Oxidative processes peculiarity in rats affected by carbon tetrachloride based on malathion intoxication

Topicality. The chemical pollution of environment and its impact on the human body is one of the problems not fully studied. It is a question of priority. Carbon and tetrachloride malathion are the most dangerous contaminative elements considered to be the risk factors for many environmentally dependent diseases. Therefore, it is actual to study the characteristics and mechanisms of the aforementioned xenobiotics combined action on the body. Aim. To investigate the free radical processes activity and the endogenous markers of intoxication at the conditions of effect on the rats organism by tetrachloride bbeing affected by malathion. Materials and methods. Experiments conducted on white rats being under carbon tetrachloride intoxication influence (4th, 7th days of the affection) on the basis of thirtieth days malathion affection . Rats were taken out of the experiment by the thiopental anesthesia. Results and discussion. The experimental data proved the malathion and carbon tetrachloride toxic effect, which increases by the conditions of their combined using. Conclusions. It was established, that the maximum activation of free radical processes and deepening endogenous intoxication observed on the seventh day affection by the carbon tetrachloride on on the basis of thirtieth days malathion intoxication.

Ключевые слова: тетрахлорметан; карбофос; крысы; эндогенная интоксикация; свободнорадикальные процессы INTRODUCTION The study of combined effect characteristics and mechanisms of the most common xenobiotics as the main factors of the many environmentally dependent diseases development is the most perspective biology and medicine direction in the environmental pollution conditions. These toxins are organophosphorus compounds (OPs), and chlorinated hydrocarbons.
The body contamination with OPs leads to the development of the oxidative stress, which themself are significant toxicity agents [1,2]. OPs characterized by a high biological activity. OPs include malathion with a middle degree of toxicity.
One of the most dangerous xenobiotics are chlorinated hydrocarbons including carbon tetrachloride (CCl 4 ), which is quite widely used at workplaces, homes and agriculture. Carbon tetrachloride known as a classical hepatotropic poison and its toxic effects on the liver was well studied in experimental models [3,4,5].
Usually it often happens the combined effect of several toxic agents, which can disrupt the balance of oxidative processes in the body and cause serious diseases.
Objective. The aim of the presented study was to investigate the free radical activity and the level of endogenous intoxication in rats under simultaneous affection of malathion and carbon tetrachloride.

MATERIALS AND METHODS
Experiments were conducted on white rats with the weight 175-200 g. The animals were kept on a standard food allowance at the vivarium of I. Horbachevsky Ternopil State Medical University. All experiments on animals were carried out in compliance with the general rules that were approved at the National Congress on Bioethics (Kiev, Ukraine 2001) and the 1-st Ukrainian National Congress on Bioethics subjected to the NIH Guide for the Care and Use of Laboratory Animals (Guide., 2011).
The animals were divided into five groups: the 1-stintact control; the 2-nd -the animals were affected with malathion for 10 days and on the 4-th day the rats were affected with tetrachloromethane, the 3-d group of animals -10 days affecting by malathion and on the 7-th day intoxication with ССl 4, , the 4-th group -the rats were affected by malathion for 30 days and on the 4-th days was caused the development of tetrachloromethane hepatitis, the 5-th group of animals -30 days affecting with malathion and on the 7-th day affecting with ССl 4 .
Malathion was daily administrated intragastrically in the form of the aqueous solution of 20 mg/kg on the weight of a rat, that is equal 1/10 from the LD 50 . Carbon tetrachloride was administrated intragastrically, twice, in a day duration in the form of 50 % oil solution in the dose of 1.0 ml/kg calculated on a weight of the animal.
The rats were euthanized with sodium thiopental. The investigation objects were a blood serum, a myocardium and a liver of the animals.
The activity of the free radical processes in the rats bodies was evaluated concerning to the amount of TBAactive products in a blood serum, homogenates of myocardium and liver of the animals [6,7] and the oxidation modification of protein by 2,4-Dinitrophenylhydrazine (2,4 DNPh) [8].
The level of the endogenous intoxication studied according to the content of molecules of the average weight (MAW) in a blood serum (BМ 1 fraction BМ 2 ) [9] and the degree of damage of the erythrocyte membrane (ЕІІ) [10,11].
The statistic processing of the received results were carried outby the "Statistica 6,0" with the applying of Student's t-criterion. Results were considered significant at p ≤ 0.05.

RESULTS AND DISCUSSION
The conducted experiments showed that, after administration of xenobiotic into the rats bodies it was observed the activation of lipid peroxidation. One of the main product of LPO is TBA-AP which allowed us to judge the intensity of these processes (Tab. 1).
The content of TBA-AP increased on 42 % in a blood serum, 35 % -in a liver, 14 % -in a myocardium after

THE CONTENT OF ТBA -ACTIVE PRODUCTS IN A BLOOD SERUM (µmol/L), LIVER (µmol/kg) AND MYOCARDIUM (µmol/kg) OF THE RATS, AFTER COMBINED AFFECTING BY TETRACHLOROMETHANE AND MALATHION (M ± m; n = 30)
Groups of animals Term of the investigation, days 10 + 4ССІ 4 10 +  the administration of the toxins on the 10-th and 4-th day of affection. At ten days intoxication by malathion and on the 7-th day after the administration of carbon tetrachloride, this index increased in a blood serum on 85 %, 41 %in a liver, 21 % -in a myocardium. After thirty days administration of malathion and on the 4-th day of intoxication by ССl 4 , the content of TBA-AP increased on 185 % in a blood serum, 60 % -in a liver, 39 % -in a myocardium regarding to the level of the intact animals. On the 30-th and the 7-th days of the administration of toxins, the level of TBA-AP increased on 234 % in a blood serum, 84 % -in a liver, 48 % -in a myocardium regarding to the standard.
The obtained results indicate a significant activation of lipid peroxidation in the bodies of the rats at the combined affection by malathion and carbon tetrachloride. The simultaneous administration of the toxins has a synergistic effect.
Excessive formation of ROS in the affected body by toxicants not only leads to the activation of lipid peroxidation, bus intensifies the processes of oxidative modification of proteins [8,12]. The study of the OMP indicators showed that a blood serum and liver of the rats after the intoxication by xenobiotics increased the level of 2,4-DNPh neutral (370 nm) and basic character (430 nm).
The content of 2,4-DNPh neutral in a blood serum and a liver of the affected animals increases on 25 % and 31 % respectively at ten days administration of malathion and on the 4-th day after the intoxication by ССl 4 , data indicated in Tab. 2.
A similar tendency to rising it was also observed for 2,4-DNPh basic at the affecting animals. At ten days intoxication by malathion and on the 7-th day after the administration of carbon tetrachloride, the amount of 2,4-DNPh neutral in a blood serum increased on 25 %, 38 % -in a liver; 2,4-DNPh basic increased on 32 % in a blood serum and in a liver regarding to its level in the intact animals.
After 30 days administration of malathion and on the 4-th day intoxication by ССl 4 the level of 2,4-DNPh neutral increased on 33 % in a blood serum, on 46 % in a liver of the affected animals. In the same period the content of 2,4-DNPh basic character increased on 38 % in a blood serum and on 42 % in a liver.
On the 30-th and 7-th days of the intoxication by the toxins the content of 2,4-DNPh neutral character increased on 50 % in a blood serum of the affected animals, on 54 % in a liver. The amount of 2,4-DNPh basic character increased on 38 % in a blood serum, on 33 %in the liver.
At the combined affection by malathion and carbon tetrachloride it was established the activation of the free radical processes and formation of the toxic products, causing a destructive impact on bio-macromolecules, these lead to development of endogenous intoxication of the body, the level of which is evaluated by the content of MAW.
After the intoxication we observed a significant increasing of the content of fractions MAW 1 (dominated by aliphatic amino acids) and MAW 2 (dominated by aromatic amino acids) in a blood serum of the experimental rats during a whole experiment (Tab. 3).
On the 30-th and 7-th day of the experiments under the affecting of toxins the content of MAW 1 increased on 132 % in a blood serum, the content of MAW 2 increased on 96 % in a blood serum, relatively to the level of the intact control.
Toxic products were formed as a result of the activation of lipid peroxidation process and the accumulation of endogenous toxins formed by ROS activity that can cause destruction of cell membranes and change their permeability [13,14].
We studied the permeability of the erythrocyte membrane at combined action of the studied toxins (Tab. 4).
During the experiments it was detected the relative increasing of EII after the affecting of the rats by malathion

THE CONTENT OF THE MOLECULES OF THE AVERAGE WEIGHT (MAW) IN A BLOOD SERUM (unit/L) OF THE RATS AFTER COMBINED AFFECTING BY TETRACHLORMETHANE AND MALATHION (M ± m; n = 30)
Group of animals МСМ 1 (254 nm) МСМ 2 (280 nm) A blood serum A blood serum Intact control 0.99 ± 0.10 1.11 ± 0.16 Affected 10 + 4ССl 4 1.62 ± 0.02* 1.68 ± 0.04* Affected 10 + 7ССl 4 1.79 ± 0.04* 1.90 ± 0.05* Affected 30 + 4ССl 4 2.21 ± 0.04* 2.13 ± 0.03* Affected 30 + 7ССl 4 2.30 ± 0.05* 2.18 ± 0.03* Український біофармацевтичний журнал, № 2 (49) 2017 and carbontetrachloride. It was established that the permeability of the erythrocyte membrane highly increased and till the end of the experiment it was 16 % above the norm. Our results showed that the intoxication of the rats by carbon tetrachloride become stronger under the action of malathion on the body. Lipid and protein peroxidation processes significantly intensified that evidenced by the veritable increasing of TBA -active products and 2,4-DNPh in all investigated rat's tissues.It was established that the simultaneous affection of the rats by malathion and carbon tetrachloride leads to the development of endogenous intoxication and accumulation of the high amount of endogenous toxins especially the content of molecules of the average weight that can be served as a marker for assessing the degree of intoxication in the affected body.
The obtained results indicate the toxicity effect of the mentioned xenobiotics on erythrocyte membrane, that improved by the percentage increasing of their permeability and as a result can cause the destructive influence of toxicants on the structural components of cell membranes.

CONCLUSIONS
The lipid peroxidation activation and proteins oxidative modification caused by the combined carbon tetrachloride and malathion affection on the body leads to the development of endogenous intoxication. At the same condition it was observed the veritably of increased molecules average weight content in a blood serum. Exogenous and endogenous toxins perform a destructive influence on cell membranes, including red blood cells, as evidenced by the increasing of erythrocyte index of intoxication after affecting.
Conflicts of Interest: authors have no conflict of interest to declare.