THE DEVELOPMENT OF MEMBRANOUS PATHOLOGY OF HEPATOCYTES THE INFLUENCE OF INTOXICATION *

We studied the subtoxic effect of small doses of sodium fluoride on the activity of microsomal hepatocytes on 30 Wistar rats' populations in subacute experiment. The intensity of lipid peroxidation (LPO) in liver of rats which were administered sodium fluoride orally for a long time at doses of 1/10 and 1/100 LD50, was evaluated by the content of its molecular products diene conjugates (DC), and MDA-reagents Schiff bases. We found that oral administration of sodium fluoride to rats at doses of 1/10 and 1/100 LD50 promotes a statistically significant increase (r≤0,002) relative to the control group of animals in DC content during the entire period of observation. At a dose 1/10 LD50 we observed the most significant increase in this indicator on the 10th day of the experiment at 265%, and at a dose 1/100 LD50 – on the 20th day an average of 234%. In rats’ liver by the action of subtoxical dose of sodium fluoride at a dose of 1/10 LD50, starting from the 20th day, we detected a gradual increase (r≤0.001) of TBA-reagents relative to control for 27, 41, 78, 133%. Secondary end products and lipid peroxidation, which are defined under the long-term of sodium fluoride, somehow contribute to the disruption of the microstructure of hepatocytes membranes, their permeability, reduce their division, regeneration and inhibition of mitochondrial respiratory chain enzymes and microsomal monooxygenase system.


Introduction
The main biological role of fluoride and its compounds consists in bone formation, formation of dentin, enamel, preventing the development of senile osteoporosis.High concentrations of fluoride stimulate lipid peroxidation and inhibit antioxidant defense system.It belongs to the elements of the first class of danger -especially hazardous chemicals.Prolonged excessive intake of fluoride compounds in the composition of the body can cause pathological condition -fluorosis.Despite the significant content of fluoride in different tissues of the human body, its physiological role so far has not been clarified.
The processes of free radical oxidation constitute an important part of nonspecific metabolic component of adaptation to the effects of stress factors, including chemi-cal origin.Evidence of this position is confirmed by the nature of radical systems, especially on electron transport chain of endoplasmic reticulum and mitochondria.Adaptive adjustment of the oxygen metabolism due, for example, to stress, xenobiotics biotransformation accompanied usually by certain shifts in the mode of formation of free radical intermediates [1,2,3].
The aim of this study is to investigate the state of free radical processes and lipid peroxidation in the liver of rats with chronic fluoride intoxication.

Material and methods
Studies were conducted on mature rats of Wistar line weighing 180-220 g, which were kept at hospital vivarium.Rats were subjected to oral seed probe using aqueous solutions of sodium fluoride (FS) once daily for 60 days at doses of 1/10, 1/100 and 1/1000 LD50, which were under 20 mg/kg, 2 mg/kg and 0.2 mg/kg body weight (FS average lethal dose for rats received orally, is 200 mg/kg).The animals in the control group were injected with appropriate amounts of drinking water.Research conducted indices 10, 20, 30, 50 and 60 days after the start of the experiment.Each group had 10 animals.Rats were euthanized by decapitation with guillotine knife, pre-anesthetized with thiopental sodium 50 mg/kg.
The content of diene conjugates in rats' liver homogenate was evaluated by spectrophotometer at 233 nm with pre-extraction with heptane izopropanol mixture [2,3,4].The content of diene conjugates was calculated based on the molar extinction coefficient ε=2,2•105 mol-1cm-1.The content of TBA-reagents in rats' liver homogenate was determined by reaction between malondialdehyde and tiobarbiturats acid (TBA), which at high temperature and acidic environment is to form trymetynes colored complex with maximum absorption at a wavelength of 532 nm [5].Number of TBA-calculated reagents was based on the molar extinction coefficient ε=1,56•105 mol-1cm-1.The level of Schiff bases -products of interaction of carbonyl compounds and amins groups of proteins, amino acids, nucleic acids -was measured in the liver homogenate by spectrofluorometer at a wave length of 360 nm, excitation and emissionwave length of 430 nm from the previous extraction by Folcha mixture (chloroform-methanol) [6].
Statistical analysis of the results was carried out using a computer application package for processing of statistical information Statistica 6.1 (StatSoft, Inc., USA).

Results and discussion
The level of free radical processes in the liver of rats, which during the 60 days were administered in doses of FS 1/10 and 1/100 LD50 was evaluated in the intensity of H2O2-induced chemiluminescence (CL).On the 10th and 20th day of the experiment, we observed a statistically significant increase (p<0.001) relative to the control rate of case of FS action at a dose 1/10 LD50, respectively, 37 and 134%.On the 30th day we registered a slight decrease (37%) of CL intensity in relation to the value of the previous observation period, but compared to controlincrease (p<0.001) by 66%.It is necessary to point out that on the 60th day of oral administration of FS to rats at doses 1/10 LD50 there was a statistically significant (p<0.001)reduction of 33% of CL intensity in the liver of rats as compared with control.Action of FS at a dose 1/100 LD50 was accompanied by a rather significant increase (r≤0.002) when compared to control intensity su-perweak glow in all periods of observation, especially pronounced in the 30th day -an average of 85%.In terms of level of intensity, following chemiluminescence in the liver of rats it gradually decreased and on the 60th day of FS administration it was 19%.
It is proved that any disruption of dynamic equilibrium between pro-and antioxidants in many pathological conditions is displayed on CL intensity biological substrates [4,5].Thus, the increase in intensity of rat liver homogenate CL for actions in the UNFPA doses of 1/10 and 1/100 LD50 can be explained by a shift prooxidantantioxidant balance toward increased formation of prooxidant, including the AFC.However, recently the scientists have widely discussed the involvement of ROS in signal transduction from the receptors to the cell nucleus and ROS regulation being dependent on redox signaling system [6,7,8].
The peculiarity of the latter is a property of ROS to cause the expression of genes, the products of which have antioxidant activity, which leads to increased buffer capacity and restoring 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 of 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 acts as inducers of oxidative damage to macromolecules basic cell, primarily proteins, in disruption of their functional activity [9].Changing any component of redox homeostasis leading to its imbalance of compensatory reactions on the local level, which probably arises under conditions of FS effects on rats showed increased intensity and refrigerator.The prevalence of nonspecific oxidative stress in cells entails the use of energy substrates and proteins are important to balance the redox system.Depending on the length of oxidative stress and antioxidant system functional reserves depletion occurring in reparation and adaptation of the organism, which is probably on the 60th day of oral dose of FS 1/10 LD50 and is confirmed by a reduction in the intensity of CL homogenate in rats' liver.
The intensity of LPO in liver of rats which were administered FS orally for a long time at doses 1/10 and 1/100 LD50, was evaluated by the content of its molecular products -diene conjugates (DC), TBA-reagents and Schiff bases.The results showed that oral administration to rats in doses of FS 1/10 and 1/100 LD50 promotes statistically significant (r≤0.002)relative to the control group of animals increase in GC content during the entire monitoring period.If the dose was 1/10 LD50, the most significant increase in this indicator was observed on the 10th day of the experiment -at 265%, and if the dose was 1/100 LD50 on the 20th day -an average of 234%.In the liver of rats FS toxification was also increasing the content of TBAreagents.On the 10th day of the experiment, there was an increase when compared with the value in control was unreliable for both doses (p=0.059 and p=0.199).If the dose 1/10 LD50, starting from the 20th day, clearly defined gradual increase (r≤0.001) of TBA-reagents relative to control -for 27, 41, 78, 133%.Similar dynamics was characterized by change and action to FS in a dose 1/100 LD50-an increase of TBA-reagents was 19, 73, 70 and 99%, respectively, on the 20th, 30th, 50th and 60th day of observation.On the 10th and 20th day of FS action at doses 1/10 and 1/100 LD50 when compared to control statistically significant changes in the content of the end products of lipid peroxidation -Schiff bases were observed.The results showed a significant gradual increase (p<0.001) of this indicator on the 30th, 50th and 60th day -172, 370 and 380% respectively.A similar, but less pronounced dynamics was found to 1/100 the dose LD50 -103, 173 and 169%.
In general, the results obtained reflect the initiation of LPO process that can be regarded as a mechanism response on organism rats on long-term effect of FS.Dynamics of LPO products content has a certain dependence on the term of the FS.In the initial stages (10 and 20 days) oral doses of FS 1/10 and 1/100 LD50 recorded a marked increase in primary products of lipid peroxidation -DK, indicating the initial activation process.DK is the most volatile products of lipid peroxidation, increase of their level usually reflects the significant impact of the intensity of the pro-oxidant, such as ROS [10,11].
The logical consequence of activation of lipid peroxidation in the liver of rats under FS action is to increase secondary products -TBA-reagents (especially since the 30th day), indicating a more intense and deep course of the process.But in rat liver, beginning with the 50th day, FS LPO activation is more pronounced at the formation of end products -Schiff bases, which are regarded as indicators of chronic processes of free radical oxidation.Secondary end products and lipid peroxidation, which are under long-term FS somehow contribute to the disruption of the microstructure of hepatocytes membranes their permeability, reduce their division, regeneration and inhibition of mitochondrial respiratory chain enzymes and microsomal monooxygenase system [12][13][14][15].
To confirm the orientation of the dynamics of lipid peroxidation in rats under conditions of prolonged exposure to doses of FS 1/10 and 1/100 LD50 calculated the ratio Schiff bases/(DC+TBA-reagents).It proved statistically significant (r≤0.016)relative to control reduction coefficient of 10 and 20 th day dose in steps UNFPA 1/10 LD50 respectively 63 and 59%, while 30, 50 and 60th day, on the contrary, increased respectively by 16, 92 and 120%.In oral administration at a dose of FS 1/100 LD50 value ratio decreased on the 10th, 20th and 30th day of the experiment (respectively 69, 70 and 11%) and on the 50th and 60th day -increased to 17 and 32% relative to control values.

Conclusion.
The observed increase in the coefficient of correlation Schiff bases/(dienes+TBA-reagents) in the liver under the action of UNFPA clearly indicates the direction of LPO towards the formation of toxic end products -Schiff bases and reduce the activation of POL at the formation of primary and secondary products.