The ability to effectively monitor gene mutation and micronucleated reticulocyte (MN-RET) frequency in short-term and repeated dosing schedules was investigated using the recently developed flow cytometric Pig-a mutation assay and flow cytometric micronucleus analysis. Eight reference genotoxicants and three presumed nongenotoxic compounds were studied: chlorambucil, melphalan, thiotepa, cyclophosphamide, azathioprine, 2-acetylaminofluorene, hydroxyurea, methyl methanesulfonate, o-anthranilic acid, sulfisoxazole, and sodium chloride. These experiments extend previously published results with seven other chemicals. Male Sprague Dawley rats were treated via gavage for 3 or 28 consecutive days with several dose levels of each chemical up to the maximum tolerated dose. Blood samples were collected at several time points up to day 45 and were analyzed for Pig-a mutation with a dual-labeling method that facilitates mutant cell frequency measurements in both total erythrocytes and the reticulocyte subpopulation. An immunomagnetic separation technique was used to increase the efficiency of scoring mutant cells. Blood samples collected on day 4, and day 29 for the 28-day study, were evaluated for MN-RET frequency. The three nongenotoxicants did not induce Pig-a or MN-RET responses. All genotoxicants except hydroxyurea increased the frequency of Pig-a mutant reticulocytes and erythrocytes. Significant increases in MN-RET frequency were observed for each of the genotoxicants at both time points. Whereas the highest Pig-a responses tended to occur in the 28-day studies, when total dose was greatest, the highest induction of MN-RET was observed in the 3-day studies, when dose per day was greatest. There was no clear relationship between the maximal Pig-a response of a given chemical and its corresponding maximal MN-RET response, despite the fact that both endpoints were determined in the same cell lineage. Taken with other previously published results, these data demonstrate the value of integrating Pig-a and micronucleus endpoints into in vivo toxicology studies, thereby providing information about mutagenesis and chromosomal damage in the same animals from which toxicity, toxicokinetics, and metabolism data are obtained.

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.

The development of automated flow cytometric (FCM) methods for evaluating micronucleus (MN) frequencies in erythrocytes has great potential for improving the sensitivity, reproducibility, and throughput of the traditional in vivo rodent MN assay that uses microscopy-based methods for data collection. Although some validation studies of the FCM evaluation methods have been performed, a comprehensive comparison of these two data collection methods under routine testing conditions with a variety of compounds in multiple species has not been conducted. Therefore, to determine if FCM evaluation of MN frequencies in rodents was an acceptable alternative to traditional manual scoring methods in our laboratory, we conducted a comparative evaluation of MN-reticulocyte (MN-RET) frequencies determined by FCM- and microscopy-based scoring of peripheral blood and bone marrow samples from B6C3F1 mice and Fisher 344 rats. Four known inducers of MN (cyclophosphamide, ethyl methanesulfonate, vincristine sulfate, acrylamide) were assayed in bone marrow and peripheral blood of both mice and rats. In addition, MN-RET frequencies were measured in bone marrow (microscopy) and peripheral blood (FCM) of mice treated with five nongenotoxic chemicals (S-adenosylmethionine chloride, cefuroxime, diphenolic acid, 3-amino-6-methylphenol, pentabromodiphenyl oxide). No significant differences were observed between results obtained by the two methods in either species. These results support the use of FCM for determining MN-RET frequency in rodents after chemical exposure.

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.

Experiments described herein were designed to evaluate the performance characteristics of a flow cytometry-based system that scores the incidence of peripheral blood micronucleated reticulocytes (MN-RETs). These procedures represent the continued refinement of a previously reported anti-CD71-based method (Dertinger et al. [1996]: Mutat Res 371:283-292), with the following modifications: incorporation of a third fluorescent label to exclude platelets from the MN-RET region, and use of a CD71-associated fluorescence thresholding technique to increase data acquisition rates. Mouse, rat, and human blood samples were analyzed using both the previously described two-color procedure (anti-CD71-FITC and propidium iodide) and a newly developed three-color technique (which adds an antiplatelet-PE antibody). The rodent specimens were also evaluated by standard microscopy procedures (acridine orange staining). Mouse blood was collected via heart puncture of vehicle- and 5-fluorouracil-treated CD-1 mice; blood samples from saline-treated Sprague-Dawley rats were collected from the tail vein and via heart puncture. Rodent blood samples were analyzed by both the two- and three-color methods. Human blood specimens, obtained via arm venipuncture from cancer patients undergoing radiation therapy, were analyzed for MN-RETs using the two-color method. Subsequently, blood samples from a single chemotherapy patient were analyzed by both the two- and three-color methods. Finally, the chemotherapy patient blood samples and blood samples from 15 healthy volunteers were evaluated at very high densities in conjunction with a CD71-associated fluorescence thresholding technique. Results of these investigations showed that data from mouse blood analyzed by the two- and three-color procedures correlated well with microscopy data (r values = 0.917 and 0.937 for the two- and three-color methods, respectively); all three methods confirmed the genotoxicity of 5-FU. Data from rat tail vein samples showed improved reproducibility with the three-color technique, but no significant difference between the two techniques was seen with the heart puncture specimens. Human blood analyzed according to the two-color procedure produced unreliable results, as platelets and platelet aggregates impacted the rare MN-RET scoring region. The three-color technique effectively overcame this problem and produced reproducible measurements that fell within expected ranges. For human blood analyses, the high cell density/CD71-thresholding technique provided significant improvements over the low-density technique, as it allowed data acquisition to occur approximately six times faster with no loss of sensitivity.