Mouse Pig‐a and micronucleus assays respond to N‐ethyl‐N‐nitrosourea, benzo[a]pyrene, and ethyl carbamate, but not pyrene or methyl carbamate

Abstract

This laboratory previously described a method for scoring the incidence of peripheral blood Pig-a mutant phenotype rat erythrocytes using immunomagnetic separation in conjunction with flow cytometric analysis (In Vivo MutaFlow®). The current work extends the method to mouse blood, using the frequency of CD24-negative reticulocytes (RET(CD24-)) and erythrocytes (RBC(CD24-)) as phenotypic reporters of Pig-a gene mutation. Following assay optimization, reconstruction experiments demonstrated the ability of the methodology to return expected values. Subsequently, the responsiveness of the assay to the genotoxic carcinogens N-ethyl-N-nitrosourea, benzo[a]pyrene, and ethyl carbamate was studied in male CD-1 mice exposed for 3 days to several dose levels via oral gavage. Blood samples were collected on Day 4 for micronucleated reticulocyte analyses, and on Days 15 and 30 for determination of RET(CD24-) and RBC(CD24-) frequencies. The same design was used to study pyrene, with benzo[a]pyrene as a concurrent positive control, and methyl carbamate, with ethyl carbamate as a concurrent positive control. The three genotoxicants produced marked dose-related increases in the frequencies of Pig-a mutant phenotype cells and micronucleated reticulocytes. Ethyl carbamate exposure resulted in moderately higher micronucleated reticulocyte frequencies relative to N-ethyl-N-nitrosourea or benzo[a]pyrene (mean ± SEM = 3.0 ± 0.36, 2.3 ± 0.17, and 2.3 ± 0.49%, respectively, vs. an aggregate vehicle control frequency of 0.18 ± 0.01%). However, it was considerably less effective at inducing Pig-a mutant cells (e.g., Day 15 mean no. RET(CD24-) per 1 million reticulocytes = 7.6 ± 3, 150 ± 9, and 152 ± 43 × 10(-6), respectively, vs. an aggregate vehicle control frequency of 0.6 ± 0.13 × 10(-6)). Pyrene and methyl carbamate, tested to maximum tolerated dose or limit dose levels, had no effect on mutant cell or micronucleated reticulocyte frequencies. Collectively, these results demonstrate the utility of the cross-species Pig-a and micronucleated reticulocyte assays, and add further support to the value of studying both endpoints in order to cover two distinct genotoxic modes of action.

Differential genotoxicity of acrylamide in the micronucleus andPig-a gene mutation assays in F344 rats and B6C3F1 mice

“…The increase in micronuclei in mice in the present study was highly consistent with results of a previous multiple dose micronucleus assay conducted in our laboratory (21). For the present study of rats and mice exposed to acrylamide we assessed the induction of chromosomal damage by using the in vivo micronucleus assay and mutation of an endogenous gene using a novel flow cytometry based assay that can detect mutation at the Pig-a locus in the blood of mice, rats and humans (38,(48)(49)(50). The Pig-a gene mutation assay is based on the loss of Pig-a, a gene whose product is essential for the synthesis of glycosylphosphatidylinositol (GPI) anchors on the cell surface of reticulocytes and red blood cells (51).…”

Integrated In Vivo Genotoxicity Assessment of Procarbazine Hydrochloride Demonstrates Induction of Pig‐a and LacZ Mutations, and Micronuclei, in MutaMouse Hematopoietic Cells

“…These results show that PCH elicits a robust mutagenic response in the bone marrow even at doses that do not produce cytotoxicity. While Phonethepswath et al () also reported PCH‐induced Pig‐a mutation in mice, it is not productive comparing those results with data reported herein because the previous study utilized an early mouse blood labeling protocol that is now understood to systematically underestimate mutant cell frequency (Labash et al ). Finally, Chen et al () recently reported strong induction of Pig‐a mutations following a 28‐day exposure regimen in rats using daily doses higher than those used here.…”

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

“…To the best of our knowledge, the in vivo mutagenic evaluation of Pyr has been conducted in the following 2 studies: rat Pig-a assay by Torous et al [16], and mouse Pig-a assay by Labash et al [24]. In both Pig-a studies, Pyr was reported to show no mutagenic effects.…”