Pig-a Gene Mutation and Micronucleus Induction in Rat Peripheral Blood by Methyl Methanesulfonate

Abstract

A recent report on an interlaboratory trial on the Pig-a mutation assays using an antibody against the erythroid marker (HIS49 Pig-a assays) has shown transferability and reproducibility of the assays. In this report, methyl methanesulfonate (MMS) was used to further evaluate the ability of HIS49 Pig-a assays in different study designs (28-day repeated dose and single dose). MMS was administered to male Sprague Dawley (SD) rats orally for 28 days at a dose of 7.5, 15, or 30 mg/kg/day or by single oral gavage at 50, 100 and 200 mg/kg in 28-day study and single dose study, respectively. Pig-a assays for total red blood cells (RBC Pig-a assay), for reticulocytes (PIGRET assay) and micronucleated reticulocytes (MN-RET) measurement were performed. In the 28-day study, there were significant increases in the Pig-a mutant frequency (Pig-a MF) in PIGRET assay at 30 mg/kg/day on Days 8, 15 and 29 with the maximum Pig-a MF on Day 29. On the contrary, there was no change in the Pig-a MF in RBC Pig-a assay. In the single dose study, MMS resulted in a steady increase in Pig-a MF in PIGRET assay from Day 8 through to Day 29 and a delayed increase in Pig-a MF in RBC Pig-a assay at Day 15 and 29 with maximam Pig-a MF on Day 29. MMS also induced significant increases in the MN-RET frequency on Day 4 of the 28-day study and on Day 3 of the single dose study. These results suggest that the PIGRET assay has the following advantages. The accumulated properties with repeated doses would be useful when the assay is integrated with a repeated dose general toxicity study, and the rapid detection of mutagenesis using a single dose protocol would be useful for early assessment of the in vivo mutagenicity of chemicals.

Pyrene did not induce gene mutation in red blood cell Pig-a assay and PIGRET assay in rats

“…The sample preparation and the gating strategy for the RBC Pig-a assay and PIGRET assay were described previously [18][19][20][21][22]. In brief, 3 L of blood in heparin were mixed with 0.2 mL PBS, and then the cells were labeled with anti-rat CD59 (1 g) and anti-rat erythroid marker (0.133 g) antibodies.…”

“…After gating for single cells, approximately 1 × 10 6 erythroid marker (HIS49)-positive cells were analyzed for the presence of surface CD59, and the Pig-a MFs were calculated as previously described. Briefly, the FCM gating strategy to count CD59-negative cells has been described previously [18][19][20][21][22]. Before analyzing the experimental samples, all gates to count the CD59-negative RBCs and RETs were set using unstained and single-stained samples.…”

“…The enriched samples were incubated for 30 min in the dark at room temperature to label with anti-CD59 and anti-HIS49 antibodies for total RBC labeling. Finally, the cells were resuspended in 0.5 mL PBS and the Pig-a MFs using CD71-and HIS49-positive RETs were examined using a BD FACSCant TM II flow cytometer as previously described [18][19][20][21][22].…”

“…This assay was named as the PIGRET assay. Recently, a collaborative study by the Japan Health Sciences Foundation validated the single dose PIGRET assay [18][19][20][21][22]. Based on the findings from this collaborative study, it was suggested that the single dose PIGRET assay may detect the in vivo mutagenicity of test chemicals earlier than the RBC Pig-a assay.…”