EA016530B1 - Method of cytogenetic and cytotoxic effects detection of environmental factors in thyroid cells - Google Patents

Abstract

The invention aims to simplify and reduce the cost of method, increase reliability, informatively and performance evaluation of various factors on thyroid gland cells of experimental animals and humans. The technical result is achieved by the cells preparation for the study carried out by fixing the organ in formalin, the cells dissociation with 50% KOH, application to dry slides, staining with azure-eosin by Romanovsky. Determine 4 cytogenetic indexes (cells with micronuclei, cells with nuclear protrusions, cells with internuclear bridges and the amount of cells with cytogenetic disorders) and 11 cytotoxic indexes (cells with atypical nuclei, dual- nuclei cells, triple- nuclei cells, cells with double nuclei, the amount of cells with three or more nuclei, mitosis, chromatin condensation, pyknosis, karyorrhexis, karyolysis, apoptosis and vacuolization of the cytoplasm) by light microscopy analysis of 1000 follicular thyrocytes (A-cells).

Description

The present invention relates to medicine and can be used for the diagnosis of mutagenic (cytogenetic) and cytotoxic effects of various exogenous and endogenous factors on the human and animal body, as well as for in-depth morphological analysis.

The thyroid gland is actively involved in the functioning of all organs and systems of the human body. In addition, it is one of the first to respond to changes of both endogenous and exogenous nature. In this regard, research on the study of the influence of the environment on a given organ is so important.

In various pathological processes or exposure of the human body to ionizing radiation, chemical compounds, viruses in humans, various forms of cell nucleus abnormalities are observed, which are analyzed mainly in peripheral blood lymphocytes and polychromatophilic erythrocytes of the red bone marrow. Thyroid cells are rarely used for cytogenetic and cytotoxic analysis. At the same time, many carcinogens are tropic to the thyroid gland. Evaluation of cytogenetic and cytotoxic indicators along with morphological research significantly expands the possibilities for evaluating the mutagenic and toxic effects of the studied factors. In addition, this approach allows us to predict a carcinogenic effect, since cytogenetic disorders are a prognostic criterion for neoplasms.

In most studies of various organs, only the frequency of cells with micronuclei and in rare cases other indicators determine the cytogenetic damage.

There is a method for detecting cytogenetic and cytotoxic effects of environmental factors in thyroid cells, including obtaining separate thyroid cells, applying to dry slides, staining and counting cells with micronuclei (Gansburg MA Analysis of cells with micronuclei in the evaluation of epithelial proliferation of the thyroid glands // Abstract of thesis, Candidate of Medical Science, Yaroslavl, 2005).

The disadvantages of this method are the use of collagenase for dissociation of thyroid cells, since this reagent is expensive, working with it requires certain conditions, and its enzymatic activity decreases during prolonged storage;

the use of this method in conducting large-scale experiments and screening of the population is economically disadvantageous;

possibility of imitation of micronuclei by some strains of microbes when stained according to Felgen (No. 1 and. е1 а1., 1991);

coloring of preparations according to the Feelgen method makes it difficult to carry out the morphological differentiation of various types of thyroid cells;

assessment of the cytogenetic effect of the studied factors is carried out on only one indicator - the registration of cells with micronuclei;

assessment is carried out without taking into account the morphology of the cells of the thyroid gland;

Only 1 indicator of the cytotoxic effect of the test substance - cell ploidy is taken into account.

The technical objectives of the present invention are to simplify and reduce the cost of the method of obtaining cells, increasing the reliability, informativeness and overall effectiveness of assessing the effect of various factors on the thyroid cells of experimental animals and humans.

These technical results are achieved by the fact that in the method of detecting cytogenetic and cytotoxic effects of environmental factors in thyroid cells, including obtaining separate cells of the thyroid gland, applying to dry slides, staining and counting of cells with micronuclei, preparations of thyroid cells are obtained by fixing body in 10% formalin, alkaline dissociation with 50% KOH, applied by smear on a dry glass slide, dried in air, stained with azure-eosin in P Omanovsky, washed with distilled water, dried in air, using a light microscope, quantitatively determined by analyzing 1000 follicular thyrocytes (A-cells), 4 indicators of cytogenetic action (cells with micronuclei, cells with nuclear protrusions, cells with internuclear bridges, the sum of cells with these indicators ) and 11 indicators of cytotoxic action (cells with an atypical shape of the nucleus, dual-core cells, trinuclear cells, cells with dual nuclei, the sum of cells with three or more nuclei, mitosis, condensation xp Matin, karyopyknosis, karyolysis, karyorhexis, apoptotic index and vacuolization of cytoplasm). The main differences of the proposed method are as follows.

1. To obtain drugs, the organ is fixed in 10% formalin, and then individual cells are obtained using alkaline dissociation (50% KOH).

2. Cells are stained with azure-eosin according to Romanovsky.

3. Takes into account additional cytogenetic indices (cells with protrusions, cells with internuclear bridges, sum of cytogenetic indices).

4. Takes into account 11 indicators of cytotoxic action (cells with an atypical shape of the nucleus, dual-core cells, trinuclear cells, cells with dual nuclei, the sum of cells with three or more nuclei

- 1 016530 mi, mitosis, chromatin condensation, karyripnosis, karyolysis, karyorexis, apoptotic index and vacuolization of the cytoplasm).

The proposed method allows to overcome the above disadvantages of the prototype:

replace cell dissociation with collagenase by dissociation using inexpensive, widely available KOH after routine fixation of the organ in formalin;

replace the multistage Felgen staining with the simple common method of painting with azureosin according to Romanovsky.

The proposed method of preparing the material for microscopic examination is convenient, simple in execution, economical, does not require special equipment. Azov-eosin coloring according to Romanovsky allows:

to distinguish the considered changes in the nucleus from microbes, cytoplasmic inclusions or detritus; differentiate three types of thyroid cells according to their individual characteristics: staining of the nucleus and cytoplasm;

instead of one, take into account four cytogenetic indicators: micronuclei, protrusions, internuclear bridges and the sum of these indicators, which significantly increases the information content of the proposed method;

consider 11 additional indicators of cytotoxic action.

Microkernels reflect the presence of chromosomal and genomic mutations. The appearance of micronuclei can be associated either with DNA damage (formed by a fragment of the chromosome), or with damage to the spindle of division (formed by one or more outdated chromosomes that are not included in the main nucleus).

Protrusions are DNA-containing formations tightly bound to the nucleus, having various shapes. They reflect the presence of aberrant chromosomes in the interphase nucleus or, according to some authors, are regarded as an intermediate stage before the formation of a microkernel.

Internuclear bridges appear as a result of nondisjunction of chromosomes during mitosis. Microkernels, protrusions and bridges are markers of mutagenic and carcinogenic effects.

The presence of a cytogenetic effect of the factor under study is diagnosed if it causes a statistically significant increase in the frequency of thyroid cells with micronuclei, protrusions, inter-nuclear bridges and a total of all three indicators (the sum of cytogenetic disorders) in the exposure group compared to the control.

The nuclei of an atypical form (nuclei with an abnormal shape of the nucleus, atypical cells) differ in shape and size from the nuclei of the cells of their population. This feature is widely used in morphological and cytological studies and is one of the prognostic signs of neoplasms.

Accounting for the number of mitoses and cells with two, three nuclei, dual nuclei allows to evaluate the proliferative processes in the cell population. Changes in proliferative activity under the action of exogenous or endogenous factors characterize their cytotoxic effect.

Dual-core cells are most common. They are formed by dividing the nucleus without separating the cytoplasm and contain a double set of genetic material. Also formed cells with three or more nuclei, which characterizes the pathological process of mitosis. Along with individual indicators of proliferation, it is important to evaluate the total indicator of cells with two or more nuclei.

Mitoses are well visualized when conducting this analysis and significantly complement the overall picture of the state of proliferation.

This method also allows to evaluate the destruction of cells (apoptosis and necrosis). Apoptosis is a programmed cell death. This process maintains cellular homeostasis by removing damaged and old cells. Apoptosis is characterized by a decrease in cell volume, wrinkling, chromatin condensation, karyorrhexis (the formation of individual chromatin clumps), karyopynosis (compression and compaction of the nucleus), karyolysis (first partial, and then complete dissolution of chromatin). In our proposed method, you can take into account all these indicators, as well as their sum - the apoptotic index.

Another type of cell death is necrosis. Necrosis is characterized by rupture of the cytoplasmic and intracellular membranes, which leads to the destruction of organelles, the release of lysosomal enzymes and the release of the contents of the cytoplasm into the intercellular space. An increase in the frequency of cells with vacuolation of the cytoplasm characterizes necrosis and is also an indicator of cytotoxic action.

The effectiveness of this approach was tested in the study of the cytogenetic and cytotoxic effects of acrylamide. Acrylamide is a low molecular weight chemical compound that is widely used in the chemical industry and can be formed during cooking. The material for the study was obtained and analyzed by the method indicated above. The studied parameters are confirmed by examples 1 and 2, the results of which are presented in table. 1 and 2.

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Example 1

In tab. 1 presents the results of a study of the cytogenetic and cytotoxic effects of acrylamide. Two groups of animals were compared: control and experimental, the animals of which were injected with acrylamide at a dose of 0.5 mg / kg. The cytogenetic effect of acrylamide was revealed by comparison with the control by increasing the frequency of protrusions of cells by 9 times, the sum of cells with cytogenetic disorders by 8 times. The cytotoxic effect of acrylamide was detected compared to the control by increasing the frequency of cells with atypical nuclei 4 times, dual core cells 1.8 times, cells with a double nucleus 2.4 times, the sum of cells with two or more nuclei 2 times, cells in mitosis - 3.7 times, cells with chromatin condensation - 27 times, cells with karyopynosis - 2.2 times, apoptotic index - 2.2 times.

Table 1

Cytogenetic and cytotoxic effect of acrylamide on thyroid cells of laboratory rats

Indicators The frequency of cells with the studied parameters in% about x _C p. ± t Control Acrylamide dose 0.5 mg / kg Indicators of cytogenetic action Microkernels 0 0.57 ± 0.3 Protrusions 0.83 ± 0.48 7.71 ± 1.84 *** Internuclear bridges 0.33 ± 0.21 1,14 ± 0,4 The amount of cytogenetic disorders 1.16 ± 0.54 9.43 ± 2.23 *** Indicators of cytotoxic action Atypical nuclei 3.33 ± 1.02 13.43 ± 4.39 *** Two cores 10.17 ± 2.10 18 ± 2.98 *** Three cores 0 0.57 ± 0.37 Dual cores 3 ± 1.24 7.29 ± 2.43 *** The sum of cells with two or more nuclei 13.17 ± 3.24 25.86 ± 5.23 *** Mitosis 0.5 ± 0.22 1.86 ± 0.74 * Chromatin vacuolization 0 0 Chromatin condensation 0.17 ± 017 4.57 ± 1.09 *** Cariopicnosis 3.5 ± 1.65 7.57 ± 1.39 ** Cariolysis 0 0 Cariorexis 0 0.14 ± 0.14 Apoptotic index 3.5 ± 1.65 7.71 ± 1.43 ** Cytoplasm vacuolization 3.17 ± 1.8 3.86 ± 1.45

Note: * p <0.05; ** p <0.005; *** p <0.001 - significant differences from the conditionally control group when comparing data on χ ² .

Example 2

A comparison of the cytogenetic and cytotoxic effects of acrylamide with its single and triple administration to laboratory rats at a dose of 12.4 mg / kg (Table 2). A more pronounced cytogenetic effect of acrylamide was shown with a threefold administration compared to a single cell frequency with inter-core bridges (4.4-fold increase); cytotoxic effects on the frequency of dual-core cells (4-fold increase), cells with dual nuclei (4.2-fold increase), total of cells with two or more nuclei (4-fold increase), cell frequency in mitosis (5-fold decrease) , cells with karyopicnosis (2.4 times increase), apoptotic index (2.4 times increase).

At the same time, the method outlined in the prototype, aimed at counting cells with micronuclei, would not allow in these experiments to identify the cytogenetic effect of acrylamide or show a more pronounced threefold effect of this substance, since the differences in the frequency of cells with micronuclei considered in the prototype were not statistically authentic.

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table 2

Comparison of the cytogenetic and cytotoxic effects of acrylamide in two groups of rats after a single and three times the introduction

Indicators The proportion of cells with the studied parameters in% about X er. ± t A single introduction of acrylamide Three times the introduction of acrylamide Indicators of cytogenetic action Microkernels 0.86 ± 0.26 0.57 ± 0.3 Protrusions 3.86 ± 0.91 3.85 ± 1.53 Internuclear bridges 0.29 ± 0.29 1.28 + 0.36 * The amount of cytogenetic disorders 5.0 ± 1.11 5.71 + 1.71 Indicators of cytotoxic action Atypical nuclei 12.57 ± 3.39 9.14 + 2.87 * Dual core cells 6.43 ± 0.78 26.29 + 10.9 *** Trinuclear cells 0.29 ± 0.18 0.71 + 0.36 Dual cores 1.29 ± 0.64 5.43 + 1.88 *** The sum of cells with two or more nuclei 8.0 ± 1.29 32.43 + 12.45 *** Mitosis 2.86 ± 1.08 0.57 + 0.3 ** Chromatin vacuolization 0.14 ± 0.14 0 Condensation chromatin 5.14 ± 2.10 3.14 + 1.14 Cariopicnosis 6.86 ± 1.5b 16.29 + 4.98 *** Cariolysis 0 0 Cariorexis 0.14 ± 0.14 0.29 + 0.18 Apoptotic index 7.0 ± 1.63 16.57 + 5.04 *** Cytoplasm vacuolization 3.29 + 0.94 5.14 + 2.59

Note: * p <0.05; ** p <0.005; *** p <0.001 - significant differences from the conditionally control group when comparing data on χ ² .

Thus, the proposed method of detecting cytogenetic and cytotoxic effects of environmental factors in the cells of the thyroid gland is more informative, simple to perform and cost-effective.

Claims (1)

CLAIM A method for detecting the cytogenetic and cytotoxic effects of environmental factors in thyroid cells, including isolation of thyroid cells, preparation of smears of cells, staining, determination and evaluation of cells with micronuclei, characterized in that the isolated thyroid cells are obtained from a fixed in 10% formalin solution organ tissue by alkaline dissociation with a 50% potassium hydroxide solution, staining with azure-eosin according to Romanovsky, and then using a light micro by quantitatively analyzing 1000 follicular thyrocytes, cells with nuclear protrusions, cells with internuclear bridges and the sum of the mentioned cells are additionally determined and evaluated as indicators of cytogenetic action, and cells with an atypical form of the nucleus, binuclear cells, tricyclic cells, cells with dual cells are also determined and evaluated nuclei, the sum of cells with three or more nuclei, mitosis, chromatin condensation, karyopyknosis, karyolysis, karyorexis, apoptotic index and cytoplasm vacuolation as an indicator s cytotoxic effect. 4 ^ 8) Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2