Flow cytometric analysis of micronuclei in peripheral blood reticulocytes IV: an index of chromosomal damage in the rhesus monkey (Macaca mulatta).
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.
Hotchkiss
Charlotte E
CE
The Bionetics Corporation, Jefferson, Arkansas 72079, USA.
Bishop
Michelle E
ME
Dertinger
Stephen D
SD
Slikker
William
W
Moore
Martha M
MM
Macgregor
James T
JT
D000818:Animals; D000970:Antineoplastic Agents / Q000493:pharmacokinetics / Q000633:toxicity; D001854:Bone Marrow Cells / Q000187:drug effects / Q000473:pathology; D002869:Chromosome Aberrations / Q000139:chemically induced*; D003520:Cyclophosphamide / Q000493:pharmacokinetics / Q000633:toxicity; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D007275:Injections, Intravenous; D007700:Kinetics; D008253:Macaca mulatta; D008297:Male; D048629:Micronuclei, Chromosome-Defective / Q000187:drug effects*; D015162:Micronucleus Tests / Q000379:methods*; D009153:Mutagens / Q000493:pharmacokinetics / Q000633:toxicity*; D012016:Reference Values; D012156:Reticulocytes / Q000187:drug effects* / Q000473:pathology
D000970:Antineoplastic Agents; D009153:Mutagens; D003520:Cyclophosphamide