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ToxSci Advance Access published online on January 21, 2008
Toxicological Sciences, doi:10.1093/toxsci/kfn013
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Published by Oxford University Press 2008.

Flow Cytometric Analysis of Micronuclei in Peripheral Blood Reticulocytes IV: An index of chromosomal damage in the rhesus monkey (Macaca mulatta)a

Charlotte E. Hotchkiss1, Michelle E. Bishop2, Stephen D. Dertinger3, William Slikker, Jr.2, Martha M. Moore2 and James T. MacGregor4,5

1 The Bionetics Corporation, Jefferson, AR 2 FDA National Center for Toxicological Research, Jefferson, AR 3 Litron Laboratories, Rochester, NY 4 FDA National Center for Toxicological Research, Rockville MD 5 presently Toxicology Consulting Services, Arnold, MD

Corresponding author: James T. MacGregor, Toxicology Consulting Services, 201 Nomini Drive, Arnold, MD 21012, Phone: 410-975-0480, Fax: 410-975-0481, E-mail: jtmacgregor{at}earthlink.net

Received November 16, 2007; revision received January 2, 2008; accepted January 10, 2008


  Abstract

We report evaluation in rhesus monkeys of a flow cytometric procedure (MicroFlow®) that has previously been shown to allow assessment of micronucleated reticulocytes (MN-RETs) in the peripheral blood of rats and dogs. Reticulocytes (RETs) were labeled with anti-CD71-FITC, DNA was stained with propidium iodide using RNase treatment, and anti-CD61-PE was used to reduce interference from platelets. Flow cytometric data were compared with microscopic scores of peripheral blood and bone marrow using standard acridine orange staining. A single intravenous administration of cyclophosphamide (CP, 5 mg/kg) induced an approximately 10-fold increase in blood MN-RET frequency, with the peak occurring two days after administration. After daily CP treatment to approximate a steady-state condition, the frequency of MN-RETs in peripheral blood was approximately 25% of that in bone marrow, indicating strong selection against micronucleated erythrocytes. Nonetheless, CP-treated animals exhibited markedly elevated blood MN-RET values (2.45 - 3.99%, n=3; compared to a mean baseline of 0.12%, n=6). These measurements closely reflected the increased frequencies observed in the bone marrow compartment (Spearman's correlation coefficient = 0.9856, n = 6). These data suggest that MN-RET measurements in blood are suitable for assessing chemical-induced chromosomal damage and can be readily integrated into routine toxicity tests, allowing genotoxicity data to be obtained as an integral part of toxicity evaluations. Microscopy-based scoring is challenging due to the low frequency of reticulocytes and MN-RET in monkeys, but sufficient numbers of cells are easily scored with the flow cytometric procedure.

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

a The views presented in this article do not necessarily reflect those of the U.S. Food and Drug Administration


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