Abstract
During the last two decades the micronucleus (MN) test has been extensively used as a genotoxicity screening tool of chemicals and in a variety of exploratory and mechanistic investigations. The MN is a biomarker for chromosomal damage or mitotic abnormalities since it can originate from chromosome fragments or whole chromosomes that fail to be incorporated into daughter nuclei during mitosis (Fenech et al., Mutagenesis 26: 125–132, 2011; Kirsch-Volders et al., Arch Toxicol 85: 873–899, 2011). The simplicity of scoring, accuracy, amenability to automation by image analysis or flow cytometry and the readiness to be applied to a variety of cell types either in vitro or in vivo made it a versatile tool that contributed to a large extent in our understanding of key toxicological issues related to genotoxins and their effects at the cellular and organism levels. Recently, the final acceptance of the in vitro MN test Organization for Economic Cooperation and Development (OECD) guideline 487 (OECD, Guideline for testing of chemicals: in vitro mammalian cell micronucleus test 487: in vitro mammalian cell micronucleus test (MNVIT). Organization for Economic Cooperation and Development, Paris, 2010) together with the standard in vivo MN test OECD guideline 474 (OECD, Guideline for the testing of chemicals no. 474 mammalian erythrocyte micronucleus test. Organization for Economic Cooperation and Development, Paris, 1997) further positioned the assay as a key driver in the determination of the genotoxicity potential in exploratory research as well as in the regulatory environment. This book chapter covers to some extent the protocol designs and experimental steps necessary for a successful performance of the MN test and an accurate analysis of the MN by the flow cytometry technique.
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References
Fenech M, Kirsch-Volders M, Natarajan AT et al (2011) Molecular mechanisms of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells. Mutagenesis 26:125–132
Kirsch-Volders M, Plas G, Elhajouji A et al (2011) The in vitro MN assay in 2011: origin and fate, biological significance, protocols, high throughput methodologies and toxicological relevance. Arch Toxicol 85:873–899
OECD (2010) Guideline for testing of chemicals: in vitro mammalian cell micronucleus test 487: in vitro mammalian cell micronucleus test (MNVIT). Organization for Economic Cooperation and Development, Paris
OECD (1997) Guideline for the testing of chemicals no. 474 mammalian erythrocyte micronucleus test. Organization for Economic Cooperation and Development, Paris
International Conference on Harmonization (2008) Guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use S2(R1). http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Safety/S2_R1/Step4/S2R1_Step4.pdf. Accessed on 5 Mar 2012, Geneva
Corvi R, Albertini S, Hartung T et al (2008) ECVAM retrospective validation of in vitro micronucleus test (MNT). Mutagenesis 23:271–283
Fenech M, Morley AA (1985) Measurement of micronuclei in lymphocytes. Mutat Res 147:29–36
Carter SB (1967) Effects of cytochalasins on mammalian cells. Nature 213:261–264
Elhajouji A, Cunha M, Kirsch-Volders M (1998) Spindle poisons can induce polyploidy by mitotic slippage and micronucleate mononucleates in the cytokinesis-block assay. Mutagenesis 13:193–198
Decordier I, Cundari E, Kirsch-Volders M (2008) Mitotic checkpoints and the maintenance of the chromosome karyotype. Mutat Res 651:3–13
Kirsch-Volders M, Sofuni T, Aardema M et al (2003) Report from the in vitro micronucleus assay working group. Mutat Res 540:153–163
Fenech M (2006) Cytokinesis-block micronucleus assay evolves into a “cytome” assay of chromosomal instability, mitotic dysfunction and cell death. Mutat Res 600:58–66
Kirkland D, Pfuhler S, Tweats D et al (2007) How to reduce false positive results when undertaking in vitro genotoxicity testing and thus avoid unnecessary follow-up animal tests: report of an ECVAM workshop. Mutat Res 628:31–55
Avlasevich SL, Bryce SM, Cairns SE et al (2006) In vitro micronucleus scoring by flow cytometry: differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability. Environ Mol Mutagen 47:56–66
Bocker W, Muller WU, Streffer C (1995) Image processing algorithms for the automated micronucleus assay in binucleated human lymphocytes. Cytometry 19:283–294
Bocker W, Streffer C, Muller WU et al (1996) Automated scoring of micronuclei in binucleated human lymphocytes. Int J Radiat Biol 70:529–537
Bryce SM, Avlasevich SL, Bemis JC et al (2008) Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay. Mutat Res 650:181–195
Bryce SM, Bemis JC, Avlasevich SL et al (2007) In vitro micronucleus assay scored by flow cytometry provides a comprehensive evaluation of cytogenetic damage and cytotoxicity. Mutat Res 630:78–91
Castelain P, Van Hummelen P, Deleener A et al (1993) Automated detection of cytochalasin-B blocked binucleated lymphocytes for scoring micronuclei. Mutagenesis 8:285–293
Decordier I, Papine A, Plas G et al (2009) Automated image analysis of cytokinesis-blocked micronuclei: an adapted protocol and a validated scoring procedure for biomonitoring. Mutagenesis 24:85–93
Frieauff W, Potter-Locher F, Cordier A et al (1998) Automatic analysis of the in vitro micronucleus test on V79 cells. Mutat Res 413:57–68
Lukamowicz M, Kirsch-Volders M, Suter W et al (2011) In vitro primary human lymphocyte flow cytometry based micronucleus assay: simultaneous assessment of cell proliferation, apoptosis and MN frequency. Mutagenesis 26:763–770
Lukamowicz M, Woodward K, Kirsch-Volders M et al (2011) A flow cytometry based in vitro micronucleus assay in TK6 cells-validation using early stage pharmaceutical development compounds. Environ Mol Mutagen 52:363–372
Lukamowicz-Rajska M, Kirsch-Volders M, Suter W et al (2012) Miniaturized flow cytometry-based in vitro primary human lymphocyte micronucleus assay-validation study. Environ Mol Mutagen 53(4):260–270
Nusse M, Kramer J (1984) Flow cytometric analysis of micronuclei found in cells after irradiation. Cytometry 5:20–25
Nusse M, Marx K (1997) Flow cytometric analysis of micronuclei in cell cultures and human lymphocytes: advantages and disadvantages. Mutat Res 392:109–115
Roman D, Locher F, Suter W et al (1998) Evaluation of a new procedure for the flow cytometric analysis of in vitro, chemically induced micronuclei in V79 cells. Environ Mol Mutagen 32:387–396
Schreiber GA, Beisker W, Bauchinger M et al (1992) Multiparametric flow cytometric analysis of radiation-induced micronuclei in mammalian cell cultures. Cytometry 13:90–102
Slavotinek A, Miller E, Taylor GM et al (1995) Micronucleus frequencies in lymphoblastoid cell lines measured with the cytokinesis-block technique and flow cytometry. Mutagenesis 10:439–445
Tates AD, van Welie MT, Ploem JS (1990) The present state of the automated micronucleus test for lymphocytes. Int J Radiat Biol 58:813–825
Varga D, Johannes T, Jainta S et al (2004) An automated scoring procedure for the micronucleus test by image analysis. Mutagenesis 19:391–397
Verhaegen F, Vral A, Seuntjens J et al (1994) Scoring of radiation-induced micronuclei in cytokinesis-blocked human lymphocytes by automated image analysis. Cytometry 17:119–127
Viaggi S, Braselmann H, Nusse M (1995) Flow cytometric analysis of micronuclei in the CD2+/− subpopulation of human lymphocytes enriched by magnetic separation. Int J Radiat Biol 67:193–202
Wessels JM, Nusse M (1995) Flow cytometric detection of micronuclei by combined staining of DNA and membranes. Cytometry 19:201–208
Soejima T, Iida K, Qin T et al (2007) Photoactivated ethidium monoazide directly cleaves bacterial DNA and is applied to PCR for discrimination of live and dead bacteria. Microbiol Immunol 51:763–775
Heddle JA (1973) A rapid in vivo test for chromosomal damage. Mutat Res 18:187–190
Schmid W (1975) The micronucleus test. Mutat Res 31:9–15
Abramsson-Zetterberg L, Grawe J, Zetterberg G (1999) The micronucleus test in rat erythrocytes from bone marrow, spleen and peripheral blood: the response to low doses of ionizing radiation, cyclophosphamide and vincristine determined by flow cytometry. Mutat Res 423:113–124
Cammerer Z, Schumacher MM, Kirsch-Volders M et al (2010) Flow cytometry peripheral blood micronucleus test in vivo: determination of potential thresholds for aneuploidy induced by spindle poisons. Environ Mol Mutagen 51:278–284
Torous DK, Dertinger SD, Hall NE et al (2000) Enumeration of micronucleated reticulocytes in rat peripheral blood: a flow cytometric study. Mutat Res 465:91–99
Torous DK, Hall NE, Dertinger SD et al (2001) Flow cytometric enumeration of micronucleated reticulocytes: high transferability among 14 laboratories. Environ Mol Mutagen 38:59–68
Wakata A, Miyamae Y, Sato S et al (1998) Evaluation of the rat micronucleus test with bone marrow and peripheral blood: summary of the 9th collaborative study by CSGMT/JEMS. MMS. Collaborative Study Group for the Micronucleus Test. Environmental Mutagen Society of Japan. Mammalian Mutagenicity Study Group. Environ Mol Mutagen 32:84–100
Vander JB, Harris CA, Ellis SR (1963) Reticulocyte counts by means of fluorescence microscopy. J Lab Clin Med 62:132–140
Hayashi M, MacGregor JT, Gatehouse DG et al (2000) In vivo rodent erythrocyte micronucleus assay. II. Some aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring. Environ Mol Mutagen 35:234–252
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
Cammerer Z, Elhajouji A, Suter W (2007) In vivo micronucleus test with flow cytometry after acute and chronic exposures of rats to chemicals. Mutat Res 626:26–33
Tometsko AM, Torous DK, Dertinger SD (1993) Analysis of micronucleated cells by flow cytometry. 1. Achieving high resolution with a malaria model. Mutat Res 292:129–135
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Elhajouji, A., Stadelmann, P. (2019). Micronucleus Analysis by Flow Cytometry. In: Dhawan, A., Bajpayee, M. (eds) Genotoxicity Assessment. Methods in Molecular Biology, vol 2031. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9646-9_9
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DOI: https://doi.org/10.1007/978-1-4939-9646-9_9
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