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 (80 mg/kg/day), ethyl carbamate (600 mg/kg/day), or thiotepa (7.5 mg/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.

Assessment of the Pig‐a, micronucleus, and comet assay endpoints in rats treated by acute or repeated dosing protocols with procarbazine hydrochloride and ethyl carbamate

“…It is noteworthy that EC caused a significant induction of Pig‐a mutant cells: increased RET CD59− and RBC CD59− frequencies were noted in both the 3‐day and 28‐day studies. The kinetics and magnitude of the mutant cell responses in the 3‐day and 28‐day studies were very similar to those shown in an experiment conducted by Labash et al () in male and female rodents exposed to 600 mg/kg/day EC for 3 consecutive days and the research of Bemis et al () in rats dosed orally for 28 consecutive days with 250 mg/kg/day EC. However, the maximal mutation frequency of RET in our 28‐day treatment study with EC (150–300 mg/kg/day), an ~13.2‐fold increase over the control, was lower than the ~74.0‐fold increase in a similar study conducted by Stankowski et al (), where SD rats were treated orally with 25–250 mg/kg/day using the same dosing protocol; while the maximal ~26.4‐fold increase over the control for mutant RBC in our study is greater than the ~8.1‐fold increase in the study of Stankowski et al ().…”

“…Although the two test chemicals in our study, PCZ and EC, have been investigated previously in similar integrated studies as well as assays measuring single genotoxicity endpoints, including Pig‐a gene mutation (Phonethepswath et al, ; Bemis et al, ; Labash et al, ; Stankowski et al, ; Pu et al, ; Revollo et al, ), the data reported herein allow comparisons between the responses for multiple genotoxicity endpoints as a function of two commonly employed treatment regimens (short‐term, 3‐day and subchronic, 28‐day studies). In addition to conducting multiple genotoxicity assays, analyses were made to collect data for hematology, clinical chemistry, organ weight, and other general toxicity indicators.…”

The potential application of human PIG‐A assay on azathioprine‐treated inflammatory bowel disease patients

“…No clear sex bias has been found for Pig‐a mutation in rodent studies (Labash et al ; Labash et al ), while studies in different ethnic human populations have produced inconsistent results (Dobrovolsky et al ; Dertinger et al ; Cao et al ). The Pig‐a gene is on the X chromosome, suggesting that X‐chromosome inactivation in females may affect spontaneous MFs.…”

Standard protocol for the PIGRET assay, a high-throughput reticulocyte Pig-a assay with an immunomagnetic separation, used in the interlaboratory trial organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen and Genome Society

“…Healthy young adult rats, usually 6-10 weeks old at the start of dosing, are used. Male or female rats can be used for this assay [29][30][31]. Groups of 6 rats are recommended at the 6th IWGT Workgroup meeting.…”