the level of enzyme and nonenzyme system of antioxidant defense of the body in conditions of chronic bull cadmium toxicity.

Material and methods
Research were carried on farm in village Ivanivtsi, Zhydachiv district, Lviv region with 10 bulls six months of age, black and white breed that were formed into 2 groups ( 5 animals in each): Group 1 -control (C), calves were fed with food cadmium chloride at a dose of 0.04 mg/kg body weight; Group 2 -research (R), calves fed with food cadmium chloride at a dose of 0.04 mg/kg body weight along with «Metisevit» at a dose of 0.36 g/kg feed.
When conducting research we followed the rules required when performing experiments on selection zootechnical and animal analogues in groups, technology procurement, use and accounting of consumed feed.The diet of the animals was balanced in nutrients and minerals that ensure their basic need for batteries.
Antioxidant drug «Metisevit» was developed at the Department of Pharmacology and Toxicology Lviv National Stepan Gzhytskyi University of Veterinary Medicine and Biotechnology which incorporates contains «Metifen», vitamin E and selenium.These components enhance each other's action and promote a better balance in the normalization of complex «System of antioxidant defense ↔ Peroxidate oxidation of lipids».
The experiment lasted for 30 days.Blood for analysis were taken from the jugular vein in the 1-, 8-, 16-, 24-, and 30 th day of the experiment.

Results and discussion
Glutathione peroxidase (GP) -is an enzyme that protects the body from oxidative damage.It catalyzes the recovery and restoration of lipid peroxides hydrogen and peroxide to water.In these reactions the enzyme oxidezes glutathione.Furthermore, glutathione peroxidase takes place in redox regulation of various metabolic processes.Over 70% of GP is localized in the cytosol and 25 -30% -in the mitochondrial matrix (Gutyj, 2013).
From the data presented in Table 1 it is shown that under conditions of chronic cadmium toxicity glutathione peroxidase activity in blood serum in animal control group on the first day of the experiment, has increased by 3% compared to the indices of blood taken prior after feeding with cadmium chloride.The lowest enzyme activity was on the twenty-fourth day of the experiment and was 27,7 ± 1,24 nmol NADPH/min.to 1 mg of protein.Subsequently, the research activity of the enzyme gradually increased, and on the thirtieth day was 31,1 ± 1,20 nmol NADPH/min.to 1 mg of protein.
After applying metisevit in bulls experimental group the activity of glutathione peroxidase increased, on the eighth day, according to 16%.On the sixteenth day of the experiment, the activity of the enzyme in the experimental group of calves was 36,1 ± 1,25 nmol NADPH/min.to 1 mg of protein.Later, glutathione peroxidase activity in blood serum of calves in experimental group continued to rise, and on the twenty-fourth day of the experiment it increased by 32%.
Table 1 The activity of glutathione peroxidase in the blood of bulls after feeding metisevit after chronic cadmium toxicity; (М ± m, n = 5) The degree of probability according the control group -p <0,05-*, p> 0.01 -** Thus, normalization of glutathione peroxidase in blood of calves after entering metisevit drew the first day, and the highest enzyme activity is set to the twenty-fourth and thirty-day of experiment.
Superoxide dysmutase -is one of the key enzymes of antiradical protection, which refers to the direct action of antioxidants.This enzyme rutes superoxideradical to peroxide, which is less toxic to the previous.Actually so SOD plays a crucial role in the antioxidant protection of almost all types of cells that are in contact with oxygen.SOD is primarily in the mitochondrial matrix, the cytosol and the cytoplasm (Gutyj, 2013).
For chronic cadmium toxicity in bulls on the first day of the experiment the activity of superoxide dismutase in the serum of animals in the control group increased by 10% compared to the initial values.Later in patients bulls enzyme activity began to decline according to the eighth day of the experiment it was 0,52 ± 0,011 cu / Mg protein.
The low activity of the enzyme was on the twenty-fourth day of the experiment, where a relatively initial value is decreased by 35% (Table 2).
In the bull, which together with the investigational toxin were fed drug «Metisevit», superoxide dismutase activity increased throughout the experiment on the values of the control group animals.On the eighth and sixteenth day of the experiment enzyme activity in the experimental group of animals was higher by 17 and 41% relative values of the control group animals.

Table 2
Activity of superoxide dismutase in bulls' blood after feeding them with drug «Metisevit» for chronic cadmium toxicity; (М ± m, n = 5)
Catalase recovers hydrogen peroxide to water.To the active center of the enzyme is trivalent iron protoporphyrin, which interacts with hydrogen peroxide by catalase, or peroxidase mechanism, depending on the concentration of the substrate.The enzyme is found in all tissues at a concentration of 10 -6 M. Generally, the action of catalase is reduced to decrease the concentration of cytotoxic hydroxyl radicals.The highest catalase activity was found in the red blood cells, liver, kidneys.For high intensity formation of hydrogen peroxide in the body, he cleared catalase, and the low -glutatin antioxidant system (Gutyj, 2013).
So, catalase as the mechanism of action of antioxidant system defense refers to direct action.The activity of catalase in bull's blood serum in conditions of chronic cadmium toxicity and exposure to the drug "Metisevit" is shown in Table 3.

Table 3
The activity of catalase in blood after feeding bulls with drug «Metisevit» for chronic cadmium toxicity; (М±m, n = 5)
On the twenty-fourth day of the experiment catalase activity in blood of animals which were fed with food cadmium chloride was low and accordingly was 5,65 ± 0,11 units.On the thirtieth day of experiment, the enzyme activity increased slightly, but remained at a low level.
The use of «Metisevit» contributed to the increase catalase activity in blood of experimental animals.On the eighth day of the experiment enzyme activity increased by 5% for the values of the control group animals.On the sixteenth day of the experiment catalase activity in experimental group amounted to 6,50 ± 0,15 units.On the twenty-fourth day of the experiment indicated in these animals likely increased activity of the enzyme on the values of the control group animals at 15% respectively.On the thirtieth day of the experiment catalase activity in the first experimental group ranged within physiological values.
Thus, the drug «Metisevit» contributed to increasing activity of catalase, superoxide dismutase and that in animals play an important role in lipid peroxidation.
Glutathione is a major component of glutation antioxidant defense system.It consists of three amino acids: glutamic, cysteine and glycine.Glutathione does not only protect cells from toxic compounds and free radicals, but also defines the status of intracellular redox environment.In addition, it increases the resistance of cells to the negative effects of stress factors and is involved in the removal of xenobiotics.Actually so, glutathione is a key element in antioxidant system because it is able to restore other antioxidants (Gutyj, 2013).
Table 4 shows the changes in the level of reduced glutathione in the blood of bulls in cadmium stress.As can be seen from the table data glutathione level at the beginning of the experiment was within the physiological norm values.After feeding with cadmium chloride content of reduced glutathione began to decline.The lowest indicator was on the twenty-fourth day of the experiment, which according to the original values it decreased by 8%.

Table 4
The level of reduced glutathione in the blood of bulls after feeding «Metisevit» after chronic cadmium toxicity, mg % (М ± m, n = 5) The use of animal drug «Metisevit» contributed to raising the level of reduced glutathione in the blood of experimental animals on the eighth day of the experiment by 8%, on the sixteenth day -12%.The most probable changes in the level of reduced glutathione in the experimental group of animals was founded on the twenty-fourth day of the experiment, in which the figure was 33,33 ± 0,65 mg%, and it was 13% greater than the value of the control group animals.
Equally important in antioxidant system include vitamin E, which protects cell membranes from attack of free radicals and reactive oxygen species.Its content in the blood of animals by chronic cadmium toxicity is shown in Table 5. Feeding toxicant above helped to reduce the content of vitamin E in the blood of animals throughout the experiment.So, on the eighth day of experiment vitamin content was 3,3 ± 0,11 mmol/l, which is lower to 20% compared to the initial values.On the sixteenth day of vitamin E studies continued to decline and relative quantities of blood taken at the beginning of the experiment, that is for feeding bulls with cadmium chloride, decreased to 24%, the twenty-fourth day of the experiment vitamin E decreased to 29%.
We demonstrated that after the development of chronic cadmium toxicity in young cattle, vitamin A in their blood decreases.As can be seen from the data in Table 6, vitamin A after feeding with cadmium chloride began to fall on the first day to 4%, on the eighth day -12%, on the sixteenth day -16%, on the twenty-fourth day of the experiment -27% relative initial values.
The use of experimental animals the drug of «Metisevit» boosted vitamin A in the blood of bulls under conditions of cadmium loading.Since the first day of the experiment we founded a gradual increase in the content of vitamin A in the blood of experimental animals Ratios control group of animals.On the sixteenth and twentyfourth day of the experiment vitamin A increased by 28 and 40% compared to the control group of animals.
Overall, we received the research results indicating that feeding of bulls with drug «Metisevit» positively influences the antioxidant defense system in their body.

Conclusions
1.When feeding bulls with cadmium chloride at a dose of 0.04 mg/kg body weight and level indicators of nonenzyme and enzyme systems of antioxidant defense in the blood of experimental bulls decreased throughout the experiment.The lowest level of indexes in antioxidant system installed on the twenty fourth day of the experiment, which deals with enhanced activation of lipid peroxidation and the imbalance between the activity of antioxidant system and lipid peroxidation intensity.
2. The drug «Metisevit» in cadmium stress activates the enzyme and nonenzyme system of antioxidant defense system in bull's body, as it is indicated by increased activity of glutathione peroxidase, catalase, superoxide dismutase and glutathione increase of vitamins A and E in the blood of animals.Addition of «Metisevit» in feeding prevents the oxidation stress, that cause peroxide damages in cell membranes and the intracellular biopolymers.
Prospects for further researches.The results of research will be applied in subsequently studies of antioxidant defense and processes of lipid peroxidation in the blood of the bull to develop antidote drug for the treatment of animals in cadmium toxicosis.