Evaluation of in vivo gene mutation with etoposide using Pig-a and PIGRET assays

The Pig-a assay detects the expression of the endogenous phosphatidylinositol glycan anchor biosynthesis, class A gene (Pig-a) as a reporter of mutations and shows promise for detecting mutations in vivo. This assay requires two to four weeks to detect mutations in erythrocytes after animals are administered a single dose of test compound. In contrast, the more recently developed PIGRET assay using reticulocytes detects mutation sensitively one week after dosing, which is an advantage for conducting short-term genotoxicity studies in vivo. As part of the Japanese Environmental Mutagen Society/Mammalian Mutagenicity Study Group collaborative study, we conducted Pig-a and PIGRET assays to detect Pig-a mutations in rats treated with etoposide. The DNA topoisomerase II-inhibitor, etoposide, causes DNA cleavage and subsequent protein-linked DNA double-strand breaks, induces chromosomal aberrations and micronuclei, and is classified as 2A (probably carcinogenic to humans) by IARC. Etoposide was intravenously given once to 7-week old SD rats at doses of 0, 5, 10, and 20mg/kg. Severe myelosuppression was induced 2days after dosing in all dosing groups. We conducted Pig-a mutant analysis in Pig-a and PIGRET assays 1, 2, and 4 weeks after dosing. Neither Pig-a nor PIGRET assays showed any statistically significant increases in Pig-a mutant frequency in any of the dose groups at any of the sampling times. It was concluded that etoposide was judged negative in Pig-a and PIGRET assays of bone marrow cells, and the results of the two assays showed hardly any differences.