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ToxSci Advance Access originally published online on September 13, 2007
Toxicological Sciences 2007 100(2):406-414; doi:10.1093/toxsci/kfm241
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Published by Oxford University Press 2007.

Flow Cytometric Analysis of Micronuclei in Peripheral Blood Reticulocytes III. An Efficient Method of Monitoring Chromosomal Damage in the Beagle Dog

Susan B. Harper*,¶, Stephen D. Dertinger{dagger}, Michelle E. Bishop{ddagger}, Anthony M. Lynch§, Maria Lorenzo,1, Michelle Saylor,2 and James T. MacGregor||,3,4

* Department of Veterans Affairs, Washington, DC 20422 {dagger} Litron Laboratories, Rochester, NY 14623 {ddagger} National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR 72079 § GlaxoSmithKline Research & Development, Herts, SG12 0DP, UK U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Laurel, MD 20708 || Toxicology Consulting Services, Arnold, MD 21012

4 To whom correspondence should be addressed at Toxicology Consulting Services, Arnold, MD 21012. Fax: (410) 975-0481. E-mail: jtmacgregor{at}earthlink.net.

Received June 11, 2007; accepted September 5, 2007


  Abstract

Erythrocyte-based micronucleus tests have traditionally analyzed bone marrow because splenic filtration in most species removes micronucleated cells from peripheral blood. We have evaluated a flow cytometric method for monitoring micronucleated reticulocyte frequencies (%MN-RET) in the peripheral blood of beagle dogs treated with cyclophosphamide (CP) and have found that analysis of micronucleated reticulocytes (MN-RETs) in peripheral blood is a suitable surrogate for bone marrow analysis. The three-color flow cytometric method uses anti-CD71 labeling to identify reticulocytes and Plasmodium berghei–containing erythrocytes as a calibration standard. The spontaneous %MN-RET determined by flow cytometry was 0.31 ± 0.09% (n = 22) for peripheral blood, compared with 0.38 ± 0.13% (SD, n = 12) for bone marrow, and 0.27 ± 0.08% (n = 12) for peripheral blood by microscopic scoring with acridine orange staining. The kinetics of appearance and disappearance of MN-RETs in blood were determined by collecting daily samples after iv treatment with CP. The maximum frequency occurred ~48 h after dosing. Frequencies of MN-RETs in peripheral blood at steady state following daily CP treatment were 55–68% of corresponding bone marrow values assessed by microscopy and 55–112% as assessed by flow cytometry. This difference is presumably due to splenic removal, which appears slightly less stringent than that previously reported for CP-treated Sprague-Dawley rats. Responses in bone marrow and peripheral blood were highly correlated and similar to or greater than those reported in mice and rats at equitoxic doses.

Key Words: chromosomal damage; micronucleus; flow cytometry; reticulocytes; blood; bone marrow.

1 Present address: Johns-Hopkins University, Baltimore, MD 21218.

2 Present address: U.S. Army Medical Research Institute for Infectious Diseases, Fort Detrick, MD 21702.

3 Previous address: U.S. Food and Drug Administration, National Center for Toxicological Research, Rockville, MD 20857.

Disclaimer: The contents of this article are the sole responsibility of the authors and do not necessarily reflect the views or policies of the institutions or companies with which they are affiliated.


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C. E. Hotchkiss, M. E. Bishop, S. D. Dertinger, W. Slikker Jr, M. M. Moore, and J. T. MacGregor
Flow Cytometric Analysis of Micronuclei in Peripheral Blood Reticulocytes IV: An Index of Chromosomal Damage in the Rhesus Monkey (Macaca mulatta)
Toxicol. Sci., April 1, 2008; 102(2): 352 - 358.
[Abstract] [Full Text] [PDF]


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