Comparative scoring of micronucleated reticulocytes in rat peripheral blood by flow cytometry and microscopy.

A flow cytometric technique for scoring the incidence of micronucleated reticulocytes in rat peripheral blood was compared to a standard microscopy-based procedure. For these studies, groups of five male Sprague-Dawley rats were treated with vehicle or a broad range of chemical genotoxicants: 6-thioguanine, N-methyl-N'-nitro-N-nitrosoguanidine, vincristine, methylaziridine, acetaldehyde, methyl methanesulfonate, benzene, monocrotaline, and azathioprine. Animals were treated once a day for up to 2 days, and peripheral blood was collected between 24 and 48 h after the final administration. These samples were processed for flow cytometric scoring and microscopy-based analysis using supravital acridine orange staining, and the percentage of reticulocytes and micronucleated reticulocytes was determined for each sample. The resulting data demonstrate good agreement between these scoring methodologies, although careful execution of the flow cytometric method was found to enhance the micronucleus assay by reducing both scoring time and scoring error. These data add further support to the premise that the peripheral blood compartment of rats can be used effectively to detect genotoxicant-induced micronuclei.

[1]  M. Fenech,et al.  Flow cytometric enumeration of micronucleated reticulocytes: High transferability among 14 laboratories , 2001, Environmental and molecular mutagenesis.

[2]  T. Sofuni,et al.  The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides. , 1990, Mutation research.

[3]  Csgmt.  Micronucleus test with mouse peripheral blood erythrocytes by acridine orange supravital staining: the summary report of the 5th collaborative study by CSGMT/JEMS.MMS. The Collaborative Study Group for the Micronucleus Test. , 1992, Mutation research.

[4]  C. Streffer,et al.  [The micronucleus test]. , 1988, Strahlenschutz in Forschung und Praxis.

[5]  T. Sofuni,et al.  The micronucleus assay using peripheral blood reticulocytes from mitomycin C- and cyclophosphamide-treated rats. , 1992, Mutation research.

[6]  R R Tice,et al.  In vivo rodent erythrocyte micronucleus assay. , 1994, Mutation research.

[7]  J. Bishop.  Molecular themes in oncogenesis , 1991, Cell.

[8]  G. Alink,et al.  A comparative study on the metabolic activation of 3,4-benzo[a]pyrene to mutagens by aorta smooth muscle cells of rat and rabbit. , 1990, Mutation research.

[9]  S. Dertinger,et al.  Simple and reliable enumeration of micronucleated reticulocytes with a single-laser flow cytometer. , 1996, Mutation research.

[10]  R. Schlegel,et al.  The persistence of micronucleated erythrocytes in the peripheral circulation of normal and splenectomized Fischer 344 rats: implications for cytogenetic screening. , 1984, Mutation research.

[11]  J. T. Macgregor,et al.  Clastogen-induced micronuclei in peripheral blood erythrocytes: the basis of an improved micronucleus test. , 1980, Environmental mutagenesis.

[12]  J A Heddle,et al.  A rapid in vivo test for chromosomal damage. , 1973, Mutation research.

[13]  T. Sofuni,et al.  An application of Acridine Orange fluorescent staining to the micronucleus test. , 1983, Mutation research.

[14]  M. Bergqvist,et al.  Human cytogenetic biomonitoring using flow‐cytometric analysis of micronuclei in transferrin‐positive immature peripheral blood reticulocytes , 2000, Environmental and molecular mutagenesis.

[15]  S. Dertinger,et al.  Enumeration of micronucleated CD71-positive human reticulocytes with a single-laser flow cytometer. , 2002, Mutation research.

[16]  Csgmt.  The Collaborative Study Group for the Micronucleus Test : Micronucleus test with mouse peripheral blood erythrocytes by acridine orange supravital staining : The summary report of the 5th collaborative study by CSGMT/JEMS MMS , 1992 .

[17]  L. Abramsson-Zetterberg,et al.  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. , 1999, Mutation research.

[18]  S. Dertinger,et al.  Analysis of micronucleated cells by flow cytometry. 4. Kinetic analysis of cytogenetic damage in blood. , 1995, Mutation research.

[19]  The suitability of rat peripheral blood in subchronic studies for the micronucleus assay. , 1995, Mutation research.

[20]  J. Barrett,et al.  Mechanisms of multistep carcinogenesis and carcinogen risk assessment. , 1993, Environmental health perspectives.

[21]  K. O’Neill,et al.  Analysis of single-cell gel electrophoresis using laser-scanning microscopy. , 1993, Mutation research.

[22]  Makoto Hayashi,et al.  Evaluation of the rat micronucleus test with bone marrow and peripheral blood: Summary of the 9th collaborative study by CSGMT/JEMS·MMS , 1998, Environmental and molecular mutagenesis.

[23]  G. Krishna,et al.  Practical aspects of mutagenicity testing strategy: an industrial perspective. , 2000, Mutation research.

[24]  S. Dertinger,et al.  Analysis of micronucleated cells by flow cytometry. 1. Achieving high resolution with a malaria model. , 1993, Mutation research.

[25]  J. T. Macgregor,et al.  In vivo rodent erythrocyte micronucleus assay. II. Some aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring , 2000, Environmental and Molecular Mutagenesis.

[26]  S. Dertinger,et al.  Enumeration of micronucleated reticulocytes in rat peripheral blood: a flow cytometric study. , 2000, Mutation research.

[27]  A. Wakata,et al.  Evaluation of the rodent micronucleus assay by a 28‐day treatment protocol: Summary of the 13th Collaborative Study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Environmental Mutagen Society of Japan (JEMS)–Mammalian Mutagenicity Study Group (MMS) , 2001, Environmental and molecular mutagenesis.