Manifestation and persistence of Pig-a mutant red blood cells in C57BL/6 mice following single and split doses of N-ethyl-N-nitrosourea.

ABSTRACT

Treating rats with single doses of N-ethyl-N-nitrosourea (ENU) results in a time-dependent accumulation of Pig-a-mutant phenotype peripheral red blood cells (RBCs), reaching a plateau at about 6-weeks posttreatment, with the response persisting for at least 26 weeks. In the present study, groups of 5 C57BL/6 male mice were administered single i.p. doses of up to 140 mg/kg ENU, and blood samples were collected up to 26 weeks posttreatment. The samples were analyzed by flow cytometry for the frequency of CD24-deficient (presumed Pig-a mutant) reticulocytes (RETs) and total RBCs; micronucleated RET frequencies were evaluated at 1 day posttreatment. Mean Pig-a mutant frequencies and micronucleated RET frequencies increased in a dose-responsive manner, with maximum Pig-a frequencies in RETs and RBCs observed at Week 2 and Week 4 posttreatment, respectively. Mutant frequencies in RETs and RBCs generally decreased slowly with time after reaching their maxima. In a second experiment, groups of five male C57BL/6 mice were given single i.p. injections of 8, 32, or 160 mg/kg ENU, or four weekly doses of 8 or 40 mg/kg ENU (split doses totaling 32 and 160 mg/kg, respectively). In each case the maximum RET and RBC mutant frequencies produced by the split doses were similar to but not as great as the mutant frequencies produced by the equivalent single doses. The data indicate that ENU-induced Pig-a mutant RBC frequencies accumulate in mice as they do in rats; however, mice and rats differ in the manifestation kinetics and the persistence of the responses.