Flow Cytometric Analysis of Micronuclei in Peripheral Blood Reticulocytes IV: An Index of Chromosomal Damage in the Rhesus Monkey (Macaca mulatta)

• Published in: Toxicological sciences 2008-04, Vol.102 (2), p.352-358
• Main Authors: Hotchkiss, Charlotte E., Bishop, Michelle E., Dertinger, Stephen D., Slikker, William, Moore, Martha M., MacGregor, James T.
• Format: Article
• Language: English
• Subjects: Animals; Antineoplastic Agents - pharmacokinetics; Antineoplastic Agents - toxicity; blood; bone marrow; Bone Marrow Cells - drug effects; Bone Marrow Cells - pathology; chromosomal damage; Chromosome Aberrations - chemically induced; Cyclophosphamide - pharmacokinetics; Cyclophosphamide - toxicity; Female; flow cytometry; Flow Cytometry - methods; Injections, Intravenous; Kinetics; Macaca mulatta; Male; Micronuclei, Chromosome-Defective - drug effects; micronucleus; Micronucleus Tests - methods; Mutagens - pharmacokinetics; Mutagens - toxicity; Reference Values; reticulocytes; Reticulocytes - drug effects; Reticulocytes - pathology
• ISSN: 1096-6080; 1096-0929
• DOI: 10.1093/toxsci/kfn013

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.