Comparison of male versus female responses in the Pig-a mutation assay

Validation of the Pig-a gene mutation assay has been based mainly on studies in male rodents. To determine if the mutagen-induced responses of the X-linked Pig-a gene differ in females compared to males, 7- or 14-week old male and female Sprague Dawley rats were exposed to N-ethyl-N-nitrosourea (ENU). In the study with the 7-week old rats, exposure was to 0, 1, 5 or 25mg ENU/kg/day for three consecutive days (study Days 1-3). Pig-a mutant phenotype reticulocyte (RET(CD59-)) and mutant phenotype erythrocyte (RBC(CD59-)) frequencies were determined on study Days -4, 15, 29 and 46 using immunomagnetic separation in conjunction with flow cytometric analysis (In Vivo MutaFlow®). Additionally, blood samples collected on Day 4 were analysed for micronucleated reticulocyte (MN-RET) frequency (In Vivo MicroFlow®). The percentage of reticulocytes (%RET) was markedly higher in the 7-week old males compared to females through Day 15 (2.39-fold higher on Day -4). At 25mg/kg/day, ENU reduced Day 4 RET frequencies in both sexes, and the two highest dose levels resulted in elevated MN-RET frequencies, with no sex or treatment × sex interaction. The two highest dose levels significantly elevated the frequencies of mean RET(CD59-) and RBC(CD59-) in both sexes from Day 15 onward. RET(CD59-) and RBC(CD59-) frequencies were somewhat lower for females compared to males at the highest dose level studied, and differences in RET(CD59-) resulted in a statistically significant interaction effect of treatment × sex. In the study with 14-week old rats, treatment was for 3 days with 0 or 25mg ENU/kg/day. RET frequencies differed to a lesser degree between the sexes, and in this case there was no evidence of a treatment × sex interaction. These results suggest that the slightly higher response in younger males than in the younger females may be related to differences in erythropoiesis function at that age. In conclusion, while some quantitative differences were noted, there were no qualitative differences in how males and females responded to a prototypical mutagen, and support the contention that both sexes are equally acceptable for Pig-a gene mutation studies.