Cross‐Species Genetic Toxicity Assessment Accomplished by Flow Cytometric Analysis of Blood

Jeffrey C. Bemis1, Dorothea K. Torous1, Carol R. Tometsko1, Stephen D. Dertinger1

1 Litron Laboratories, Rochester, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 2.14
DOI:  10.1002/0471140856.tx0214s36
Online Posting Date:  May, 2008
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The formation of micronuclei in blood cells has been an established indicator of genotoxicity for decades. Standard microscopy methods are time‐consuming and lack the objectivity that fully automated methods can provide. The ability of flow cytometric technology to rapidly and objectively survey thousands of cells for micronuclei can significantly improve the value of this endpoint. In addition, since many more cells can be scored, and specific populations can be targeted, species that historically have been difficult to obtain micronucleus data from, such as humans, can now be readily evaluated using this methodology. This unit describes a procedure for fixation, staining, and analysis of blood samples using materials supplied in MicroFlow kits (Litron Laboratories) and a single‐laser flow cytometer. This methodology provides a reliable, robust assessment of chromosome damage that is used in basic science research as well as drug‐safety screening programs at large pharmaceutical and chemical companies. Curr. Protoc. Toxicol. 36:2.14.1‐2.14.15. © 2008 by John Wiley & Sons, Inc.

Keywords: genotoxicity; chromosome damage; micronuclei; reticulocytes; erythrocytes; CD71‐defined antigen; CD61‐defined antigen; cross species

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Table of Contents

  • Introduction
  • Basic Protocol 1: Genetic Toxicity Assessment in Mouse Blood
  • Alternate Protocol 1: Genetic Toxicity Assessment Using High‐Density Rat Blood Samples
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Genetic Toxicity Assessment in Mouse Blood

  • MicroFlowPLUS kit (Litron Laboratories) specific for the species in use and containing:
    • Fixative (solution A)
    • Anticoagulant (solution B)
    • Washing/diluent buffer (solution C), ice cold
    • RNase solution (solution D)
    • Anti‐CD71‐FITC (solution E)
    • Propidium iodide (solution F)
    • Anti‐CD61‐PE (solution G)
    • Malaria biostandard
  • Peripheral blood sample (e.g., mouse, rat, canine, nonhuman primate, human) treated according to experimental design
  • Labeling solution I (see recipe)
  • Labeling solution II (see recipe)
  • DNA staining solution (see recipe)
  • 2.5‐ml microcentrifuge tubes
  • 15‐ml screw‐cap polypropylene tubes
  • –85°C chest freezer
  • Refrigerated centrifuge
  • 12 × 75–mm polypropylene or polystyrene flow cytometry tubes
  • Aluminum foil
  • Flow cytometer equipped with 488‐nm excitation and appropriate filters (e.g., 530‐ ±30‐nm band‐pass filter and 650‐nm long‐pass filter for green and red fluorescence, respectively)
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Literature Cited

Literature Cited
   Abramsson‐Zetterberg, L., Grawe, J., and 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.
   Dertinger, S.D., Torous, D.K., Hall, N., Tometsko, C.R., and Gasiewicz, T.A. 2000. Malaria‐infected erythrocytes serve as biological standards to ensure reliable and consistent scoring of micronucleated erythrocytes by flow cytometry. Mutat. Res. 464: 195‐200.
   Dertinger, S.D., Torous, D.K., Hall, N., and Tometsko, C.R. 2003. Measurement of cytogenetic damage in rodent blood with a single‐laser flow cytometer. Curr. Protoc. Cytometry 23: 7.21.1‐7.21.9.
   Dertinger, S.D., Camphausen, K., Macgregor, J.T., Bishop, M.E., Torous, D.K., Avlasevich, S., Cairns, S., Tometsko, C.R., Menard, C., Muanza, T., Chen, Y., Miller, R.K., Cederbrant, K., Sandelin, K., Ponten, I., and Bolcsfoldi, G. 2004. Three‐color labeling method for flow cytometric measurement of cytogenetic damage in rodent and human blood. Environ. Mol. Mutagen. 44: 427‐435.
   Hayashi, M., Kodama, Y., Awogi, T., Suzuki, T., Asita, A.O., and Sofuni, T. 1992. The micronucleus assay using peripheral blood reticulocytes from mitomycin C‐ and cyclophosphamide‐treated rats. Mutat. Res. 278: 209‐213.
   Hayashi, M., MacGregor, J.T., Gatehouse, D.G., Adler, I.‐D., Blakey, D.H., Dertinger, S.D., Krishna, G., Morita, T., Russo, A., and Sutou, S. 2000. In vivo rodent erythrocyte micronucleus assay: Aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring. A report from the International Workshop on Genotoxicity Test Procedures (IWGTP). Environ. Mol. Mutagen. 35: 234‐252.
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   Honda, K.I., Ishiko, O., Hato, F., Kitagawa, S., Jikihara, I., Yoshida, H., and Ogita, S. 2001. CD71 antibody enhances iron uptake by mouse bone marrow cells and the survival potential of erythroid progenitor cells. Int. J. Mol. Med. 8: 135‐140.
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   Organisation for Economic Cooperation and Development (OECD). 1997. OECD guidelines for the testing of chemicals: Mammalian Erythrocyte Micronucleus Test, Section 4, Guideline 474 (Draft: February 1997).
   Tometsko, A.M., Torous, D.K., and Dertinger, S.D. 1993. Analysis of micronucleated cells by flow cytometry. 1. Achieving high resolution with a malaria model. Mutat. Res. 292: 129‐135.
   Torous, D.K., Hall, N.E., Dertinger, S.D., Diehl, M.S., Illi‐Love, A.H., Cederbrant, K., Sandelin, K., Bolcsfoldi, G., Ferguson, L.R., Pearson, A., Majeska, J.B., Tarca, J.P., Hewish, D.R., Doughty, L., Fenech, M., Weaver, J.L., Broud, D.D., Gatehouse, D.G., Hynes, G.M., Kwanyuen, P., McLean, J., McNamee, J.P., Parenteau, M., Van Hoof, V., Vanparys, P., Lenarczyk, M., Siennicka, J., Litwinska, B., Slowikowska, M.G., Harbach, P.R., Johnson, C.W., Zhao, S., Aaron, C.S., Lynch, A.M., Marshall, I.C., Rodgers, B., and Tometsko, C.R. 2001. Flow cytometric enumeration of micronucleated reticulocytes: High transferability among 14 laboratories. Environ. Mol. Mutagen. 38: 59‐68.
   Tweats, D.J., Blakey, D., Heflich, R.H., Jacobs, A., Jacobsen, S.D., Morita, T., Nohmi, T., O'Donovan, M.R., Sasaki, Y.F., Sofuni, T., and Tice, R.; IWGT Working Group. 2007. Report of the IWGT working group on strategies and interpretation of regulatory in vivo tests I. Increases in micronucleated bone marrow cells in rodents that do not indicate genotoxic hazards. Mutat. Res. 627: 78‐91.
   Udroiu, I. 2006. Feasibility of conducting the micronucleus test in circulating erythrocytes from different mammalian species: An anatomical perspective. Environ. Mol. Mutagen. 47: 643‐646.
   Wakata, A., Miyamae, Y., Sato, S., Suzuki, T., Morita, T., Asano, N., Awogi, T., Kondo, K., and Hayashi, M. 1998. Evaluation of the rat micronucleus test with bone marrow and peripheral blood: Summary of the 9th collaborative study by CSGMT/JEMS‐MMS. Environ. Mol. Mutagen. 32: 84‐100.
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