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The Lambda Select cII Mutation Detection System
Published on: April 26, 2018
PIGRET assay can detect mutagenicity of ethyl methanesulfonate much earlier than RBC Pig-a assay
Satoru Itoh1, Chiharu Hattori2, Shiho Nakayama1
1Medicinal Safety Research Laboratories, Daiichi Sankyo Co., Ltd., 1-16-13, Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan.
Abstract:
The comparison between the original red blood cell (RBC) Pig-a assay, which measures Pig-a mutant RBCs, and the PIGRET assay, which uses reticulocytes, was conducted using in vivo mutagenesis by ethyl methanesulfonate (EMS) as a part of a collaborative study by the Mammalian Mutagenicity Study Group in the Japanese Environmental Mutagen Society. Three dose levels of EMS (180, 360, and 720mg/kg) were administered once by oral gavage to 8-week-old male Crl:CD(SD) rats, and peripheral blood was sampled at 0 (1 day before dosing), 1, 2, and 4 weeks after dosing with EMS. As a result, a statistically significant increase in the mutant frequency of the Pig-a gene was observed from 2 weeks after dosing and a higher value was obtained on week 4 at the highest dose only in the RBC Pig-a assay. In the PIGRET assay, on the other hand, a statistically significant increase in Pig-a mutant frequency was obtained at the highest dose from 1 week after dosing, and it decreased on weeks 2 and 4 compared to the value at week 1. The Pig-a mutant frequency appeared to reach a plateau 1 week after dosing in the PIGRET assay and it might continue to increase even after week 4 in the RBC Pig-a assay. These results indicate that the PIGRET assay can detect Pig-a mutants much earlier than the original RBC Pig-a assay, and it can enable judgement of mutagenicity of EMS within 1 week after a single dosing.
Insights
The PIGRET assay detects Pig-a gene mutations earlier than the traditional RBC Pig-a assay. This new method allows for faster assessment of mutagenicity from chemicals like ethyl methanesulfonate (EMS).
Area of Science:
- Toxicology
- Genetics
- Biotechnology
Background:
- The Pig-a gene assay is crucial for evaluating mutagenicity.
- Comparing the traditional red blood cell (RBC) Pig-a assay with the newer PIGRET assay is important for improving detection methods.
Purpose of the Study:
- To compare the sensitivity and speed of the RBC Pig-a assay and the PIGRET assay in detecting in vivo mutagenicity.
- To evaluate the PIGRET assay's potential for early detection of Pig-a gene mutations.
Main Methods:
- In vivo mutagenesis study using ethyl methanesulfonate (EMS) in male rats.
- Administration of three dose levels of EMS via oral gavage.
- Measurement of Pig-a mutant frequency in both RBCs and reticulocytes (PIGRET assay) at multiple time points post-dosing.
Main Results:
- The PIGRET assay detected a statistically significant increase in Pig-a mutant frequency one week after EMS administration at the highest dose.
- The RBC Pig-a assay showed a significant increase in mutant frequency starting from two weeks post-dosing, with higher values at four weeks only at the highest dose.
- Pig-a mutant frequency in the PIGRET assay appeared to plateau by one week, while it might continue to increase beyond four weeks in the RBC Pig-a assay.
Conclusions:
- The PIGRET assay offers earlier detection of Pig-a mutants compared to the conventional RBC Pig-a assay.
- The PIGRET assay enables a more rapid assessment of mutagenicity, potentially within one week after a single exposure to mutagens like EMS.
