Simultaneous Measurement of Benzo[a]pyrene-induced Pig-a and lacZ Mutations, Micronuclei and DNA Adducts in Muta™ Mouse

In this study we compared the response of the Pig-a gene mutation assay to that of the lacZ transgenic rodent mutation assay, and demonstrated that multiple endpoints can be measured in a 28-day repeat dose study. Muta™Mouse were dosed daily for 28 days with benzo[a]pyrene (BaP; 0, 25, 50 and 75 mg/kg body weight/day) by oral gavage. Micronucleus (MN) frequency was determined in reticulocytes (RETs) 48 hr following the last dose. 72 h following the last dose, mice were euthanized, and tissues (glandular stomach, small intestine, bone marrow and liver) were collected for lacZ mutation and DNA adduct analysis, and blood was evaluated for Pig-a mutants. BaP-derived DNA adducts were detected in all tissues examined and significant dose-dependent increases in mutant Pig-a phenotypes (i.e., RETCD24- and RBC CD24-) and lacZ mutants were observed. We estimate that mutagenic efficiency (i.e., rate of conversion of adducts into mutations) was much lower for Pig-a compared to lacZ, and speculate that this difference is likely explained by differences in repair capacity between the gene targets, and differences in the cell populations sampled for Pig-a versus lacZ. The BaP doubling doses for both gene targets, however, were comparable, suggesting that similar mechanisms are involved in the accumulation of gene mutations. Significant dose-related increases in % MN were also observed; however, the doubling dose was considerably higher for this endpoint. The similarity in dose response kinetics of Pig-a and lacZ provides further evidence for the mutational origin of glycosylphosphatidylinositol (GPI)-anchor deficiencies detected in the Pig-a assay. Environ. Mol. Mutagen. 2011. © 2011 Wiley-Liss, Inc

Integration of Mutation and Chromosomal Damage Endpoints into 28-Day Repeat Dose Toxicology Studies

Two endpoints of genetic toxicity, mutation at the X-linked Pig-a gene and chromosomal damage in the form of micronucleated reticulocytes (MN-RETs), were evaluated in blood samples obtained from 28-day repeat-dosing studies typical of those employed in toxicity evaluations. Male Wistar Han rats were treated at 24-h intervals on days 1 through 28 with one of five prototypical genotoxicants: N-ethyl-N-nitrosourea, 7,12-dimethyl-12-benz[a]anthracene, 4-nitroquinoline-1-oxide (4NQO), benzo(a)pyrene, and N-methyl-N-nitrosourea. Flow cytometric scoring of CD59-negative erythrocytes (indicative of glycosylphosphatidylinositol anchor deficiency and hence Pig-a mutation) was performed using blood specimens obtained on days −1, 15, 29, and 56. Blood specimens collected on days 4 and 29 were evaluated for MN-RET frequency using flow cytometry–based MicroFlow Kits. With the exception of 4NQO, each chemical induced significant increases in the frequency of MN-RETs on days 4 and 29. All five agents increased the frequency of mutant phenotype (CD59 negative) reticulocytes (RETs) and erythrocytes. Mutation responses in RETs occurred earlier than in erythrocytes and tended to peak, or nearly peak, at day 29. In contrast, the mutant phenotype erythrocyte responses were modest on day 29 and required additional time to reach their maximal value. The observed kinetics were expected based on the known turnover of RETs and erythrocytes. The data show that RETs can serve as an appropriate indicator cell population for 28-day studies. Collectively, these data suggest that blood-based genotoxicity endpoints can be effectively incorporated into routine toxicology studies, a strategy that would reduce animal usage while providing valuable genetic toxicity information within the context of other toxicological endpoints

Pig-a Mutation: Kinetics in Rat Erythrocytes Following Exposure to Five Prototypical Mutagens

An in vivo mutation assay has been developed based on flow cytometric enumeration of glycosylphosphatidylinositol (GPI) anchor–deficient rat erythrocytes. With this method, blood is incubated with anti-CD59-PE and SYTO 13 dye, and flow cytometry is used to score the frequency of CD59-negative erythrocytes. The experiments described herein were designed to define the kinetics of mutant erythrocyte appearance and disappearance from peripheral blood to support appropriate treatment and sampling designs for the assay. Wistar Han rats were treated with one of five prototypical mutagens: N-ethyl-N-nitrosourea (ENU); 7,12-dimethyl-1,2-benz[a]anthracene (DMBA); 4-nitroquinoline-1-oxide; benzo[a]pyrene; and N-methyl-N-nitrosourea. ENU and DMBA were also evaluated in Sprague Dawley rats. Animals were treated on three consecutive days (days 1–3) via oral gavage, and blood specimens were obtained on days −1, 4, 15, 30, 45, and 90 (and day 180 for ENU). A second endpoint of genotoxicity, the frequency of peripheral blood micronucleated reticulocytes, was measured on day 4. Each chemical induced micronuclei and the GPI anchor–deficient phenotype. Increased mutant cell frequencies were evident at day 15. Mutant reticulocyte frequencies remained relatively stable for some chemicals, but others peaked and then dropped significantly. The differences in kinetics observed are presumably related to the degree to which mutation occurs in hematopoietic stem cells versus more committed cells with limited self-renewal capacity. Collectively, the results suggest that enumerating GPI anchor–deficient erythrocytes is an efficient means of evaluating the in vivo mutagenic potential of chemicals. The kinetics and ease of scoring this blood-based endpoint suggest that integration into routine toxicology studies will be feasible

International Pig-a gene mutation assay trial: Evaluation of transferability across fourteen laboratories

Experiments described herein were designed to evaluate the reproducibility and transferability of an in vivo mutation assay based on the enumeration of CD59-negative rat erythrocytes, a phenotype that is indicative of Pig-a gene mutation. Fourteen laboratories participated in this study, where anti-CD59-PE and SYTO 13 dye were used to label leukocyte-depleted blood samples, and the frequency of CD59-negative erythrocytes (RBCCD59-) and CD59-negative reticulocytes (RETCD59-) were determined via flow cytometric analysis. To provide samples with a range of mutant phenotype cell frequencies, male rats were exposed to the prototypical mutagen N-ethyl-N-nitrosourea (ENU) via oral gavage for three consecutive days (Days 1-3). Each laboratory studied 0, 20 and 40 mg ENU/kg/day (n = 5 per group). Three sites also evaluated 4 mg/kg/day. At a minimum, blood samples were collected three times: pre-dosing and on Days 15 and 30. Blood samples were processed according to standardized sample processing and data acquisition protocols, and three endpoints were measured: %reticulocytes, frequency of RETCD59-, and frequency of RBCCD59-. As illustrated by the analysis of technical replicates, the methodology was found to be highly reproducible, as experimental coefficients of variation approached theoretical values. Good transferability was evident from the similar kinetics and magnitude of the responses that were observed among different laboratories. Dose-related increases in the frequency of RETCD59- and RBCCD59- were consistently observed on Day 15. Whereas maximal RETCD59- responses tended to occur by Day 15, peak RBCCD59- responses occurred at approximately Day 45. Elevated mutant phenotype cell frequencies were maintained through the latest time-point studied (Day 90). High concordance correlation coefficients show a remarkable level of agreement between the reference site and the test sites. Collectively, these data demonstrate that with adequate training of personnel, flow cytometric analysis is capable of reliably enumerating mutant phenotype erythrocytes, thereby providing a robust in vivo mutation assay that is readily transferable across laboratories