Accumulation and persistence of Pig-A mutant peripheral red blood cells following treatment of rats with single and split doses of N-ethyl- N-nitrosourea

Abstract

We previously reported the development of an in vivo gene mutation assay using the phosphatidylinositol glycan complementation group A gene ( Pig-A) as an endogenous reporter. The assay quantifies mutation in rat peripheral red blood cells (RBCs) by flow cytometric detection of cells negative for glycosylphosphatidyl inositol (GPI)-anchored protein surface markers. In this study, we examined the accumulation and persistence of Pig-A mutant RBCs in rats treated with N-ethyl- N-nitrosourea (ENU) using two dosing schedules. Male F344 rats were given single i.p. injections of 8.9, 35.6, or 142.4 mg/kg ENU or four equal weekly doses totaling 35.6 or 142.4 mg/kg ENU (8.9 mg/kg × 4 or 35.6 mg/kg × 4; split-dose groups). Before the treatment and through 26 weeks after the single dose or beginning the split-dose regimen, peripheral RBCs were collected and Pig-A mutant frequencies measured as RBCs negative for the GPI-anchored protein, CD59. Mean CD59-negative RBC frequencies in negative control rats ranged from 3.9 × 10 −6 to 28.7 × 10 −6 and displayed no time-related trend. With single ENU doses, CD59-negative RBC frequencies increased in a time- and dose-related manner. Maximum responses were observed beginning at 6 weeks post-treatment (57.3 × 10 −6 in the 8.9 mg/kg group; 186.9 × 10 −6 in the 35.6 mg/kg group; 759.2 × 10 −6 in the 142.4 mg/kg group), and these elevated mutant frequencies persisted to the last sampling time. In addition, splitting the dose of ENU into four weekly doses produced nearly the same mutant frequency as when given as a single dose: the maximum responses after four weekly doses of 8.9 or 35.6 mg/kg were 176.8 × 10 −6 and 683.3 × 10 −6, respectively. These results indicate that ENU-induced Pig-A mutant RBCs accumulate in a near additive fashion in rats, and once present in the peripheral blood, persist for at least 6 months. These characteristics of Pig-A mutation could be important for detecting weak mutagens by repeated or subchronic/chronic dosing protocols.