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Alok Dhawan and Mahima Bajpayee (eds.), Genotoxicity Assessment: Methods and Protocols, Methods in Molecular Biology, vol. 1044, DOI 10.1007/978-1-62703-529-3_9, © Springer Science+Business Media New York 2013

Chapter 9

In Vivo Micronucleus Assay in Mouse Bone Marrow and Peripheral Blood

Sawako Kasamoto , Shoji Masumori , and Makoto Hayashi

Abstract

The rodent micronucleus assay has been most widely and frequently used as a representative in vivo assay system to assess mutagenicity of chemicals, regardless of endpoint of mutagenicity. The micronucleus has been developed to assess induction of structural and numerical chromosomal aberrations of target chemical. In this chapter, we describe the standard protocols of the assay using mouse bone marrow and peripheral blood. These methods are basically applicable to other rodents. The methodology of the micronucleus assay is rapidly developing, especially automatic analysis by fl ow cytometry (see also Chapter 11 ). Also we have to pay attention to the animal welfare, for example integration into repeat dose toxicity assay, combi-nation of the micronucleus assay and Comet assay, and also omission of concurrent positive control group. Therefore, modifi cation of the standard protocol is necessary for the actual assay on a case-by-case basis.

Key words Chromosomal aberration , Micronucleus , In vivo assay , Bone marrow , Peripheral blood

1 Introduction

Chromosomal aberration is one of the main endpoints of genotoxicity as well as gene mutation to assess the mutagenicity of chemicals. To assess chromosomal aberration in vitro, established cell lines (e.g., CHL/IU, CHO) or primary culture of human lympho-cytes are mainly used for the assay. For the in vivo assay, which is more important for the risk characterization, the rodent erythro-cyte micronucleus assay is most widely and frequently used to assess induction ability of chromosomal aberration of chemicals in the body of animals.

The micronucleus assay was developed as an assay system in the 1970s by Heddle [ 1 ] and Schmid [ 2 ] using mouse bone marrow erythropoietic cells. A good correlation was shown between chro-mosomal aberration induction and micronucleus induction [ 3 ]. The Collaborative Study Group of Micronucleus Test (CSGMT) of Mammalian Mutagenicity Study (MMS) group which belongs to the Japanese Environmental Mutagen Society (JEMS) has

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widely studied the many factors that might affect the result of the assay, e.g., sex difference [ 4 ], strain difference [ 5 ], dose adminis-tration route [ 6 ], number of treatments [ 7 ], and evaluation of the assay sensitivity [ 8 , 9 ]. These outcomes were refl ected in the inter-nationally accepted guidelines such as OECD test guideline TG474 [ 10 ] and ICH guideline [ICH S2(R1); [ 11 ]]. The micronucleus assay has been harmonized mainly because of methodological aspects reviewed by the International Workshop on Genotoxicity Testing (IWGT) [ 12 – 14 ].

The main target tissue of the micronucleus assay has been the bone marrow of mouse and rat. The use of peripheral blood instead of bone marrow erythropoietic cells was introduced by MacGregor et al. [ 15 ]. The Collaborative Study Group for the Micronucleus Test [ 8 ] and Hayashi et al. [ 16 ] using acridine orange supravital staining. There are several advantages of using peripheral blood instead of bone marrow, for example, only tiny amount of blood is enough for analysis and then there is no need to sacrifi ce the ani-mals, as well as samples can be periodically obtained from the same animals. On the other hand, the most important disadvantage exists, which is the elimination of micronucleated erythrocytes by spleen of many species, e.g., human, monkey, and rat [ 17 ], except in the mouse. Therefore, we have to select the assay system on a case-by-case basis according to the purpose of the study.

Here, we describe the standard practical methods of the micro-nucleus assay. Of course these are examples that can be modifi ed according to the purpose of the study. Based on the animal welfare, several attempts have been started, which are trying to reduce ani-mal usage: (1) integration of micronucleus endpoint into repeat dose toxicological study [ 18 ], (2) combination of study of micro-nucleus and Comet assays as one study [ 19 ], and (3) omitting the concurrent positive control group.

2 Materials

To perform the micronucleus assay, we need the following materials and working solutions.

1. Negative control: The solvent/vehicle that is used to prepare dosing formulations is used as the negative control ( see Note 1 ). Examples of negative control include: (a) Water. (b) Physiological saline. (c) Methylcellulose solution. (d) Carboxymethyl cellulose sodium salt solution. (e) Olive oil.

2.1 Controls

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2. Positive control: Positive controls should produce micronuclei in vivo at exposure levels expected to give a detectable increase over the background. Examples of positive control substances include:

(a) Ethyl methanesulfonate. (b) Ethyl nitrosourea. (c) Mitomycin C. (d) Cyclophosphamide (monohydrate). (e) Triethylenemelamine.

1. Fetal calf serum. 2. Methanol. 3. 1/100 mol/L sodium phosphate buffer (pH 6.8). 4. Staining solution: 3 % Giemsa’s solution prepared in

1/100 mol/L sodium phosphate buffer. 5. 0.001 % citric acid solution.

1. Fetal calf serum. 2. Acridine orange (AO)-coated slide: 20 μL of 0.1 % acridine

orange in ethanol is placed on a cleaned glass slide (end frosted slides can also be used) and covered with another slide. Move these slides right and left, separate, and then air-dry. The AO-coated slides are stored in a refrigerator or a freezer. The preparation of AO-coated slide refers to the original method described in the literature ( see also ref. 16 ).

3. Sealing agent, e.g., Permount.

3 Methods

1. Commonly used laboratory strains of young healthy animals (6–10 weeks old at the start of the treatment schedule) should be employed.

2. In general, only one sex, usually male, is used ( see Note 2 ). However, if there is evidence indicating a relevant difference in toxicity or metabolism between males and females, then both sexes of the animals should be used.

3. Animals are housed in a temperature- and humidity-controlled room. Lighting should be artifi cial, the sequence being 12-h light, and 12-h dark. For feeding, conventional laboratory diets can be used with an unlimited supply of drinking water.

4. Animals are housed individually, or caged in small groups of the same sex.

2.2 Micronucleus Assay in Mouse Bone Marrow

2.3 Micronucleus Assay in Mouse Peripheral Blood

3.1 Animal Maintenance

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5. Following arrival at the testing facility, each animal should be monitored for at least 5 days to ensure that it is healthy (no abnormalities) and growing normally; animals are acclimated to the laboratory environment.

6. Animals are randomly assigned to the control and treatment groups.

7. The animals are identifi ed uniquely.

1. Each treated and control group must include at least 5 analyzable animals per sex.

2. The study may be performed in two ways: (a) Animals are treated with the test substance once. Samples

of bone marrow are taken at least twice, starting not ear-lier than 24 h after treatment, but not extending beyond 48 h after treatment with appropriate interval(s) between samples. The use of sampling times earlier than 24 h after treatment should be justifi ed. Samples of peripheral blood are taken at least twice, starting not earlier than 36 h after treatment, with appropriate intervals following the fi rst sample, but not extending beyond 72 h. When a positive response is recognized at one sampling time, additional sampling is not required.

(b) If two or more daily treatments are used (e.g., two or more treatments at 24-h intervals), samples should be col-lected once between 18 and 24 h following the fi nal treat-ment for the bone marrow and once between 36 and 48 h following the fi nal treatment for the peripheral blood.

3. A dose around the maximal tolerated dose or 2,000 mg/kg, whichever is lower, will be selected as the high dosage level, and two additional lower doses separated by a factor less than square root of 10 will be selected as the middle and lower dos-age levels. The highest dose may also be defi ned as a dose that produces some indication of toxicity of the bone marrow (e.g., a reduction of immature erythrocytes among total erythro-cytes in the bone marrow or the peripheral blood).

4. The test substance is usually administered by gavage using a stomach tube or a suitable intubation cannula, or by intraperi-toneal injection. Other routes of exposure may be acceptable where they can be justifi ed. The maximum volume of liquid that can be administered by gavage or injection at one time depends on the size of the test animal. The volume should not exceed 2 mL/100 g body weight.

5. The body weight of each animal is measured at the time of assign-ment to groups and just before sampling. Animals are assigned randomly considering keeping similar average of body weight.

6. Clinical signs of animals are examined at appropriate period after the dosing and just before sampling.

3.2 Study Design and Chemical Treatment

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1. Euthanize the animals before sampling. 2. Remove one femur, trim the muscle around it, and cut both

ends of the femur to collect the bone marrow cells. 3. Flush the bone marrow cells out with 0.5 mL of fetal calf

serum into centrifuge tubes using a 1-mL syringe fi tted with a 22 G needle.

4. Centrifuge the tubes at approximately 200 × g in a microcentrifuge for 5 min.

5. Remove the supernatant, and resuspend the cells with the remaining serum.

6. Drop a small amount of the cell suspension on the end of a glass slide and spread it by pulling the material behind a pol-ished cover glass held at an angle of approximately 45° ( see Note 3 ).

7. Allow the slides to air-dry and fi x them in methanol for 3 min. 8. Stain the slides with 3 % Giemsa solution diluted and prepared

with 1/100 mol/L sodium phosphate buffer (pH 6.8) for 30 min. 9. Rinse the slides with 1/100 mol/L sodium phosphate buffer

and then with purifi ed water, and dry them. 10. Rinse the slides with 0.001 % citric acid solution ( see Note 4 ),

and then with purifi ed water, and dry again.

1. At the time of sampling, pierce a tail blood vessel and collect about 5 μL of peripheral blood with a micropipette.

2. Mix the blood with 5 μL of fetal bovine serum and slightly stir (Fig. 1 ).

3.3 Preparation of Bone Marrow Smears

3.4 Preparation of Peripheral Blood Smears

Fig. 1 Approximately 5 μL of peripheral blood are mixed well with the same volume of fetal bovine serum by pipetting

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3. Drop 5–7 μL of the serum-mixed blood on a 24 × 40 mm coverslip and place on it an AO-coated slide ( see preparation in Subheading 2.3 ; Figs. 2, 3 and 4 ).

4. Seal the peripheral blood smear with a sealing, e.g., Permount ( see Note 5 ).

5. Slides should be allowed to stand for a few hours or overnight to allow cells to settle and to maximize staining, and examined with a microscope within 5 days after preparation. The prepa-rations can be stored for about a week in a refrigerator or a couple of months in a deep freezer.

Fig. 2 Drop the diluted peripheral blood onto coverslip (approximately 7 μL)

Fig. 3 Cover with glass slide that was pre-coated with acridine orange

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1. The slides are coded so as not to reveal the treatment groups to the scorer.

2. First the slides are scanned at low or medium magnifi cation looking for region of suitable technical quality, where the cells are well spread without overlapping, undamaged, and clearly stained to distinguish young and mature erythrocytes.

3. The erythrocytes should be well spread, neither globular nor having slurred contours.

4. Their staining has to be vigorous, pink in mature erythrocytes (normochromatic erythrocytes: NCE), and with a bluish tint in the immature forms (polychromatic erythrocytes: PCE) (Fig. 5 ).

3.5 Microscopic Observation of Bone Marrow Smears

Fig. 4 Spread blood fi lm and let stay for staining before microscopy

Fig. 5 Bone marrow slide (×100: low magnifi cation)

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5. As an indicator of cell toxicity in bone marrow, the number of PCEs is counted by examining at least 200 erythrocytes (PCE + NCE) per animal under a microscope (e.g., ×1,000) with immersion oil. Then, the ratio of PCE to total erythro-cytes is calculated as a percent ( see step 3 of Subheading 3.7).

6. At least 2,000 PCEs per animal are examined and the number of micronucleated polychromatic erythrocytes (MNPCE) is recorded. Micronuclei are identifi ed according to the criteria established by Schmid [ 2 ] and are darkly stained (purple) and generally round or almond shaped, although lightly stained, ring-shaped micronuclei occasionally occur (Fig. 6 ). Micronuclei have sharp borders and are generally 5–20 % the size of the PCEs and may occur in either PCE or NCE. However, only MNPCE is counted ( see Note 6 ). Then, the incidence of MNPCE to total PCEs is calculated as a percent.

1. The slides are coded to avoid the bias of the scorer who is aware of the treatment.

2. First the slides are scanned at low or medium magnifi cation looking for region of suitable technical quality, where the cells are well spread, undamaged, and clearly stained (Fig. 7 ).

3. Cells should be intact and the nuclei of nucleated cells and the reticulum structure of reticulocytes (RET; immature cell) should fl uoresce strongly green-yellow and red, respectively, using a fl uorescent microscope (e.g., ×800) equipped with a blue excitation fi lter and a green barrier fi lter ( see Note 7 ).

4. As an indicator of cell toxicity in bone marrow, the number of RET is determined by examining at least 1,000 erythrocytes (RET + mature cells) per animal using a fl uorescent microscope

3.6 Microscopic Observation of Peripheral Blood Smears

Fig. 6 Bone marrow slide (×1,000: high magnifi cation)

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(e.g., ×800) equipped with a blue excitation fi lter and a green barrier fi lter. Then, the ratio of RET to total erythrocytes is calculated as a percent.

5. At least 2,000 RET per animal are analyzed, and the number of micronucleated reticulocytes (MNRET) is recorded. Micronuclei are round in shape and fl uoresce green- yellow ( see Notes 6 and 8 ; Fig. 8 ). Then, the incidence of MNRET to total RET is calculated as a percent.

1. The defi nition of a positive call should be predetermined, for example, increment of incidence of micronucleated imma-ture erythrocytes is statistically signifi cant compared to that

3.7 Evaluation of Results

Fig. 7 Peripheral blood slide (×100: low magnifi cation)

Fig. 8 Peripheral blood slide (×800: high magnifi cation)

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of the negative control group. The biological relevance, of course, should be included, namely, the response is dose dependent and reproducible among animals within the treat-ment group.

2. The negative call can be defi ned as not positive, i.e., no signifi -cant increase over negative control. If the result is not clear and equivocal, we recommend engaging a repeat or a confi rmatory test.

3. Statistically signifi cant reduction of the ratio of PCE or RET as compared with the negative control is used as an indication of toxicity (inhibition of cell proliferation) in bone marrow.

4 Notes

1. If peripheral blood is used, a pretreatment sample may also be acceptable as a concurrent negative control to reduce the total number of animals to be used.

2. Extensive studies of the activity of known clastogens in the mouse bone marrow micronucleus assay have shown that, in general, male mice are more sensitive than female mice for micronucleus induction [ 4 ]. Quantitative differences in micro-nucleus induction have been identifi ed between the sexes, but no qualitative differences have been described.

3. To prepare good smears of the bone marrow, all favorable con-ditions including concentration and volume of the cell suspen-sion, angle of the cover glass, and speed of pulling the cover glass are required.

4. Slides are immersed in 0.001 % citric acid solution for a few seconds to bleach the Giemsa stain slightly. This procedure allows us to make easy differentiation of the PCE and NCE. If the NCE cannot be identifi ed, slides are immersed in the citric acid solution again for another few more seconds. On the other hand, if the PCE cannot be identifi ed, which would be caused by overbleaching, they should be re-stained after fully removing Giemsa stain by immersing in the ethanol.

5. If the slides are not sealed, air comes in between a slide and cover glass and makes it hard to clearly observe the slides.

6. Only typical micronuclei should be counted. To confi rm that micronuclei are present inside of a cell, change the microscopic focus up and down. If white-shining granules can be seen, then these should not be counted as micronuclei because they are artifi cial particles attached on the cells.

7. Acridine orange stains both RNA and DNA fl uorescing differ-ently, i.e., RNA fl uoresces red and DNA appears green-yellow

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when viewed under fl orescent light. RET contain RNA in their cytoplasm and can be distinguished easily from mature erythrocytes, which do not fl uoresce because they lack RNA.

8. Bacterial contamination is suspected when moving green-fl uorescing particles are observed in the slides.

References

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2. Schmid W (1975) The micronucleus test. Mutat Res 31:9–15

3. Hayashi M, Sofuni T, Ishidate M Jr (1984) Kinetics of micronucleus formation in relation to chromosomal aberrations in mouse bone marrow. Mutat Res 127:129–137

4. Sutou S, Hayashi M, Nishi Y et al (1986) Sex difference in the micronucleus test. The col-laborative study group for the micronucleus test. Mutat Res 172:151–163

5. Sutou S, Hayashi M, Shimada H et al (1988) Strain difference in the micronucleus test. The Collaborative Study Group for the Micronucleus Test. Mutat Res 204:307–316

6. Hayashi M, Sutou S, Shimada H et al (1989) Difference between intraperitoneal and oral gavage application in the micronucleus test: the 3rd collaborative study by CSGMT/JEMS.MMS. Mutat Res 223:329–344

7. Collaborative Study Group for the Micronucleus Test, the Mammalian Mutagenesis Study group of the Environmental Mutagen Society, Japan (CSGMT/JEMS⋅MMS) (1990) Single versus multiple dosing in the micronucleus test: the summary of the fourth collaborative study by CSGMT/JEMS⋅MMS. Mutat Res 234:205–222

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9. Morita T, Asano N, Awogi T et al (1997) Evaluation of the rodent micronucleus assay in the screening of IARC carcinogens (Groups 1, 2A, and 2B). The summary report of the 6th collaborative study by CSGMT/JEMS⋅MMS. Mutat Res 389:3–122

10. Organization for Economic Co-operation and Development (OECD) (1997) OECD Guideline for the Testing of Chemicals 474 Mammalian Erythrocyte Micronucleus Test

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