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Mouse Pig‐a and micronucleus assays respond to N ‐ethyl‐ N ‐nitrosourea, benzo[ a ]pyrene, and ethyl carbamate, but not pyrene or methyl carbamate
Author(s) -
Labash Carson,
Avlasevich Svetlana L.,
Carlson Kristine,
Berg Ariel,
Torous Dorothea K.,
Bryce Steven M.,
Bemis Jeffrey C.,
MacGregor James T.,
Dertinger Stephen D.
Publication year - 2016
Publication title -
environmental and molecular mutagenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1
H-Index - 87
eISSN - 1098-2280
pISSN - 0893-6692
DOI - 10.1002/em.21965
Subject(s) - ethyl carbamate , reticulocyte , micronucleus test , benzo(a)pyrene , microbiology and biotechnology , pyrene , chemistry , carcinogen , micronucleus , biology , biochemistry , toxicity , gene , organic chemistry , messenger rna , food science , wine
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. Environ. Mol. Mutagen. 57:28–40, 2016. © 2015 Wiley Periodicals, Inc.