RU2627444C1 - Method for simulation of subacute liver exposure to toxic formalin - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: formalin (CH₂O) is used, which is injected orally to male rats every day, at the rate of 0.1 ml per 100 g of animal weight, for 14 days. On day 14, animals are decapitate with subsequent laboratory tests and liver study. The model of subacute lesions caused by the oral introduction of formalin (CH₂O) is obtained, represented by increased degenerative processes in the liver, evolving creatininemia, increased transaminase level, development of cholestasis, and histologically proven by expressed degenerative damages of almost all sites of the liver.

EFFECT: method allows to obtain a model of subacute liver damage to be used in an experiment.

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Description

The invention relates to medicine, namely to experimental biology, toxicology, and can be used to study the formation and progression of changes in the liver that occur under the influence of toxic-chemical damaging factor.

One of the leading places in occupational pathology among employees of enterprises with particularly harmful and hazardous working conditions is toxic liver damage. Hepatotropic substances used in production, for example, formalin, are absorbed and deposited in the liver, undergoing methylation, amination, carboxylation, and oxidation with the participation of enzymes in the hepatobiliary system [TB. Popova, P.N. Lyubchenko, 1996; L.B. Lazebnik, E.Ya. Selezneva, 2004]. At the same time, poisons and products of their detoxification have an adverse effect on the pigment, protein, carbohydrate and enzymatic functions of the liver, which is accompanied by certain histomorphological changes that manifest themselves in an inflammatory reaction - toxic liver damage.

The use of formaldehyde in the production of resins as a bactericidal and balsamic agent, fungicide - as intermediates in the production of chemicals and as a component of the end use of consumer goods gives this substance a certain toxicological value in experimental modeling of toxic liver lesions.

The following methods are known for simulating toxic liver damage in an experiment: daily use of 10% ethanol solution (C ₂ H ₅ OH) instead of drinking water with free watering, with oral administration of formalin (CH ₂ O), to male rats, every other day, at the rate of 0 , 2 ml per 100 g of animal mass, for 21 days, followed by laboratory tests and liver tests (Kozlova V.V., Kotova M.E., Reps V.F. Method for modeling toxic hepatitis with formalin and ethyl alcohol in the experiment. Invention No. 2564758 from October 10, 2015); subcutaneous administration to rats of a 50% oil solution of carbon tetrachloride (Litvinova, E.S., Pharmacological correction of immune homeostasis disturbances due to combined exposure to hepatotropic agents and a constant magnetic field: abstract of dissertation, Candidate of Medical Sciences: 14.00.25. / Litvinova Elena Sergeevna. - Kursk, 2005 .-- S. 22)

The disadvantages of these methods include the introduction of carbon tetrachloride in a solvent, subcutaneously, although most occupational diseases are formed upon the ingress of toxic substances by inhalation and per os, the use of a toxic compound for modeling, as well as the combined use of two toxicants, which somewhat complicates the modeling process.

Closest to the technical nature of the present invention is a method of initiating toxic liver damage by oral administration to male rats of carbon tetrachloride (CCl ₄ ) grade ChP every other day, at the rate of 0.1 ml per 100 g of animal weight, for 21 days, followed by laboratory tests and liver tests (Kozlova V.V., Reps V.F., Kotova M.E. A method for modeling toxic damage by carbon tetrachloride in a toxicological experiment. Invention Patent No. 2487421 dated July 10, 2013).

The disadvantage of this method is the relative duration of the creation of an experimental pathological model of subacute lesion (21 days), as well as the use of carbon tetrachloride grade ChP, which is dangerous for the health of experimenters and has side effects on the nervous, respiratory and genitourinary systems of experimental animals, reducing the survival rate. It should be noted that the time indicator for creating experimental models of subacute toxic hepatitis with formalin for our modeling option is 14 days and with the daily introduction of a toxicant, it allows us to achieve the desired result in a shorter time.

The technical result of the proposed method is to solve the problem of creating a model of subacute toxic liver damage with formalin in a shorter time, daily oral administration, with an appropriate picture of the lesion, in a physiological way, with high reproducibility and less toxicity for the experimenter.

The advantages of the proposed model is that it is easily reproducible, less toxic for the experimenter, and the result is achieved in a shorter period of time compared to the prototype. Increasing the reproducibility of the formation of a subacute pathological process in the liver is achieved due to the daily oral administration of formalin to experimental animals and reducing the duration of the experiment to 14 days.

The specified technical result is achieved by the fact that daily formalin (CH ₂ O) was administered orally to male rats at the rate of 0.1 ml per 100 g of animal weight for 14 days, followed by laboratory tests.

To achieve the adequacy of the proposed model, 50 male Wistar rats weighing 200-215 g, divided into groups, were studied. The first group consisted of 10 intact animals. 20 rats, representing the second group, were daily orally administered formalin (CH ₂ O) at the rate of 0.1 ml per 100 g of animal weight for 14 days. To 20 rats of the third group, toxic damage was initiated by oral administration to male rats of carbon tetrachloride (CCl ₄ ) of grade ChC every other day, at the rate of 0.1 ml per 100 g of animal weight, for 21 days, according to the prototype method.

On days 14 and 21, the animals were decapitated, followed by laboratory tests and pathomorphological and histological studies of the liver. The serum levels of aspartate aminotransferase (ACT) and alanine aminotransferase (ALT) in U / L, creatinine (KR) in micromol / L, alkaline phosphatase (ALP) in IU / L, cholesterol (X) in mmol / L, triglycerides (TG) were studied. ) in mmol / l.

The method was carried out as follows. A group of animals was orally administered formalin (CH ₂ O) daily for 14 days at the rate of 0.1 ml of solution per 100 g of animal weight. Reproduction of the model was ascertained by a change in biochemical parameters, such as serum aspartate aminotransferase (ACT), alanine aminotransferase (ALT), creatinine (CR), alkaline phosphatase (ALP), cholesterol (X), triglycerides (TG).

Formalin (CH ₂ O) was prepared by dissolving formaldehyde in water to a concentration of 40%.

The method was tested in the conditions of the department for studying the mechanisms of the action of physical factors (OIMDFF) FSBI PGNIIK FMBA of Russia.

The object of the study was 50 Wistar male rats, divided into 3 groups - intact, with a model of subacute formalin intoxication and with a prototype model. At the end of the experiment, the resulting pattern of pathogenetic reactions in systems that control metabolic reactions was compared.

The method can be illustrated by the following examples.

An intact male Wistar rat No. 3, body weight 200 g. No additional effects. The study of the animal was carried out simultaneously with the animals of the experimental group. The blood content of KR is 78.5 μmol / L, alkaline phosphatase is 392.9 IU / L, ACT is 137.1 U / L, ALT is 80.0 U / L, X is 1.8 mmol / L, and TG is 2.1 mmol / L.

Male Wistar rat No. 11, body weight - 205 g. The animal was administered daily oral administration of formalin (CH ₂ O) at the rate of 0.1 ml per 100 g of animal weight for 14 days. KR - 110.8 μmol / L, alkaline phosphatase - 454.2 IU / L, ACT - 202.0 U / L, ALT - 110.0 U / L, X - 0.8 mmol / L, TG - 1.7 mmol / l. The result is regarded as subacute poisoning.

Male rat No. 47 of the Wistar line, body weight 210 g. After a day, 1 time per day, for 21 days, 0.1 ml of carbon tetrachloride grade CC (CCl ₄ ) per 100 g of animal weight was injected orally with a syringe. The blood content of KR is 96.7 μmol / L, alkaline phosphatase - 500.2 IU / L, ACT - 299.5 U / L, ALT - 138.4 U / L X - 1.7 mmol / L, TG - 1 9 mmol / L. The result is regarded as subacute poisoning.

From the table it follows that the increase in the content of ACT; ALT (P <0.001), as well as an increase in CR (P <0.001) in blood serum indicate an increase in the degree of liver damage and indicate damage to hepatocytes, a significant decrease in X and TG (P <0.001, P <0.01) indicates a decrease in the synthetic function of the liver, and an increase in the level of alkaline phosphatase (P <0.001) - for the development of cholestasis.

To confirm the reliability of changes in the obtained biochemical parameters, pathological and histological studies were additionally carried out, for which macro- and micropreparations were studied.

Macro preparations

Group 1. In a pathomorphological study, the liver of rats of normal sizes 2.5 ± 0.5 cm by 2.42 ± 0.3 cm, average weight 6.65 ± 0.3 g. The capsule of the liver is dense, smooth, shiny; the consistency is dense. All rats had a liver with a red-brown incision, without pathological foci.

Group 2. In a pathomorphological study, the liver of some rats was ocher-red, light brown with multiple hemorrhages. The organ is hypertrophied, in most cases loose, in some places with densified areas; dimensions 4.3 ± 0.4 cm by 6.6 ± 0.5 cm; average weight 10.23 ± 0.7 g. In most rats, the liver on the incision is dry, blood does not protrude, in some cases the blood on the incision protrudes moderately.

Subacute lesion is confirmed by an increase in liver size by 3.58 g or 53.83%. The hepatotoxic effect of formalin while taking 10% ethyl alcohol on rat liver was confirmed with a reliable probability (P <0.001).

Group 1. Microscopic picture of the liver of intact animals without pathology. The beam structure of the liver is not broken. Hapatocytes contain oval nuclei with well visible nucleoli. The presence of fatty degeneration was detected in single hepatocytes.

Group 2. During a histological examination of the liver of 20 poisonous rats, hepatocyte dystrophy is determined in the form of swelling, their cytoplasm is cloudy, the cell borders are fuzzy, and the nuclei are also swollen. Individual cells are very large and represent a continuous vacuole. The main localization of altered hepatocytes is mainly in the periportal zone, discomplexation of hepatic beams and proliferation of connective tissue in portal tracts with thickening of fibrous cords are immediately formed. In the described areas - lymphoplasmacytic infiltration with an admixture of single segmented white blood cells. In 30% of cases, small foci of necrosis of various sizes were found, in which the structural elements of the cells are not visualized, and the liver tissue is a homogeneous structureless mass. Among the cells, a large number of leukocytes, macrophages. In general, the vast majority of rats showed a toxic hepatitis pattern with a fairly high rate of tissue damage. The detected changes indicate the development of a typical toxic liver dystrophy in animals.

Thus, a model of subacute intoxication caused by formalin was obtained, which manifests itself in an increase in degenerative processes in the liver, developing creatininemia, the development of cholestasis and histologically confirmed in severe degenerative lesions of almost all parts of the liver, i.e. the proposed modeling method is quite informative and shows the effectiveness of its use in the experiment.

Claims (1)

A method for modeling subacute toxic liver damage, characterized in that formalin (CH ₂ O) is used, which is administered to male rats orally, daily, at a rate of 0.1 ml per 100 g of animal weight, for 14 days, followed by laboratory tests .