Induction of Pig-a mutant erythrocytes in male and female rats exposed to 1,3-propane sultone, ethyl carbamate, or thiotepa

Abstract

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, groups of five male and female Sprague Dawley rats were exposed to the mutagens 1,3-propane sultone (80mg/kg/day), ethyl carbamate (600mg/kg/day), or thiotepa (7.5mg/kg/day) for three consecutive days (study days 1–3). Pig-a mutant phenotype reticulocyte (RETCD59−) and mutant phenotype erythrocyte (RBCCD59−) frequencies were determined on study days −4, 15, 30 and 46 using immunomagnetic separation in conjunction with flow cytometric analysis (In Vivo MutaFlow®). While the percentage of reticulocytes (%RET) was markedly higher for pre-treatment blood samples from males compared to females (6.6% vs. 3.5%), this sex effect was slight or nonexistent at later time points. Treatment-related effects to %RET were generally modest owing to the 12-day interval between last exposure and blood sampling. Mean RETCD59− and RBCCD59− frequencies were consistently low in vehicle control animals of both sexes, with 77% of samples exhibiting mutant cell frequencies ≤1×10−6 over study days 15–46. Treatment with each mutagen caused significant increases to mean RETCD59− and RBCCD59− frequencies. Whereas genotoxicant-induced RETCD59− values were maximal on day 15, induced RBCCD59− frequencies were highest at the last sampling time. Sex did not affect 1,3-propane sultone- or thiotepa-induced mutant cell frequencies. While ethyl carbamate-exposed females exhibited higher mean mutant cell frequencies compared to like-treated males, statistical significance was achieved only for RBCCD59− at one time point (7.6±1.0×10−6 compared to 4.7±0.6×10−6 on day 30). Thus, while some quantitative differences were evident, there were no qualitative differences in how males and females responded to three diverse mutagens. These data support the use of both sexes for Pig-a gene mutation studies.