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Labash C.,Litron LaboratoriesRochester | Avlasevich S.L.,Litron LaboratoriesRochester | Carlson K.,Litron LaboratoriesRochester | Berg A.,Litron LaboratoriesRochester | And 5 more authors.
Environmental and Molecular Mutagenesis | Year: 2015

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 (RETCD24-) and erythrocytes (RBCCD24-) 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 RETCD24- and RBCCD24- 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. RETCD24- 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. © 2015 Wiley Periodicals, Inc.


Bernacki D.T.,Litron LaboratoriesRochester | Bryce S.M.,Litron LaboratoriesRochester | Bemis J.C.,Litron LaboratoriesRochester | Kirkland D.,Kirkland Consulting | Dertinger S.D.,Litron LaboratoriesRochester
Environmental and Molecular Mutagenesis | Year: 2016

Previous work with a diverse set of reference chemicals suggests that an in vitro multiplexed flow cytometry-based assay (MultiFlow™ DNA Damage Kit-p53, γH2AX, Phospho-Histone H3) can distinguish direct-acting clastogens and aneugens from nongenotoxicants (Bryce SM et al. []: Environ Mol Mutagen 57:171-189). This work extends this line of investigation to include compounds that require metabolic activation to form reactive electrophiles. For these experiments, TK6 cells were exposed to 11 promutagens and 37 presumed nongenotoxicants in 96 well plates. Unless precipitation or foreknowledge about cytotoxicity suggested otherwise, the highest concentration was 1 mM. Exposure occurred for 4 hr after which time cells were washed to remove S9 and test article. Immediately following the wash and again at 24 hr, cell aliquots were added to wells of a microtiter plate containing the working detergent/stain/antibody cocktail. After a brief incubation, robotic sampling was employed for walk-away flow cytometric data acquisition. Univariate logistic regression analyses indicated that γH2AX induction and p53 activation provide the greatest degree of discrimination between clastogens and nongenotoxicants. Multivariate prediction algorithms that incorporated both of these endpoints, in each combination of time points, were evaluated. The best performing models correctly predicted 9 clastogens out of 11 and 36 nongenotoxicants out of 37. These results are encouraging as they suggest that an efficient and highly scalable multiplexed assay can effectively identify clastogenic chemicals that require bioactivation. More work is planned with a broader range of chemicals, additional cell lines, and other laboratories to further evaluate the merits and limitations of this approach. Environ. Mol. Mutagen., 2016. © 2016 Wiley Periodicals, Inc.

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