RU2232387C1 - Method for evaluation of general toxic effect of medicinal agents on body - Google Patents

Abstract

FIELD: medicine, laboratory methods of analysis.

SUBSTANCE: method involves microscopic analysis of serum blood. Sample of serum blood from intact animals and animals exposed to a medicinal agent to be study is applied on the slide surface in the amount 20 mcl, covered by cover glass, dried at room temperature for 48-72 h and comparative analysis of anisotropic morphotypes is carried out. The conclusion about the unfavorable effect of analyzed medicinal agent on body is done in the case of elevating content of secondary morphotypes in experimental samples in combination with simultaneous reducing number of normal morphotypes as compared with control. The conclusion about toxic effect of drug is done in the case of presence of atypical morphotypes or amorphization state in experimental samples. Method is simple by technical performance, shows economy and doesn't require expensive equipment and reagents.

EFFECT: improved evaluation method.

7 tbl, 7 dwg, 4 ex

Description

The invention relates to medicine, namely to laboratory research methods, and can be used, in particular, to assess the general toxic effect of drugs on the body.

A known method for evaluating the general toxic effect of drugs, consisting in the study of biochemical parameters of blood serum characterizing the functional state of internal organs and systems: indicators of bromosulfalein test, determination of total protein, protein fractions, total serum cholesterol, alkaline phosphatase activity, transaminases, blood glucose , bile acids, determination of creatinine, residual nitrogen, urea, chlorides in the blood and urine (Study of “chronic” toxicity and // Methodological recommendations for the study of the general toxic effects of pharmacological agents / Compiled by E.V. Arzamastsev, T.A. Guskova, S.S. Liborman, B.I. Lyubimov, A.G. Rudakov, O.L. Verstakova. - M., 1997 .-- 10 p.).

However, the method has significant drawbacks that limit its use in the practical activities of the laboratory - the need for a large amount of the test material (at least 2 ml), conducting blood serum studies in the first hours after its receipt, the use of expensive reagents.

There is also a method for assessing the toxic effects of drugs based on the determination of a number of hematological parameters: hemoglobin content, number of red blood cells, white blood cells, platelets, reticulocytes, leukocyte composition, blood coagulation rate, red blood cell resistance, hematocrit (Timofievskaya L.A., Evtushenko G. Yu. Methods of researching the blood system and blood formation // Methods for determining the toxicity and hazard of chemicals. - M.: “Medicine”, 1970. - S. 219-225). The disadvantages of this method are the multi-stage implementation of the method and the need to use expensive equipment and reagents.

Closest to the proposed method is a method for assessing the toxic effect of drugs, which consists in a pathological study of biological tissues of the body (Morphological studies. // Hygienic criteria for environmental conditions, 6. - Part 1. Principles and methods for assessing the toxicity of chemicals. - Geneva: WHO , 1981. - S. 204-216). The similarity of this method with the proposed one lies in the fact that both of them belong to laboratory research methods and are based on a microscopic study of the morphological parameters of the obtained samples.

The disadvantages of this method are:

- multi-stage (taking material, fixing, dehydration, pouring, preparation of sections, their coloring);

- the duration of the study (at least 10 days from the moment of taking the material);

- the need to use expensive, toxic reagents and dyes;

- expensive technical support of the method.

The present invention solves the main problem - the simplification of the method. The task was to eliminate these shortcomings and create a diagnostic method that would allow in a short time on small volumes of biological fluids and economically establish signs of the general toxic effect of drugs on the body. This is achieved by the fact that in the method for evaluating the general toxic effect of drugs on the body, including microscopic examination of dried blood serum, the blood serum of intact animals and animals receiving the test drug in the amount of 20 μl is applied to the surface of a glass slide, covered with a coverslip, dried with room temperature for 48-72 hours, perform a comparative analysis of anisotropic morphotypes and with an increase in the content of secondary morphotytes in experimental samples s, along with simultaneous reduction of morphotypes norm as compared to the control judged on the adverse effects on the organism of study drug, and in the presence of test samples atypical morphotypes amorphization or condition judge its toxic action.

Blood serum is an organism’s tissue, but highly mobile tissue due to weak intermolecular bonds between its elements. The structure of these bonds can be identified by their fixation - slow dehydration.

The essence of the invention is illustrated by photographs, where:

Figure 1 - Basic morphotypes:

a) spherulites; b) filiform

Figure 2 - Secondary morphotypes:

a) polymorphic; b) fan;

Figure 3 - Atypical morphotypes:

a) colored needles; b) spherulites with the presence of foreign inserts; c) amorphization state

The method is as follows.

The blood serum of intact animals and animals receiving an intragastric drug in the amount of 20 μl is applied to the surface of a standard slide of 75 × 25 mm, covered with a slide of 18 × 18 mm and dried at room temperature 18-25 ° C, relative humidity 50- 70% within 48-72 hours. Preliminarily, the glasses are soaked for 24-48 hours in a detergent solution, then washed in running water for 10 minutes and placed in a Nikiforov mixture consisting of equal proportions of alcohol and ether for 30 minutes. Before applying the sample, wipe the glass with a dry, lint-free cloth. The samples obtained are viewed in polarized light, the shape of the anisotropic structures, their size, color, quantity, type of morphotypes are evaluated as basic, secondary, atypical, and then a comparative analysis of the experimental and control preparations is performed. An increase in the content of secondary morphotypes with a simultaneous decrease in the number of basic morphotypes in experimental samples as compared with the control indicates an adverse effect of the studied drugs on the body. The appearance of atypical forms of crystals or a state of amorphization makes it possible to evaluate a drug as a drug with a toxic effect.

The proposed method was successfully tested at the Research Institute for the Study of Leprosy of the Ministry of Health of the Russian Federation during 2000-2001. on 350 mice of the CBA line.

Below are the results of testing.

Example 1

Twenty CBA mice were injected intragastrally with a probe at a dose of 25 mg / kg of body weight diaminodiphenylsulfone (DDS). The control group included intact animals. After 6 months, the animals were removed from the experiment by decapitation. A pathomorphological study of the internal organs (liver, kidneys), determination of serum biochemical parameters (ACT, ALT, bilirubin, creatinine, urea) were performed, and the composition of blood serum morphotypes was evaluated.

The microscopic structure of the liver in mice treated with DDS did not differ significantly from normal. In a number of cases, hepatocytes with hypertrophied basophilic nuclei and an increase in the number of binuclear cells compared with the control group were observed. A histological examination of the kidney tissue of animals from the experimental group revealed no pathological changes.

According to table 1, the quantitative indicators of the biochemical parameters of the experimental group did not differ significantly from the control, which indicated the absence of toxic effects of the drug.

The crystallogram of the blood serum of intact animals was represented by basic (filamentous and small spherulites), as well as secondary (needle and polymorphic) morphotypes. In the blood serum of animals treated with DDS, mainly basic morphotypes were recorded. Against their background, single secondary morphotypes were found (table 2).

Thus, the identity of the quantitative and qualitative composition of morphotypes of the experimental and control groups allows us to conclude that the studied drug is not toxic.

Example 2

Twenty mice of CBA line were intragastrally administered with a probe at a dose of 40 mg / kg daily for 6 months using a probe. The controls were intact animals. At the end of the experiment, a pathomorphological study of the internal organs (liver, kidneys), determination of biochemical parameters of blood serum (ACT, ALT, bilirubin, creatinine, urea) were performed, and the composition of blood serum morphotypes was evaluated.

During histological examination of the liver tissue of mice treated with hydrobunide, focal granular dystrophy and necrosis of single hepatocytes, as well as concomitant lymphomacrophage infiltration, were noted. A microscopic study of the kidney tissue of mice of the experimental group showed moderate dystrophy of the convoluted tubule epithelium.

According to the results of biochemical studies presented in table 3, the amount of bilirubin in the experimental group exceeded the same indicator in the control, the increase in the content of ALT, creatinine and urea in the animals treated with the drug was reliable. The data obtained indicate an adverse effect of the drug on the hepatobiliary and urinary systems.

Basal and secondary morphotypes were observed in the blood serum of intact animals. The composition of the blood serum morphotypes of the experimental group of animals was represented by a single base and a large number of secondary ones (table 4).

From the presented data it can be seen that an increase in the content of secondary morphotypes in blood serum with a simultaneous decrease in the number of basic morphotypes as compared with the control serves as a criterion for the adverse effect of the drug on the body.

Example 3

Twenty mice of CBA line were injected intragastrally through a probe for 6 months daily with furazonal at a dose of 0.75 g / kg. The controls were intact animals. At the end of the experiment, a pathomorphological study of the internal organs (liver, kidneys), determination of serum biochemical parameters (ACT, ALT, bilirubin, creatinine, urea) were performed, and the composition of blood serum morphotypes was evaluated.

Histological examination of the liver showed marked changes: diffuse granular dystrophy and necrosis of whole groups of hepatocytes located both in the central and peripheral sections of the hepatic lobule. Microscopic examination of the kidney tissue of mice of the experimental groups showed inflammatory infiltration of the stroma of the pyramids with lymphocytes, plasma cells mixed with eosinophils and necrosis of the epithelium of the convoluted tubules of the kidney.

In the blood serum of animals treated with furazonal, a significant increase in the studied biochemical parameters was noted, the difference in the content of ALT, ACT, bilirubin, and urea was significant, which indicates the presence of side effects of the drug (table 5).

In the serum of intact animals, mainly basic morphotypes were recorded.

The atypical composition of morphotypes (colored needles, spherulites with the presence of foreign inserts) prevailed in the blood serum of the experimental group of animals. An insignificant number of basic and secondary morphotypes was also recorded (table 6).

The appearance of atypical morphotypes in the blood serum of animals treated with furazonal correlates with pathomorphological studies of internal organs and significant deviations of biochemical parameters, which indicates the toxicity of the drug.

Example 4

A combination of preparations was administered intragastrically to 20 mice of CBA line using a probe for 6 months daily: furazonal at a dose of 0.25 g / kg, DDS at a dose of 10 mg / kg and rifampicin at a dose of 0.25 g / kg. The controls were intact animals. At the end of the experiment, a pathomorphological study of the internal organs (liver, kidneys), determination of serum biochemical parameters (ACT, ALT, bilirubin, creatinine, urea) were performed, and the composition of blood serum morphotypes was evaluated.

During a histological examination of the internal organs, changes characteristic of toxic hepatitis were observed (degeneration and necrosis of hepatocytes, concomitant inflammatory reaction).

Microscopic examination of the kidneys along with necrosis of the tubular epithelium revealed edema and cell infiltration of the interstitial tissue.

The results of biochemical studies, presented in table 7, indicate a significant increase in the studied parameters in the experimental group compared to intact animals, which indicates the presence of toxic effects of the used complex of drugs.

When viewing the blood serum samples of animals treated with the indicated complex of drugs, a dark isotropic field (amorphization state) was detected in polarized light, which is a consequence of inhibition of structuring and characterizes the state of homeostasis as decompensated.

Thus, the absence of anisotropic structures in the blood serum of experimental animals correlates with significant deviations of the biochemical parameters from the control, which indicates the pronounced toxicity of the drugs used.

Using the proposed method in comparison with the prototype method, a positive result is achieved, which consists in simplifying the method. The method is simple in technical execution and analysis of the results, there is no multi-stage process, the need to evaluate a complex of biochemical parameters is eliminated, the method is feasible using a small amount of blood serum, which is important for experimental studies, it is possible to use the test material one year after storage at -18 ° C. The method is economical, does not require a specialized laboratory, the use of expensive equipment, reagents, dyes.

The proposed method can be recommended for use in morphological, biochemical, microbiological laboratories of research institutions.

Claims (1)

A method for evaluating the general toxic effect of drugs on the body, including a microscopic examination of blood serum, characterized in that the blood serum of intact animals and animals receiving the test drug in an amount of 20 μl is applied to the surface of a glass slide, covered with a coverslip, dried at room temperature in during 48-72 hours, a comparative analysis of anisotropic morphotypes is performed and with an increase in the content of secondary morphotypes in experimental samples along with simultaneous nnym decrease in the number of morphotypes standards compared to the control trial of adverse effects on the body of the study drug, and in the presence of test samples atypical morphotypes or state amorphization judged on its toxic effects.

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