ABSTRACT

The liver, as the central metabolic organ, plays an important role in metabolism of carbohy­drates, lipids and proteins. In the environment, there are many hepatotoxic xenobiotics. CCl4 is a free radical toxic for organelles of hepatocytes. The presented work was studying the character of ultrastructural changes in CCl4-intoxicated hepatocytes as well as the tissue after treat­ment by grape seed extract (GSE), α-tocopherol (α-T). As a result of cell biore­mediation effect and reactions against corrup­tive factors, the liver has a high regeneration ability.

KEYWORDS

CCl4; GSE; α-Tocopherol; Ultra­struc­tu­ral Changes; Intoxicated Hepato­cytes; Electron Microscopy

1. INTRODUCTION

The liver, as human organ with high metabolic activity, plays an important role in metabolism of carbohydrates, lipids and pro­teins. As a result of cell protective coopera­tion and reactions against corruptive factors, liver also has a high regeneration ability [1,2]. In spite of considerable progress in hepatol­ogy [3-5], the study of corruption and re­generation mechanisms of hepatocytes on tissue, cellular and molecular levels remains to be very actual. Among toxins, which are widely used for modulating some damage of liver (liver cirrhosis—LC, hepatitis), the most popular is carbon tetrachloride (CCl4) [6].

Aim of the presented work was to study the character of ultrastructural changes in CCl4-intoxicated hepatocytes as well as the tissue after treatment by grape seed extract (GSE), α-tocopherol (α-T), as potential pro­phylaxis and treatment agents with antioxi­dant action at liver injury, mainly in experi­mental model of LC.

2. MATERIAL AND METHODS

2.1. Experimental and Preparation to Assays

In this study, we used white male rats weighting between 180 - 200 g, which were subdivided into four groups: I-control group (intact animals); II-group, animals with experimental liver damages; III-group, animals with experimental liver damages with GSE injection; IV-group, animals whit experimental liver damages with α-T. We induced liver damages by intraperitoneal injection of CCl4 at a dose of 150 µg per 100 g of body weight.

2.2. Electron Microscopy Аssays

For transmission electron microscope study we use liver tissue examples from control group as well as from experimental groups after CCl4, GSE, α-T influence. Preliminary fixation of the samples was carried out in 2.5% glutaraldehyde solution in phosphate buffer, and post fixation—in 1% solution of osmium tetroxide in the same buffer [7]. The samples were dehy­drated in ethanol solutions with increasing concentration, poured and soaked by aral­dite mixtures of resins. After polymeriza­tion, ultrathin sections were prepared for microtome “Reichert-Ultracut” consistent with their staining solution uranyl acetate and lead citrate [8].

The study of made ​​preparations and them pho­tomicrography was carried out using a transmission electron microscope “Tesla BS-500” in a voltage of 80 kV.

3. RESULTS AND DISCUSSION

Ultrastructural analysis of hepatocytes in the control group white rats liver has set a characteristic feature of hepatocytes ex­pressed the depositors function. In the cy­toplasm we can see a large number of polysomes, granular endoplasmic reticu­lum, vesicles, rosette-shaped glycogen granules, which are found in the area of lipid droplets without bounding mem­branes, lysosomes, rarely orthodox con­figuration mitochondria with the normal structure of the matrix and crista as well as heterochromatin and nucleolus in the nu­cleus of hepatocytes, granular-fibrillar structure with normal configuration of si­nusoidal capillaries, the structure of Kupffer cells and space of Disse (Figures 1(a) and (b)).

Electron microscopic study of the liver of albino rats after induction of liver toxic­ity revealed nonspecific damage of the mitochondrial membrane, mitochondrial swelling with fragmentation and destruc­tion of their

crista (Figure 2(a)). In the cyto­plasm of hepatocytes observed vesicula­tion, expansion of tanks of the endoplas­mic reticulum and perinuclear space with a pyknotic changed nucleus and marginated chromatin. (Figure 2(a)), as well as hyperplasia of the granular endoplasmic reticulum (Figure 2(b)) and the simultaneous loss of ri­bosomes, which results in the transforma­tion of granular endoplasmic reticulum in smooth (Figure 2(b)).

In the hepatocytes of the experimental animals in the areas of glycogen identified lipid droplets of various sizes and quanti­ties, evidence of fat and vacuolar degen­eration of the body. It was reveals a sharp expansion of sinusoidal space, increase in cellular structures of connective tissue, in­cluding collagen fibrils, bundles of which fill the intercellular space, indicating the onset of the initial stage of LC (Figure 3).

The ultrastructural changes are indi­cators of the activation energy, glycogen and protein synthesis processes in hepato­cytes in response to a damaging factor [9].

GSE influence manifested itself in a protective-restorative effect on the ultra­structure of mitochondria with

a decrease in the number of hepatocytes with lipid in­clusions and vacuolization (Figures 4(a) and (b)).

The ultrastructure of hepatocytes and or­ganization of sinusoidal cell space after GSE, as well as α-T was closer to the pic­ture of intact cells (Figure 5).

4. CONCLUSIONS

Thus, the model of liver toxicity and the LC, based on the nature of ultrastruc­tural changes of mitochondria and other compartments of hepatocytes, GSE, as well as α-T and STS, have corrective effect.

TEM ultrastructural characterization of the GSE influence is evidence of its success cytoprotective and antitoxic fea­tures as well as hepatocytes from xenobi­oticCCl4 induced by cirrhosis rat liver.

ACKNOWLEDGEMENTS

The study was done within the frame­work supported by Ministry of Education and Science of the Republic of Arme­nia (Basic support).

REFERENCES