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Ta 6i0TexH0iroriH iMeHi C.3. f^H^Koro Scientific Messenger of Lviv National University of Veterinary Medicine and Biotechnologies named after S.Z. Gzhytskyj
ISSN 2518-7554 print ISSN 2518-1327 online
http://nvlvet.com.ua/
UDC 636.09: 615.9: 636.2
Impact of antioxidants on enzym activities of glutatione system of bulls bodies antioxidant defense under acute nitrate and nitrite toxicity
V. Huberuk, B. Gutyj, D. Gufriy, V. Binkevych, I. Hariv, O. Binkevych, R. Salata
Lviv National University of Veterinary Medicine and Biotechnologies named after S.Z. Gzhytskyj,
Pekarska Str., 50, Lviv, 79010, Ukraine
The article presented the results of studies of the effect of sodium nitrate on indexes of glutatione system of antioxidant defense system in young cattle, such as the activity of glutathionereductase, glutathioneperoxidase and glucose-6-phosphate dehydrogenase. It was founded that feeding bulls with the toxicants at a dose 0,45 N03 / kg enzyme activity in the blood of experimental animals throughout the experiment decreased. After using the nitrate load of young cattle it was used drugs Ursovit-ADES and sodium selenite. It was founded stimulatory effects on activity of glutathione system of antioxidant defense. Specifically,it was founded significant activity increase of glutathionereductase, glutathioneperoxidase and glucose-6-phosphatedehydrogenase. in the blood of young cattle, which were conducted with nitrate loading.
Key words: nitrates, bulls, antioxidants, toxicology, antioxidant system.
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Вплив антиоксидантш на актившсть ензимш глутатюново1 системи антиоксидантного захисту оргашзму бичк1в за гострого штратно-штритного токсикозу
В.О. Губерук, Б.В. Гутий, Д.Ф. Гуфрш, В.Я. Бшкевич, I.I. XapiB, О.М. Бiнкевич, Р.В. Салата
Львiвський нщюнальнийунгверситет ветеринарногмедицини та бготехнологгй iMeHi С.З. Гжицького,
вул. Пекарська, 50, м. Львiв, 79010, Украна
У статтг наведено результати дослгджень впливу нгтрату натрiю на показники глутатгоновог системи антиоксидантного захисту оргашзму молодняку великог рогатог худоби. Дослгди проводились на бичках шестимгсячного вгку, чорно-рябог породи. Активнгсть глутатгонпероксидази (К.Ф.1.11.1.9.) та глутатгонредуктази (К.Ф.1.6.4.2.) визначали за методом В.В. Лемешко i спгвавт., актившсть глюкозо-6-фосфатдеггдрогенази (К.Ф.1.1.1.49.) - за методом N.Z. Baquezetal. Венозну кров вгдбирали на початку дослгду та через 3 години пгсля згодовування бичкам нгтрату натргю, а також через 1, 2, 3, 6, 9, 12 годин пгсля введення вгтамгнних препаратгв.
Встановлено, що за умов згодовування бичкам нгтрату натргю у дозг 0,45 N03 /кг маси тгла актившсть ензимгв глу-татгоновог системи кровг тварин упродовж усього дослгду знижувалась. За умов нгтратного навантаження, молодняку великог рогатог худоби застосовували препарати урсовгт АДЕС та селенгт натргю. Застосування препаратгв-антиоксидантгв за умов розвитку гострого штратно-штритного токсикозу у бичкгв сприяли пгдвищенню активностг ензимног ланки глутатгоновог системи антиоксидантного захисту (глутатгонредуктази, глутатгонпероксидази, глюкозо-6-фосфатдеггдрогенази) у кровг дослгдних тварин. Сукупне введення урсовгту-АДЕС та селенгту натргю проявляло кращу дгю на глутатгонову систему антиоксидантного захисту органгзму бичкгв нгж застосування лише урсовгту-АДЕС.
Ключовi слова: нгтрати, бички, антиоксиданти, токсикологгя, антиоксидантна система.
Citation:
Huberuk, V., Gutyj, B., Gufriy, D., Binkevych, V., Hariv, I., Binkevych, O., Salata, R. (2017). Impact of antioxidants on enzym activities of glutatione system of bulls bodies antioxidant defense under acute nitrate and nitrite toxicity, 19(77), 220-224.
Introduction
In modern toxicology an activation of lipid peroxidation is taken as a universal response of a living organism to the action of extreme factors (Baglaj et al., 2010; Staryk et al., 2012; Gutyj, 2013; Guberuk et al., 205; Nazaruk et al., 2016; Gutyj et al., 2017). Overall, prooxidant and antioxidant status of animals reflects a balance between two oppositely directed actions in the body, namely antioxidant properties (defense) and the formation of free radicals (damaging). The influence of extreme factors including toxins, leads to an imbalance between them to prooxidant direction and develops so-called «oxidative stress» (Antonjak et al., 2000; Nazaruk et al., 2012; Nazaruk et al., 2015; Martyshuk et al., 2016; Khariv et al., 2016; Khariv and Gutyj, 2017).
Environmental contaminations with nitrates and nitrites and their negative impact on the animals make the problem of studying the mechanism of nitrate and nitrite toxicity on farm animals particularly relevant, which has theoretical and practical importance.
Aaccording to published scientific papers about the study of nitrate-nitrite toxicity in farm animals there are some reports that claim that the toxic effect of nitrates is manifested in two interrelated ways. The first step gets methemohlobinmaking and activation of free radicals which initiate the second phase of lipid peroxidation (Gunchak et al., 2010; Guberuk et al., 2012). The intensity of free radical peroxidation in animals depends on the concentration of oxygen in the tissues, and the activity of the enzyme and nonenzyme systems.
Therefore, the aim of our research was to study the effect of Ursovit-ADES and sodium selenite on activity of the enzymatic antioxidant protecting system of young cattle bodies under acute nitrate and nitrite toxicity.
Material and methods
Experiments were conducted on six- month old bulls, black and white breed, they were formed into 3 groups, 5 animals each.
Bulls in control group were fed with food once with sodium nitrate in a dose 0.45 N03 / kg of body weight. Bulls in the first experimental group were fed once with sodium nitrate in a dose 0.45 N03 / Kg and in three hours later they were injected intramuscularly with Ursovit-ADES at a dose of 4 ml / animal. Bulls in the second experimental group were fed with sodium nitrate in a dose 0.45 N03 / Kg and in three hours later they were injected intramuscularly with Ursovit -ADES at a dose of 4 ml and 0.5% solution of sodium selenite at a dose of 1 ml. Venous blood were taken at the beginning of the experiment and in 3 hours after feeding bulls with sodium nitrate, and in 1, 2, 3, 6, 9, 12 hours after administration of vitamin preparations.
The activity of glutathione peroxidase (GP) (K.F.1.11.1.9.) and glutathionereductase (Gr) (K.F. 1.6.4.2.) it was determined by the method of V. Lemeshko et al. (Lemeshko et al., 1985), the activity of glucose-6-phosphatedehydrogenase (G-6-FDG)
(K.F.1.1.1.49.) - method of N.Z. Baquezetal (Baquezetal et al., 1967).
Results and discussion
In the pathogenesis of various toxicosis it was involved lipid peroxidation and antioxidant system in physiological conditions, maintain a system of regulation of cellular homeostasis (Hutyi, 2005; Hutyi and Hufrii, 2005). So, under acute nitrate and nitrite toxicosis we used drugs that have both direct and indirect antioxidant effects on reactive oxygen species and lipid peroxidation products.
An important antioxidant defense system is glutatione system that is represented with enzyme system: glututionreduktase, glutathioneperoxidase, glucose-6-phosphatedehydrogenase.
The activity changes of glututionreduktase under acute nitrate and nitrite toxicosis of young cattle and influence of vitamin drug and sodium selenite are given in Table 1.
At the beginning of the experiment, the activity of GH in bulls' serum were within physiological values. After feeding sodium nitrate the activity of this enzyme in the control group began to fluctuate within 1.71 ± 0.045 -1.20 ± 0.033 nmol NADPH / min. to 1 mg of protein. Only in three hours it was marked the activation of the enzyme, which compared with control values, the enzyme activity increased to 9.6, 6.3 and 4.3%.
After administration of drugs in the patient bulls enzyme activity GH in serum increased. When using vitamin-drug Ursovit-ADES animal group D1 in two hours of experiment GR activity increased by 4%, while in six hours it increased respectively by 22%. In eight hours GH activity reached physiological limits values. When combined use of Ursovit-ADES and sodium selenite GR enzyme activity was within the values of 1.62 ± 0.048 - 1.51 ± 0.035 nmol NADPH / min. to 1 mg of protein during the experiment.
The second important enzyme of glutatione system is a glutatioperoksydasa that catalyzes the schedule by a moderate form of lipid hydroperoxides using glutathione. The activity of this enzyme in serum of bulls blood after application of Ursovit-ADES and sodium selenite under acute nitrate and nitrite toxicity is given in Table 2.
Before feeding with sodium nitrate GP activity in blood serum of experimental groups of animals was within 37.10 ± 1.1 - 37.18 ± 1.2 nmol NADPH / min. to 1 mg of protein. After feeding bulls with sodium nitrate, we have noted in three hours of the experiment, a slight increase, but in the control group of animals GP activity further decreased to 28.25 ± 1.1 nmol NADPH / min. to 1 mg of protein. After introduction of the drug and vitamin sodium selenite in bulls research groups, enzyme activity was increased comparing to control group. Thus, in the experimental group D1 bulls enzyme activity in three hours of the experiment increased to 19%, in six hours - to 28%, while the experimental group D2 activity grew to 24 and 31% relative values of the control group animals.
Table 1
Glutathionereductase activity in serum of bulls blood after application Ursovit ADES and sodium selenite under
Indicators of animal blood (hours) Groups of animals
Control Experimental 1 Experimental 2
Before feeding with msodium nitrate
Control 1.56 ± 0.050 1.58 ± 0.06 1.60 ± 0.056
After feeding with sodium nitrate
In three hours 1.71 ± 0.045 1.68 ± 0.057 1.67 ± 0.035
After introduction of antioxidants
— Ursovit ADES Ursovit ADES + 0.5% sodium selenite solution
In one hour 1.57 ± 0.065 1.53 ± 0.045 1.62 ± 0.048
In two hours 1.48 ± 0.048 1.54 ± 0.042* 1.59 ± 0.045*
In three hours 1.23 ± 0.024 1.47 ± 0.037* 1.54 ± 0.035**
In six hours 1.20 ± 0.033 1.47 ± 0.031** 1.53 ± 0.025**
In eight hours 1.25 ± 0.026 1.50 ± 0.034** 1.57 ± 0.035**
Note. The degree of probability compared with the control group data - P < 0.05-*, P < 0.001-**
Table 2
The activity of glutathioneperoxidase in serum of bulls blood after application Ursovit-ADES and sodium
Indicators of animal blood (hours) Groups of animals
Control Experimental 1 Experimental 2
Before feeding with sodium nitrate
Control 37.17 ± 1.2 37.18 ± 1.2 37,10 ± 1.1
After feeding with sodium nitrate
In three hours 38.10 ± 1.1 38.15 ± 1.3 38.04 ± 1.1
After introduction of antioxidants
— Ursovit-ADES Ursovit-ADES + 0.5% sodium selenite solution
In one hour 34.98 ± 1.0 35.42 ± 1.3 35.67 ± 1.4
In two hours 32.46 ± 1.1 34.32 ± 1.2 35.96 ± 2.5*
In three hours 29.43 ± 1.0 35.06 ± 1.1** 36.59 ± 1.2**
In six hours 28.25 ± 1.1 36.18 ± 1.4** 36.87 ± 1.3**
In eight hours 31.68 ± 1.2 36.44 ± 1.5* 37.05 ± 1.4*
Note. The degree of probability compared with the control group data - P < 0,05 - *, P < 0,001
Thus, the use of vitamin-drug Ursovit ADES and sodium selenite helped to normalize the activity of glutathioneperoxidase better than the use of Ursovit-ADES, possible due to the fact that sodium selenite includes important element of antioxidant selenium, and it was a part of the enzyme that was investigated.
The activity of glucose-6-phosphatedehydrogenase under acute nitrate and nitrite toxicity and the use of antioxidants is given in Table. 3. Before feeding bulls with sodium nitrate, the activity values of G-6-FDG was within 0.72 ± 0.024 - 0.73 ± 0.024 nmol NADPH / min. to 1 mg of protein.
Table 3
The activity of glucose-6-phosphatedehydrogenase in serum of bulls blood after application Ursovit-ADES and
Indicators of animal blood (hours) Groups of animals
Control Experimental 1 Experimental 2
Before feeding with sodium nitrate
Control 0.72 ± 0.025 0.73 ± 0.024 0.72 ± 0.024
After feeding with sodium nitrate
In three hours 0.83 ± 0.035 0.86 ± 0.031 0.85 ± 0.032
After introduction of antioxidants
Ursovit-ADES Ursovit-ADES + 0.5%
sodium selenite solution
In one hour 0.56 ± 0.021 0.61 ± 0.022 0.63 ± 0.021
In two hours 0.52 ± 0.021 0.56 ± 0.020 0.61 ± 0.020*
In three hours 0.50 ± 0.017 0.57 ± 0.018* 0.64 ± 0.023**
In six hours 0.44 ± 0.016 0.53 ± 0.015* 0.69 ± 0.021**
In eight hours 0,53 ± 0.013 0.57 ± 0.021** 0.73 ± 0.019**
Note. The degree of probability compared with the control group data - P < 0.05-*, P<0.001-*
After feeding the bulls with sodium nitrate forage at a dose of 0.45 g N037 kg body weight, enzyme activity began to change. In particular, in three hours of the experiment, the activity of G-6-FDG was 0.83 ± 0.035 -0.86 ± 0.031 nmol NADPH / min. to 1 mg of protein. In the control group of bulls that were not fed with antioxidants enzyme activity continued to decline in four hours of the experiment respectively decreased to 22% comparing to the initial values.
Introduction of Ursovit-ADES and sodium selenite in the patient bulls, accompanied with an increase in the activity of G-6-FDG in their blood. The most likely enzyme changes were noted in six hours of experiment where results of D1 experimental group was lower by 20% and D2 group - 57%.
Thus, the combined administration Ursovit-ADES and sodium selenite promoted better normalization of enzyme, which was associated with synergistic properties of these drugs; they increase and continue to force each other activity.
Conclusions
1. The use of antioxidant drugs iunder conditions of acute nitrate and nitrite toxicity of bulls have improved enzymatic level of glutathione system of antioxidant protection (glutathionereductase, glutathioneperoxidase, glucose-6-phosphate-dehydrogenase) in the blood of experimental animals.
2. Cumulative input of Ursovit-ADES and sodium selenite showed better effect on glutathione sstem of antioxidant defense of the bulls' bodies than applying only Ursovit-ADES.
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Стаття надiйшла до редакцН 4.04.2017
