Evaluation of Rats' In Vivo Genotoxicity Induced by N-ethyl-N-nitrosourea in the RBC Pig-a, PIGRET, and gpt Assays

Abstract

The emerging Pig-a gene mutation assay, a powerful and promising tool for evaluating in vivo genotoxicity, is based on flow cytometric enumeration of red blood cells (RBCs), which are deficient in glycosylphosphatidylinositol anchored protein. Various approaches for measuring Pig-a mutant cells have been developed, particularly those focused on peripheral RBCs and reticulocytes (RETs). Previously, it had been reported that Pig-a and gpt mutant frequencies were relatively increased in N-ethyl-N-nitrosourea (ENU)- and benzo[a]pyrene (BP)-treated mice. The capacity and characteristics of the Pig-a assay relative to transgenic rodent (TGR) mutation assays, however, are unclear in rats. Here, using transgenic gpt delta rats, we compared the in vivo genotoxicity of single oral doses of ENU (40 mg/kg) in the gpt gene mutation assay in bone marrow and liver, and Pig-a gene mutation assays on RBCs and RETs in the same animals. The Pig-a gene mutation assays were conducted at 1, 2, and 4 weeks after treatment, whereas gpt assays were conducted on tissues collected at the 4-week terminal sacrifice. Consequently, we detected that Pig-a and gpt mutant frequencies were clearly increased in ENU-treated rats, indicating that both the Pig-a and TGR gene mutation assays can detect in vivo ENU genotoxicity equally.