Use of micronucleus test for screening and monitoring of mutagens. Historical controls

  • Authors: T.V. Tkachuk, M.G. Prodanchuk
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L.I. Medved's Research Center of Preventive Toxigology, Food and Chemical Safety Ministry of Health of Ukraine, Kyiv, Ukraine

Abstract. Introduction. Analysis of mutagenic activity is a required element of toxicological assessment of pesticides. Order to provide higher reliability in assessing the results of experiments, current research data are compared with the data historical controls.
Purpose of the study. It is necessary to form a historical control data based on the results obtained during the mutagenicity investigations of generic pesticides in the micronucleus test in the bone marrow of mice in vivo and compare the results with the published data in literature.
Materials and Methods. Experimental studies of the mutagenic activity of the test substances in the micronucleus test in polychromatic erythrocytes in the bone marrow were performed by the standard method in compliance with requires of GLP, for young adult mouse species CD1 albino, males. To compare the results of own research we used data published in 2000 by the authors G. Krishna and colleagues.
Results and Discussion. In the Laboratory of Experimental Toxicology and Mutagenesis for the period 2015–2016 years we have conducted 28 experiments to create a database of positive and negative controls for this test. The average values of the levels of micronucleated bone marrow polychromatic erythrocytes in our laboratory coincides with the data obtained G. Krishna and colleagues.
Conclusions. In the Laboratory of Experimental Toxicology and Mutagenesis we validated and put into practice the micronucleus test in polychromatic erythrocytes in the mice bone marrow in vivo (OECD 474). A database of positive and negative controls was created on the research results of this test. Comparison of the levels of historical control database with the published in the literature showed that the data from our laboratory are within the same distribution. Created by us historical control can be used for reliable evaluation of research results. The levels of Historical Control database indicates the high quality of the conditions of animal housing and feeding and the high genetic quality of animals obtained from SPF nursery of Research Center of Preventive Toxicology for using in this test.
Key words: bone marrow, micronucleated polychromatic erythrocytes, historical control, mutagenicity.

The CAS chemical list contains over 350 thousand compounds, including pesticides widely used in agriculture. According to FAO, without using a chemical methods for plant protection, the population of the planet will lose half of all food in the first year. Nowadays in the world, more than 100 thousand pesticides are used on the basis of thousands of chemical compounds. However, the use of pesticides conseals a threat of contamination of the environment and products of consumption and under certain conditions can cause the occurrence of genetic disorders in the cells of organisms.

The ability of chemical compounds to cause mutations was discovered in 1932 by V. V. Sakharov. Numerous experimental data show that some chemical agents may induce mutations both in somatic cells leading to malignant tumours, premature ageing, atherogenesis, and a number of other pathological processes, and in germ cells, leading to malformations in the development of offsprings, increasing the frequency of spontaneous abortions, stillbirths, hereditary diseases, etc. [1]. This necessitates the creation of a sufficiently operational and economically feasible system for testing chemical compounds in relation to their potential genetic hazard for humans.
In 1969, W. Schmid et. al. offered a test for the study of chemical mutagenesis, based on the micronucleus count in bone marrow erythrocytes. This method is called “Micronucleus test” [3]. In 1971, the same group of scientists conducted experiments on Chinese and golden hamster, mice, rats and guinea pigs, and some experimental studies on Rhesus macaques [4]. In 1973, John Heddle et. al., independently of W. Schmid, also proposed a micronucleus test for the screening of chemical compounds. Experiments were performed on mice, the micronucleus count was performed in bone marrow erythrocytes [5].

The Organisation for Economic Co-operation and Development (OECD) adopted guidelines No. 474 to the mammalian erythrocytes micronucleus test in 1983 [2], and in 2010 No. 487 in vitro mammalian cell micronucleus test.

Today, the micronucleus count has become possible in most populations of animal and plant cells. It is used for in-depth study of toxicity in sub-chronic or chronic experiments, evaluation of genotoxicity of chemical compounds in the main barrier systems of the body, the so-called organ-specific mutagenicity: in the respiratory system (lungs), in the digestive system (esophagus, stomach, small and large intestine, liver), excretory (urinary bladder, kidneys); to count micronuclei in early male and female germ cells; foetal liver cells in the study of transplacental activity of chemicals; in the cells of buccal epithelium; human lymphocytes and others [6]. Also, the micronucleus test is used to assess the genotoxicity of not only certain chemical compounds but also various mixtures of radionuclide substances, to evaluate the efficiense of treatment of some diseases in humans, as well as to bioindicate environmental pollution in case of various man-made accidents, environmental disasters related to emissions of pollutants, in the medical examination of workers operating with harmful substances [7].

It is known that micronuclei are a pathological structure, their formation is associated with chromosomal instability, they can be observed in cells of any proliferating tissue. Micronuclei are fragments of a eukaryotic cell nucleus that does not contain an entire genome needed for cell survival. Micronuclei are formed from a chromosomal material, which fells off at the anaphase stage. During the mitosis, this material has only one of the daughter cells. It can be included in the main nucleus or form one or more small micronuclei. Visually, micronuclei are rounded chromatin formations observed in the cytoplasm of cells in the interphase period, they are much smaller than the main nucleus. Micronuclei can include both separate whole chromosome and their fragments [1,8].

Studies of mutagenicity that were carried out via the micronucleus (MN) test are aimed at determine:

- clastogenic changes, that is, structural changes in the chromosomes (chromosomal aberrations) arising as a result of DNA damage, which leads to the breakup of the double helix;
- aneugenic changes, that is, changes in the number of chromosomes that are the result of disruption of the mitotic spindle function.

If in the mitosis process the damage factor affects to tubular proteins more than to DNA, the complete chromosome does not reach to the main nucleus upon formation of the daughter cell nucleus (Fig. 1). Microtubules of mitotic spindle are attached to the centromere of the chromosome, during the cell division, the chromosome moves toward to the pole, which thebounded with chromosome by the most of microtubules. If the number of microtubules of both poles is the same, or spindle fibres are broken, chromosomes remain in place or lagging during the anaphase. This causes aneuploidy instead of structural chromosomal aberrations — this is an aneugenic effect. If the damage factor causes structural damage to the chromosomes, an acentric fragment is formed. Since it is non-centromeric, it does not move to any of the poles, therefore, it does not get to a newly formed nucleus upon the cell division, which characterizes the clastogenic effect [9].

Fig. 1. Mechanism of micronuclei formation (according to Terradas, M., et al., 2010).

Usually, the clastogenic damage factor is the most dangerous for cells that are in the S-phase of the cell cycle (Fig. 2) [10]. At this time, the cell nuclei DNA replicates in the cell, the synthesis of RNA and proteins, as well as doubling of centrioles (poles of mitotic spindle). An aneugenic damage factor is the most dangerous for cells in the period of mitosis anaphase, this time chromosomes move towards the cell poles.

Fig. 2. The scheme of the cell cycle and reasons for the formation of the micronuclei on the example of bone marrow cells (according to Jarvis, P. et al., 2011).

Recent data on aberrations in the culture of human lymphocytes indicate that fragments (presumably the cells that contain them) survive till the next mitosis in about 30 % of cases, and dicentric chromosomes — about 50 %. These estimates are made without taking into account the proliferation of normal cells, if one takes into account proliferation, the most probable value of survival of the fragment itself is approximately 80 % (taking into account both daughter cells) [11].

Aneuploidy is the most common cause of abnormality in humans. Trisomy or monosomy of large chromosomes in the germ cells leads to an early death of the embryo. However, the trisomy of small chromosomes can be compatible with survival, although with abnormal functions. For example, trisomy of chromosome 21 is Down syndrome, chromosome 18 — Edwards, 13 — Patau. Only one monosomy found among liveborn infants is sex chromosomal monosomy (XO), known as Turner syndrome. Aneuploidy in somatic cells may affect the formation and development of tumours. The vast majority of human tumours contain aneuploid cells, and up to 10 % of tumours are monosomic or trisomic for a specific chromosome, like the only visible cytogenetic changes. Among these tumours are trisomy 8,9,12 and 21 with monosomy 7, 22 and Y most common. Some tumour states, such as chronic lymphocytic leukaemia, show a high correlation (30 %) with an additional single chromosome (in this case, trisomy 12) [12].

Various studies have shown that such violations can be associated with a wide range of factors, ranging from the effects of chemicals, heavy metals, ionizing radiation, and ending with viral infections. Likewise, micronuclei can be formed as a result of programmed cell death (apoptosis) and upon acquisition of malignant properties by cells (malignancy) [13].

To date, the analysis of mutagenic activity is a mandatory element of the toxicological assessment of environmental factors upon their regulation in the environment of human life.

To validate the results of the studies, it is obligatory to validate the in vivo MN test in polychromatophilic erythrocytes (PCE) of bone marrow and to form historical control.

Materials and methods.

The Laboratory for Experimental Toxicology and Mutagenesis is accredited for conducting researches in accordance with the GLP principles (Directive 2004/10/EC of the European Parliament and the Council of 11 February 2004) [14], as evidenced by the GLP certificate “Statement of GLP compliance No. G-042” issued by SNAS on March 09, 2015 and OECD 474 (OECD Guideline for Testing of Chemicals, Mammalian Erythrocyte Micronucleus Test) [2]. The tests were controlled by the employees of the quality control department. All manipulations with animals were agreed with the Ethics Committee of Medical and Biological Researches in accordance with the provisions of the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (Strasbourg, March 18, 1986). ETS No. 123 and the requirements of “Guide for the care and use of laboratory animals” of the National Academy Press, USA, 1996 [15].

Experimental studies of the mutagenic activity of pesticides in the MN test in bone marrow erythrocytes were performed on young adult male mice (Mus Musculus l. – CD1) weighing 18–20 g obtained from the SPF animal breeding facility of small laboratory animals of the State Enterprise “L.I.Medved’s research Center of Preventive Toxicology, Food and Chemical Safety” (6 Heroiv Oborony Street, Kyiv, 03127, Ukraine), with a certificate of animal health. The animals were examined daily during the acclimatization and experiment for compliance with the requirements for biological models in the toxicological experiment and suitability for this study by the responsible veterinarian of the Center of Preventive and Regulatory Toxicology (CPRT). Acclimatization of animals in the vivarium was carried out for at least 5 days before the start of the study. The experimental and control groups consisted of 5 animals. The animals were kept in T5 cages, polychlorocarbonate, covered with removable metal lattices, per 5 animals, over the course of the study period. The bedding consisted of sterilized non-chlorinated food paper. The cages were washed and disinfected according to the standard operating procedure. In the vivarium, the sanitary and hygienic purity norms were strictly observed: regular daily, weekly and overall cleaning. The room was provided with forced ventilation (12 volumes per hour) by prepared purified air. The temperature and relative humidity were recorded daily, temperature fluctuations ranged from 19 to
25 °C, humidity was 30–70 %. The lighting was artificial (12 hours of light, 12 hours of darkness). During the entire experiment, the mice received a balanced granulated feed produced by Altromin (Germany), disinfected, purified, UV-sterilized, deionized water from glass bottles in volume of 0.35 litres through metal tips [2, 16].

Procedure for MN test in bone marrow cells in vivo:

1.    Animals of the negative control group received orally (by gavage) of 0.4 mL of purified, UV-sterilized, deionized water with addition of the emulsifier OP-10 [17]; animals of the positive control group received intraperitoneally 0.1 mL of cyclophosphamide solution (CAS No. 50-18-0), at a dose of 40 mg/kg of body weight of the animal;

2.    24 hours after the treatment of the test substance, the animals were exposed to euthanasia and the bone marrow was collected for analysis. The bone marrow was chosen as the target tissue of the study since it has a high proliferative activity and a relatively low spontaneous background.

3.    Preparation of glass slides of bone marrow was carried out according to the standard method [2,5,18,19,20,21].

4.    Bone marrow samples were stained for 5–6 minutes with eosin-methylene blue by undiluted May-Grünwald solution, then by Gimsa diluted with distilled water 1:6 during 6 hours.

5.    Counting and analysis of micronuclei were carried out on encrypted preparations, microscopy at a large 100-fold magnification (oil immersion). The absence of damaged nuclear cells was the quality parameter of the product. The preparations with properly expanded erythrocytes lying alone, whose surface does not have processes and folds are considered suitable for the analysis. Polychromatophilous erythrocytes (PCE) have a greyish-blue colour of the cytoplasm, normochromic — orange-pink. The micronuclei have a rounded form, with a clear border, with a dark staining, similar to that of the nuclei in the preparation (Fig. 3).

Fig. 3. PCE and MNPCE. Staining by May-Grünwald solution, then stained by Gimsa solution, magnification with a 100x lens with oil immersion.


6. Statistical processing of the obtained data and determination of the result. Routine methods of statistical analysis are used in the calculation of the mean of the parameter and standard error of the mean. The statistical processing of data is carried out by different methods by different authors [22,23]. We conducted a standard method, using Student’s t-test [24].

One of the clauses of OECD 474 recommendations is the “laboratory competence verification” clause, which states that the lab should demonstrate the ability to reproduce the expected results, and if the laboratory gains sufficient experience it must create a historical control database and compare it with the published data [2, 25].

Purpose of the work: To form historical control based on the results obtained during the study of mutagenic properties of test substances of generic pesticides in the MN test in bone marrow erythrocytes in vivo and to compare our results with published data.

To achieve the goal the following tasks were set:

1.    To assess by statistical methods the results of positive and negative control tests in the MN test in in vivo mouse bone marrow erythrocytes conducted in the CPRT for the period of 2015–2016.

2.    To assess the results of the historical control databases of laboratories that use the MN test in mice bone marrow cells published data.

3.    To compare the findings with those published data.

Discussion of the results of the work

During the period of 2015–2016, 28 experiments were carried out by the laboratory of experimental toxicology and mutagenesis, which allowed us to create a database of historical controls. To compare the results of our own studies, data published in 2000 by Krishna G, Urda G, Paulissen J, in Parke-Davis Pharmaceutical Research, Warner-Lambert Company unit, USA [26], were used. The laboratory also works in the GLP quality system. Experimental conditions of this laboratory are carefully described in the article and close to ours: experiments were carried out on male mice, the target tissue was bone marrow. This allowed us to compare the results of historical control data with those described in this article.

Table 1 shows that G.Krishna et al. over 12 years conducted 47 experiments on male CD1 mice 6–8 weeks received from the Charles River Breeding Laboratories (Portage, MI) nursery. The period of acclimatization was 1 week before the study, the animals were examined to make sure that they were healthy and suitable for the study. During the experiment, the animals received balanced feed and water without restriction. In rooms for animals, the automatic timer provided a 12-hour change of light and darkness. 5 animals per group were used. For negative control, distilled or deionized water, saline, mannitol or 0.5 % methylcellulose with/without polyethylene glycol or Tween 80 was used orally at a dose of 10 mg/kg. The positive control Cyclophosphamide (CAS No. 50-18-0) was injected intraperitoneally in dose 40 mg/kg body weight, was used intraperitoneally. Glass slides of bone marrow was prepared according to the standard method. The analysis was done on coded glass slides by microscopy. The number of micronucleated PCE (MNPCE) per 1,000 PCE was counted in samples of negative and positive controls. Data for each experiment was calculated using Fisher’s t-test.

Table 1

Comparison of the conditions of the experiment

Table 2 presents a comparison of historical control data of G. Krishna et al. and laboratory studies of experimental toxicology and mutagenesis. Table 2 show that the range of data in the MN test in mice bone marrow cells in vivo in our laboratory, both in negative and in positive control, is within the same distribution of data obtained by G.Krishna et al. In the laboratory of the experimental toxicology and mutagenesis the negative control MNPCE range was 0.67–1.55 per 1,000 PCE, the difference between data was 2.3 times. The positive control range of MNPCE was 18.02–21.96 per 1,000 PCE, the difference between data was 1.2 times. According to the data obtained by G.Krishna et al., the negative control range was 0.4–3.8 MNPCE per 1000 PCE, the difference between data was 9.5 times; the positive control range was 7.7–42.7MNPCE per 1000 PCE, the difference between data is 5.5 times.

Table 2

Comparison of historical control database, the range of data of MNPCE per 1,000 PCE, differences between data by control groups

Fig. 4 and 5 demonstrate graphic results of the data of the study of the sequence of the obtained data on the level of MNPCE per 1,000 PCE. Chart diagrams show the chronological order of the mean values obtained for each individual group of negative and positive controls and the overall level of mean values.

Fig. 4 and 5 demonstrate that the values of the mean frequency of MNPCE for each individual negative control study have very slight fluctuations. According to G.Krishna et al., the mean frequency of the negative control was 1.8 MNPCE per 1,000 PCE. In the laboratory of experimental toxicology and mutagenesis the mean frequency of MNPCE of negative control was 1.1 MNPCE per 1,000 PCE. A statistical comparison of MNPCE data for negative control with that of respective positive control data showed a significant increase in the MNPCE frequency in positive control groups over negative controls in all experiments (P˂0.05). The mean frequency of positive control studies according to G.Krishna et al. was 23.7 MNPCE per 1,000 PCE, the mean frequency of the positive control of our laboratory is 19.9 PCE with micronuclei per 1,000 PCE.

Fig. 4. The control database: negative and positive (Cyclophosphamide, 40 mg/kg) of G.Krishna et al.

Fig. 5. The control database: negative and positive (Cyclophosphamide, 40 mg/kg) of the laboratory of experimental toxicology and mutagenesis.


1.    An in vivo micronucleus test in mice bone marrow (OECD 474) was validated and successfully usage in the laboratory of experimental toxicology and mutagenesis.

2.    A database of positive and negative controls was created based on the results of studies for the MN test in mice bone marrow erythrocytes.

3.    Comparison of the results of the studies with the published data of the historical control databases has shown that the data from our laboratory are in the same range of distribution.

4.    The created historical control can be used for reliable evaluation of study results.

5.    The level of negative and positive controls indicates the proper genetic quality of the animals obtained from the SPF animal breeding facility of the Research Center of Preventive Toxicology and the conditions for their maintenance for use in the micronucleus test in mice bone marrow.



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