Kolisnyk I.L., Bagmut I.Yu. MORPHOFUNCTIONAL STATE OF HEPATOCYTES UNDER THE EXPOSURE TO SODIUM FLUORIDE

CHALLENGING ISSUES RELATING TO COMPREHENSIVE EXAMINATION IN MOTOR VEHICLE INJURIES: FORENSIC MEDICAL ASPECTS Zaritskyi H.


Introduction
Fluorine compounds are widespread in nature and known as industrial pollutants [1]. Fluorine does not exist in nature in a free state, but it forms inorganic and organic complex compounds, fluo-rides, whose content in the Earth's crust is approximately 0.06-0.09%. It is known that low concentrations of fluoride are necessary for normal growth and development of the body. However, in the case of excessive intake, significant dysfunctions of vital organs, cell damage, and necrosis occur [1]. Fluoride ion as a chemical agent affects metabolism that is manifested by functional and structural changes. The damaging factors include several pathogenetic mechanisms, and in particular: disruption of the energy supply of the processes taking place in cells and damage to membrane structures and enzyme systems of cells [2]. Sodium fluoride is a metabolic poison and primarily has a membrane damaging effect. In chronic chemical stress, systemic changes in the functional state of the body occur at all levels of integration, or a syndrome of an environmentally determined decrease in body resistance. The development of this syndrome results in the effect of chemical modification that is, an increase in the number of diseases of certain organs and the aggravation of their course against the background of a decrease in immune parameters and disrupted protective and adaptive capabilities [2]. With an increase in the amount of phosphorus-containing detergents in the body, diseases of the central nervous system, endocrine system, organs of the digestive system, and in particular, the liver, occur. The experiments have demonstrated that in the prolonged exposure to synthetic phosphorus-containing detergents leads to protein and fatty degeneration in the liver and lipid peroxidation products in the blood [3].
The aim of the study was to investigate the morphofunctional state of hepatocytes in the liver of white rats under the exposure to subtoxic effect of sodium fluoride.

Material and methods of the research
Mature Wistar rats (N = 17), weighing 180-210 g, were given intragastrical injections with aqueous solutions of sodium fluoride in a dose of 1/10 LD 50 at the ration of 20 mg / kg of animal body weight daily. The subacute experiment lasted 60 days, and at the end the animals were decapitated. When working with the test animals, we adhered all the requirements of the "European Convention for the Protection of Vertebrate Animals Used in Experiments and Other Scientific Purposes" (Strasbourg, 1986), Law of Ukraine No. 3447-IV as of February 21, 2006, "On the Protection of Animals from Cruelty".
To study hepatocytes in the rat liver and to assess their morphological rearrangement at the subcellular level of organization, we used electron microscopy. We fixed the pieces of the studied liver samples sized 1 mm 3 in a 2.5% solution of glutaraldehyde on 0.1M phosphate buffer of pH 7.2 and in 1% Palladian osmium fixative. After dehydration in solutions with increasing concentration of ethanol and absolute acetone, the material was embedded into epon-araldite mixture. By ultramicrotome we cut the preparations into semi-thin sections of 1-2 μm, then stained with methylene blue, and studied at the light-optical level. Then after targeted sharpening of the blocks, we obtained ultrathin sections, which were contrasted with uranium and lead salts according to Reynolds' staining technique. We studied the samples under an electron microscope at an accelerating voltage of 75 kV.

Results and Discussion
The liver is the main organ of xenobiotic metabolism. Electron microscopy of the liver taken from test animals showed a pronounced heterogeneity of hepatocytes. Along with the usual "dark" and "light" hepatocytes, "light" cells of much larger sizes were found out. These cells seemed to squeeze smaller "dark" hepatocytes. A control study of semi-thin sections made from the same blocks, using a light microscope, revealed many large cells, significantly larger than the normal ones, observed in the control group of animals, among which "dark" hepatocytes of relatively small sizes were localized. On specimens prepared by the standard histological methods for light microscopy, "light" cells were not detected. Clearing of the cytoplasm was most likely associated with the phenomena of intracellular edema, which is a sign of the disrupted water-electrolyte balance and the permeability of the cytoplasmic membranes.
The granular endoplasmic reticulum was moderately developed, but its cisterns were sharply expanded and formed large transparent vacuoles. In these cells, significant hypertrophy of the cisterns of the smooth endoplasmic reticulum was observed. The nuclei of hepatocytes remained oval, their chromatin was unevenly distributed. Lumps of chromatin were concentrated mainly along the nuclear membrane. Karyolemma was significantly loosened; the perinuclear spaces were enlarged. The lamellar cytoplasmic Golgi complex was moderately hypertrophied and consisted of stacks of smooth membranes surrounded by a large number of vesicles of various sizes. The nuclear membrane of hepatic cells became convoluted and formed folds with deep invaginations.
These changes indicate a disruption of bioenergetics of hepatocytes associated with the mitochondrial apparatus and the development of hypoxic processes, which lead to a decrease in the activity of redox reactions occurring at the level of intracellular membranes and organelles [5]. A consequence of these disorders is a decrease in the synthetic activity of hepatocytes that is structurally expressed in vacuolization of the cisterns of the granular endoplasmic reticulum, in a sharp decrease in the number of ribosomes associated with its membranes, freely lying in the cytoplasm. Electron microscopy revealed the presence of a large number of primary lysosomes at various stages of their activity, as well as autophagosomes: this indirectly indicates an increase in the concentration of metabolites in the cytoplasm of hepatocytes. A characteristic feature of the submicroscopic organi-zation of the liver under the influence of sodium fluoride is a higher intensity of changes in organelles in the cells localized in the areas adjacent to large blood vessels and less pronounced disturbances in the ultrastructures of liver cells located in the peripheral regions [6]. A manifestation of a particularly severe reaction on the part of hepatocytes is the presence of the edematous and cleared ones, with excessively swollen mitochondria, vacuolated endoplasmic reticulum, "light" hepatocytes, which seem to squeeze "dark" cells. In the cytoplasm of these cells, we found the foci of lysis of the nuclear membrane, outer membranes and cristae of mitochondria, the appearance of autophagosomes, which indicates the exhaustion of compensatory, synthetic and reparative reserves by intracellular structures and the development of catabolic processes at the membranous and macromolecular levels. The revealed submicroscopic disorders of the endothelial cells of sinusoidal capillaries indicate a disruption of the capillary wall permeability for electrolytes and metabolites. A sharp fall in the number of micropinocytic vesicles in the cytoplasm of endotheliocyte processes indicates a decrease in the intensity of transcellular transport of substances through the capillary wall [7]. It should be emphasized that the changes described above in the submicroscopic architecture of liver cells are mainly compensatory and adaptive and their intensity lies within physiological limits. They are reversible after removal of the pathogenic factor. At the same time, the signs of the development of a destructive process begin to appear in the individual cells [8].
The main enzyme systems involved in the conversion of xenobiotics are localized in hepatocytes, where, as a result of redox reactions and conjugation reactions, a foreign chemical is modified and eliminated by excretory systems. These enzyme systems are localized in mitochondria, microsomes, or hyaloplasm. The detoxification of chemical compounds can take place by the type of chemical oxidation, reduction, hydrolytic transformation, or by conjugation. The main laboratory carrying out these processes is the endoplasmic reticulum of liver cells, whose microsomes contain a significant amount of ribonucleic acids, phospholipids and proteins.

Conclusions
Thus, the study of the ultrastructural organization of the liver under the exposure of sodium fluoride revealed changes in the submicroscopic architecture characteristic of the development of dystrophic processes. Prolonged sodium fluoride intoxication causes a number of changes in the ultrastructure of the liver, expressed in the development of intracellular edema of hepatocytes, swelling of mitochondria, alterations in the density of their matrix, partial reduction and loss of cristae, vacuolization and expansion of the cisterns of the granular endoplasmic reticulum, an increase in the number of primary lysosomes, redistribution chromatin of the nucleus and a decrease in the number of ribosomes and glycogen granules. These changes indicates the disruption of bioenergetics of hepatocytes associated with the mitochondrial apparatus and the development of hypoxic processes that lead to a decline in the activity of redox reactions occurring at the level of intracellular membranes and organelles [9].
The damaging membrane trophic activity of free radicals and lipid peroxidation products develops in two main directions. The first is a disruption of the barrier properties of membranes. It is viewed as an acute injury that leads to cell death. The most important consequence of lipid peroxidation in membranes is an increase in their permeability for protons and calcium ions, a loss of membrane electrical stability that is manifested by a decrease in the electrical breakdown potential of the membrane. These changes can result in cell death. The second is a disruption of the physicochemical state of the lipid bilayer of membranes that is considered as their "soft" modification and leads to the development of chronic pathological conditions. A change in the matrix properties of membranes can be due to a reduced microviscosity of the lipid phase, an increase in negative charge, polarity, and a decrease in the hydrophobic volume.
Increased free radical processes can develop due to a variety of chemical intoxications, physical and biological harmful effects and refer to the general nonspecific reactions of the body to the damaging effect. The activation of free radical processes and lipid peroxidation act as a universal mechanism of membrane damage and is observed in different pathological conditions, including stress factors of various nature, and aging. The products of free radical oxidation, lipid peroxidation, acting on membranes, change the viscosity of the lipid bilayer, lead to the appearance of products that quench fluorescence, reduce electrical stability, hydrophobic volume, and disrupt the ionic permeability of membranes. Further investigation of the morphometric and biochemical changes in hepatocytes under the influence of synthetic phosphoruscontaining detergents to determine the intensity of damage and the possibility of correcting these changes with hepatoprotectors seems to be clinically important.