Flow cytometric analysis of micronuclei in peripheral blood reticulocytes III. An efficient method of monitoring chromosomal damage in the beagle dog.
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 approximately 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.
Harper
Susan B
SB
Department of Veterans Affairs, Washington, DC 20422, USA.
Dertinger
Stephen D
SD
Bishop
Michelle E
ME
Lynch
Anthony M
AM
Lorenzo
Maria
M
Saylor
Michelle
M
MacGregor
James T
JT
D000818:Animals; D001853:Bone Marrow / Q000187:drug effects / Q000473:pathology; D001854:Bone Marrow Cells / Q000187:drug effects / Q000473:pathology; D003520:Cyclophosphamide / Q000633:toxicity*; D004285:Dogs; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D008297:Male; D048629:Micronuclei, Chromosome-Defective / Q000139:chemically induced*; D015162:Micronucleus Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity*; D015203:Reproducibility of Results; D012156:Reticulocytes / Q000187:drug effects* / Q000473:pathology
D009153:Mutagens; D003520:Cyclophosphamide