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ToxSci Advance Access originally published online on January 21, 2008
Toxicological Sciences 2008 102(2):352-358; 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)

Charlotte E. Hotchkiss*, Michelle E. Bishop{dagger}, Stephen D. Dertinger{ddagger}, William Slikker, Jr{dagger}, Martha M. Moore{dagger} and James T. MacGregor§,1,2

* The Bionetics Corporation, Jefferson, Arkansas72079 {dagger} National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas, 72079 {ddagger} Litron Laboratories, Rochester, New York, 14623 § National Center for Toxicological Research, U.S. Food and Drug Administration, Rockville, Maryland, 20857

1 To whom correspondence should be addressed at Toxicology Consulting Services, 201 Nomini Drive, Arnold, MD 21012. Fax: (410) 975-0481. E-mail: jtmacgregor{at}earthlink.net.

Received November 16, 2007; 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-fluorescein isothiocyanate, DNA was stained with propidium iodide using RNase treatment, and anti-CD61-phycoerythrin 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 iv administration of cyclophosphamide (CP, 5 mg/kg) induced an approximately 10-fold increase in blood MN-RET frequency, with the peak occurring 2 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 MN-RETs. 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 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 RETs 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.

2 Present address: Toxicology Consulting Services, Arnold, MD 21012.


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