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

• Published in: Toxicological sciences 2007-12, Vol.100 (2), p.406-414
• Main Authors: Harper, Susan B., Dertinger, Stephen D., Bishop, Michelle E., Lynch, Anthony M., Lorenzo, Maria, Saylor, Michelle, MacGregor, James T.
• Format: Article
• Language: English
• Subjects: Animals; blood; bone marrow; Bone Marrow - drug effects; Bone Marrow - pathology; Bone Marrow Cells - drug effects; Bone Marrow Cells - pathology; chromosomal damage; Cyclophosphamide - toxicity; Dogs; Female; flow cytometry; Flow Cytometry - methods; Male; Micronuclei, Chromosome-Defective - chemically induced; micronucleus; Micronucleus Tests - methods; Mutagens - toxicity; Plasmodium; Reproducibility of Results; reticulocytes; Reticulocytes - drug effects; Reticulocytes - pathology
• ISSN: 1096-6080; 1096-0929
• DOI: 10.1093/toxsci/kfm241

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.