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ENGLISH VERSION: CONDITION OF ANTIOXIDANT ACTIVITY OF THE LIVER IN RATS UNDER THE INFLUENCE OF SMALL DOSES OF SODIUM FLUORIDE*
Kolisnyk I.L.
Kharkiv Medical Academy of Postgraduate Education (Kharkiv, Ukraine)
The effect of small subtoxic doses of sodium fluoride on the antioxidant activity of the liver has been studied in Wistar rats populations in the subacute experiment. The intensity of iipid peroxidation was estimated by the content of its molecular products - diene conjugates (DC), and TBA-reagents and schfff bases in liver homogenate of rats (N = 30) which were administered small subtoxic doses of sodium fluoride. It has been found that oral administration of sodium fluoride in rats at doses of 1/10 and 1/100 LD50 promotes diene conjugates content in all periods of observation. In rat liver, starting from the 20th day, the gradual increase of TBA-reagents and schiff bases was clearly defined. Enhanced CL Intensity estimates by 37 and 134% was detected in the case of sodium fluoride action at a dose 1/10 LD0, respectively.
Keywords: free radical processes, sodium fluoride, liver rats.
Introduction
The study is a part of research work, which is performed at the Department of Clinical Pathophysiology, Topographical Anatomy and Operative Surgery of Kharkiv Medical Academy of Postgraduate Education "Radiotoxins pathophysiological mechanisms of the body, early diagnosis and correction".
The study of subtle mechanisms of adaptation of biological systems is an important part of modern patho-physiology. System-anti-systemic interaction of biological processes regulates the body and is responsible for normal life of hematothermals. Disorder of the antioxidant system of the body is due to the formation of pathological processes. According to the opinion of many researchers, activation of free radical processes and lipid peroxidation acts in the development of metabolically caused diseases [1,2,3,4]. Disorder of the liver antioxidant system will affect the resistance to environmental factors, particularly chemical factors that can act directly as a natural factor or due to human activity and lead to the disorders of systems and functions, and create conditions for premature aging [9,10,11].
Given the above, the aim of the study was to examine the condition of system and anti-radical and antioxidant protection of Wistar rat liver populations which received small doses of sodium fluoride.
Material and methods
The work involved animals - mature Wistar rat line -weighing 180-220 g. Rats were administered aqueous solutions of sodium fluoride (SF) orally in the morning on an empty stomach, once a day, in standard vivarium conditions, using a probe, for 60 days at doses of 1/10, 1/100 and 1/1000 LD50, which amounted 20 mg / kg, 2 mg / kg and 0.2 mg / kg of the body weight, respectively (SF average lethal dose for rats received orally, is 200 mg / kg). The animals in the control group received the appropriate amounts of drinking water. Research of indices was conducted on the 10th, 20th, 30th, 50th and 60th days after the start of the experiment. Each group consisted of 10 animals. The animals were euthanized by
decapitation with guillotine knife, with pre-anesthesia of thiopental sodium 50 mg / kg.
The level of free radical processes in the liver of rats, which were administered SF during 60 days at doses of 1/10 and 1/100 LD5o, was evaluated by the intensity of H202-induced chemiluminescence (7). The content of diene conjugates in rat liver homogenate was evaluated by spectrophotometer method [5,6,7]. The content of diene conjugates were calculated based on the molar extinction coefficient £ = 2.2 ■ 105 mol"1cm"1. The content of TBA-reagents in rat liver homogenate was determined by reaction between malondialdehyde and thiobarbituric acid (TBA), which at high temperature and acidic environment forms trimethine colored complex with maximum absorption at the wavelength of 532 nm [7]. The number of TBA-reagents was calculated based on the molar extinction coefficient £ = 1.56 ■ 105 mol"1cm"1. The level of schiff basics - products of interaction of carbonyl compounds and amino groups of proteins, amino acids, nucleic acids -was measured in the liver homogenate by spectrofluoro-metry method at the excitation wavelength of 360 nm and emission wavelength of 430 nm with the preliminary extraction with Folch mixture (chloroform-methanol) [8].
Statistical analysis of the results was carried out using the computer application package for the processing of statistical information Statistica 6.1 (StatSoft, Inc., USA).
Results and discussion
The level of free radical processes was evaluated (Table 1). The intensity of H202-induced chemiluminescence (CL) was measured at the 10th and 20th day (for 60 days) of the experiment in the liver of rats which were administered SF in doses of 1/10 and 1/100 LD50. The study found the statistically significant (p <0.001) increase, relatively to the control one, of the estimate of CL intensity to the doses by 37 and 134%, respectively. Interesting was the fact that on the 60th day oral administration of SF in doses 1/10 LD50, the statistically significant (p <0.001) reduction by 33% of CL intensity in the liver of rats compared to controls was observed.
* To cite this English version: Kolisnyk I.L. Condition of antioxidant activity of the liver in rats under the influence of small doses of sodium fluoride //Problemy ekologii ta medytsyny. - 2016. - Vol 20, № 3-4. - P. 37-42.
Table 1
The intensity of H202-induced chemiluminescence of rat liver homogenate by the action of sodium fluoride in doses subtoxic
(n = 10; Me [25%; 75%] or M ± s)
Day of observation Chemiluminescence intensity, imp / s
Control Dose, LD50
1/10 1/100
10 294±23.5 403±29.5 p<0.001 398 [320 418] p=0.002
20 296 [280; 335] 707±26.2 p<0.001 428 [415 440] p<0.001
30 321 ±14.0 650 [638; 689] p=0.003 598 [575 607] p<0.001
50 317±18.1 539 [498; 550] p<0.001 445 [432 487] p<0.001
60 343±14.5 229±19.9 p<0.001 409±19.7 p<0.001
Note: p - level of statistical significance relative to control
The effect of SF in dose 1/100 LD50 was accompanied by a reliably significant (r<0.002) increase, when compared to control, in the intensity of superweak luminance in all periods of observation, which was especially pronounced at the 30th day - an average of 85%. In subsequent periods, the level of chemiluminescence intensity in the liver of rats gradually decreased and on the 60th day of SF administration it amounted to 19% of the increase as compared with the control.
It has been proved that any disorder of dynamic equilibrium between pro- and antioxidants in many pathological conditions is displayed on CL intensity of biological substrates [7,8]. Given this fact, the increase in CL intensity of rat liver homogenate under the action of SF at doses of 1/10 and 1/100 LD50 can be explained by the shift of prooxidant-antioxidant balance toward the increased formation of pro-oxidants, including ROS. However, recently in the literature the involvement of ROS in signal transduction from the receptors to the cell nucleus is widely discussed, as well as the existence of ROS dependent regulation of redox signaling system [6, 7, 8]. The peculiarity of the latter is the property of ROS to cause the expression of genes, the products of which have antioxidant activity, which leads to increased buffer capacity and restoring of redox homeostasis. However,
the continued generation of abnormally large amounts of ROS can cause persistent changes in signal transduction and gene expression, impaired redox balance in the cells of oxidative stress, apoptosis and dysregulation as a consequence of pathological conditions. One should emphasize the fact that a large number of ROS act as inducers of oxidative damage to macromolecules basic cell, primarily proteins, in disorder of their functional activity. [9]. Change in any component of redox homeostasis leads to its imbalance, compensatory reactions on the local level, which probably arises under conditions of SF effects on rats and is shown in the increased CL intensity. Nonspecific nature and prevalence of oxidative stress in cells entails the use of energy substrates and important proteins to balance the redox system. Depending on the duration of oxidative stress and functional reserves of antioxidant system, depletion of reparation and adaptation capabilities of the organism occurs, which is probably on the 60th day of oral administration of SF at a dose of 1/10 LD50 and is confirmed by reduced CL intensity in rat liver homogenate.
Dynamics of changes in the intensity of CL in rat liver as a percentage relation to control is displayed in Figure 1.
Basic Basic Bask
Basic Basic Bask Baste Basic Basic
Bask Bask Bask Basic Basic
Dav of observation
- control I
Figure 1. Dynamics of CL intensity in the liver of rats at toxification by sodium fluoride at doses of 1/10 and 1/100 LD50
The intensity of lipid peroxidation (LPO) in liver of rats which were orally administered SF at doses of 1/10 and 1/100 LD50 for a long time, was estimated by the content
of its molecular products - diene conjugates (DC), TBA-reagents and schiff bases (Table 2).
Table 2
The content of the products of lipid peroxidation in rat liver homogenate (n = 10; Me [25%; 75%] or M ± s)
Dose Day of observation diene conjugates Nm/mg of protein TBA-reagents Nm/mg of protein Schiff bases CI/mg of lipids
10 9.09±0.88 p<0.001 0.70±0.07 p=0.059 0.37 [0.28; 0.40] p=0.545
20 8.12±0.88 p<0.001 0.91±0.08 p=0.001 0.33 [0.20; 0.47] p=0.290
1/10 LD50 30 7.64±0.60 p<0.001 1.04±0.12 p=0.001 1.08 [0.90; 1.15] p<0.001
50 6.9 [6.2; 7.8] p<0.001 1.45±0.15 p<0.001 1.53±0.16 p<0.001
60 6.2 [5.9; 7.2] p<0.001 1.76±0.16 p<0.001 1.83 [1.50; 1.91] p<0.001
10 7.65 [6.94 8.5] p=0.001 0.66 [0.60; 0.70] p=0.199 0.29±0.07 p=0.791
20 8.75 [8.54 9.8] p<0.001 0.89 [0.75; 0.94] p=0.019 0.24 [0.20; 0.33] p=0.821
1/100 LD50 30 7.35 [6.64 8.0] p<0.001 1.28±0.11 p=0.002 0.79±0.15 p<0.001
50 6.8 [6.0; 7.2] p<0.001 1.38±0.04 p<0.001 0.90±0.12 p<0.001
60 6.0±0.72 p=0.002 1.49±0.12 p<0.001 0.97±0.09 p<0.001
10 2.49±0.54 0.58 [0.52; 0.69] 0.33 [0.19; 0.50]
20 2.5 [2.3; 2.8] 0.72±0.13 0.29±0.15
Control 30 2.9 [2.5; 3.2] 0.75 [0.59; 0.86] 0.39±0.09
50 2.68±0.58 0.81±0.12 0.33±0.10
60 3.15 [2.8; 3.5] 0.74 [0.65; 0.88] 0.36±0.07
Note: p - level of statistical significance relative to control
The obtained results showed that oral administration of SF to rats in doses of 1/10 and 1/100 LD50 promotes statistically significant (r<0.002) increase, relative to the control group of animals, in DC content during the entire period of observation. In case of 1/10 LD50 dose, the
*
In the liver of rats toxified with SF, there was also the increased content of TBA-reagents (Table 2). On the 10th day of the experiment, the increase of indicator when compared with the value in control was unreliable for both doses (p = 0.059 and p = 0.199). In case of dose of 1/10 LD50, starting from the 20th day, the gradual in-
most significant increase in this indicator was observed on the 10th day of the experiment - by 265%, and in case of dose 1/100 LD50 - on the 20th day by average 234% (Figure 2).
crease of TBA-reagents was clearly defined (r<0.001) relatively to control - by 27, 41, 78, 133%. Similar dynamics of change was characteristic for the action of SF at a dose 1/100 LD50 as well - the increase of TBA-reagents was 19, 73, 70 and 99%, respectively, on the 20th, 30th, 50th and 60th day of observation (Figure 3).
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Bist M: Bask
Banc
s™ Bask
BlSk
Base
Day of observation
Bub
-LKLih • J l• - LI I-
Figure 2. Dynamics of diene conjugates content in the liver of rats at toxification with sodium fluoride at doses of 1/10 and 1/100 LD50
Figure 3. Dynamics of the content of TBA-reagents in the liver of rats at toxification with sodium fluoride
at doses of 1/10 and 1/100 LD50
On the 10th and 20th day of SF action at doses 1/10 and 1/100 LD50 no statistically significant changes in the content of the end products of lipid peroxidation - schiff bases were observed when compared to control. The results showed the significant gradual increase (p <0.001)
of this indicator on the 30th, 50th and 60th day - by 172, 370 and 380% respectively. The similar, but less pronounced dynamics was found for the dose 1/100 DL50 -by 103, 173 and 169% (Figure 4).
Day of observation
I -t-nmli -*-LMnH I
Figure 4. Dynamics in the content changes of schiff bases in the liver of rats at toxification with sodium fluoride
at doses of 1/10 and 1/100 LD50
In general, the obtained results reflect the initiation of lipid peroxidation process that can be viewed as a response mechanism of the rat's body to long-term effect of SF. Dynamics of lipid peroxidation products content has a certain dependence on the period of SF effect. In the initial stages (the 10th and 20th days) of oral administrating of SF at doses of 1/10 and 1/100 LD50 a marked increase in primary products of LPO - DC was detected, indicating that the activation of initial process chain. DCs are the most volatile products of lipid peroxidation, increase is their level usually reflects the significant impact of the intensity of pro-oxidants, such as ROS [10; 11]. The logical consequence of activation of lipid peroxida-tion in the liver of rats under SF action is the increase of secondary products - TBA-reagents (especially starting with the 30th day), indicating a more intense and deep
course of the process. However, in rat liver, beginning with the 50th day of SF action, LPO activation is more pronounced at the level of formation of end products -schiff bases which are regarded as indicators of chronic processes of free radical oxidation. Secondary and end products of lipid peroxidation, which are detected under long-term effect of SF, somehow contribute to the disorder of the microstructure of hepatocytes membranes, their permeability, reduce their division and regeneration, and inhibit the mitochondrial respiratory chain enzymes and microsomal monooxygenase system.
In order to confirm the orientation of the dynamics of lipid peroxidation in rats under conditions of prolonged exposure to SF at doses of 1/10 and 1/100 LD50, the ratio of schiff bases / (DC + TBA-reagents) was calculated (Table 3).
Table 3
The correlation ratio of schiff bases / (dienes +TBA-reagents) in rat liver under the action of sodium fluoride in subtoxic doses (n = 10;
Me [25%; 75%] or M ± s)
Day of observation schiff bases /(dienes +TBA-reagents), CI
Control Dose, LD
1/10 1/100
10 0.101 ±0.043 0.037±0.010 p<0.001 0.036±0.012 p=0.001
20 0.075 [0.053;0.154] 0.038±0.014 p=0.016 0.028±0.008 p=0.005
30 0.105±0.022 0.122±0.017 p=0.096 0.093±0.021 p=0.226
50 0.096±0.036 0.191 [0.160; 0.200] p<0.001 0.112±0.021 p=0.198
60 0.084 [0.071 ;0.115] 0.202 [0.187; 0.245] p<0.001 0.126±0.014 p=0.008
Note: p - level of statistical significance relative to control
The study has proved the statistically significant (r<0.016) reduction of the coefficient, as relative to control, on the 10th and 20th day dose at a dose of 1/10 DL50 by 63 and 59%, respectively, while on the 30th, 50th and 60th day, on the contrary, there was an increase by 16, 92 and 120%, respectively. In oral administration of SF at a dose of 1/100 LD50 value ratio decreased on the 10th, 20th and 30th day of the experiment (by 69, 70 and 11%, respectively) and on the 50th and 60th day - increased by 17 and 32% relatively to control values.
Conclusions. Increased antioxidant processes are probably due to excess number of free radicals and ROS, which occurs under the influence of prolonged exposure to small subtoxic doses of sodium fluoride. The observed increase in the coefficient of correlation schiff base / (dienes + TBA-reagents) in the liver under the action of SF clearly indicates the direction of LPO towards the formation of toxic end products - schiff bases, whereas the reduction - to the activation of LPO at the level of formation of primary and secondary products. According to the literature (1-4), such dynamics in the antioxidant protection of the liver is capable of leading to polymerization, destruction, modification of structures, changes in the activity of functions and metabolism disorders.
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