Persistence of cisplatin-induced mutagenicity in hematopoietic stem cells: implications for secondary cancer risk following chemotherapy.

Cisplatin is a cytostatic agent used in the treatment of many types of cancer, but its use is associated with increased incidences of secondary leukemia. We evaluated cisplatin's in vivo genotoxic potential by analyzing peripheral blood for Pig-a mutant phenotype erythrocytes and for chromosomal damage in the form of micronuclei. Mutant phenotype reticuloyte and erythrocyte frequencies, based on anti-CD59 antibody labeling and flow cytometric analysis, were determined in male Sprague Dawley rats treated for 28 consecutive days (days 1-28) with up to 0.4 mg cisplatin/kg/day, and sampled on days -4, 15, 29, and 56. Vehicle and highest dose groups were evaluated at additional time points post-treatment up to 6 months. Day 4 and 29 blood samples were also analyzed for micronucleated reticulocyte frequency using flow cytometry and anti-CD71-based labeling. Mutant phenotype reticulocytes were significantly elevated at doses ≥0.1 mg/kg/day, and mutant phenotype erythrocytes were elevated at doses ≥0.05 mg/kg/day. In the 0.4 mg/kg/day group, these effects persisted for the 6 month observation period. Cisplatin also induced a modest but statistically significant increase in micronucleus frequency at the highest dose tested. The prolonged persistence in the production of mutant erythrocytes following cisplatin exposure suggests that this drug mutates hematopoietic stem cells and that this damage may ultimately contribute to the increased incidence of secondary leukemias seen in patients cured of primary malignancies with platinum-based regimens.

Diethylnitrosamine genotoxicity evaluated in sprague dawley rats using pig-a mutation and reticulocyte micronucleus assays.

Diethylnitrosamine (DEN) is a genotoxic carcinogen, but in vivo DNA-damaging activities are not usually evident in hematopoietic cells because the short-lived active metabolite is formed mainly in the liver. DEN therefore represented an interesting case for evaluating the performance characteristics of blood-based endpoints of genotoxicity that have been automated using flow cytometric analysis-frequency of micronucleated reticulocytes and Pig-a mutant phenotype reticulocytes (RET(CD59-) ) and erythrocytes (RBC(CD59-) ). Male Sprague Dawley rats were treated for 28 consecutive days with DEN at levels up to 12.5 mg/kg/day. Serial blood samples were collected and micronucleus frequencies were determined on Days 4 and 29, while RET(CD59-) and RBC(CD59-) frequencies were determined on Days 15, 29, and 42. The Pig-a analyses were conducted with an enrichment step based on immunomagnetic column separation to increase the statistical power of the assay. Modest but significant reductions to reticulocyte frequencies demonstrated that bone marrow was exposed to reactive intermediates. Even so, DEN did not affect micronucleus frequencies at any dose level tested. However, RET(CD59-) frequencies were significantly elevated in the high dose group on Day 29, and RBC(CD59-) were increased at this same dose level on Days 29 and 42. These results demonstrate that the Pig-a assay is sufficiently sensitive to evaluate chemicals for genotoxic potential, even in the case of a promutagen that has traditionally required direct assessment(s) of liver tissue for detection of DNA-damage.

Pig-a gene mutation and micronucleated reticulocyte induction in rats exposed to tumorigenic doses of the leukemogenic agents chlorambucil, thiotepa, melphalan, and 1,3-propane sultone.

To evaluate whether blood-based genotoxicity endpoints can provide temporal and dose-response data within the low-dose carcinogenic range that could contribute to carcinogenic mode of action (MoA) assessments, we evaluated the sensitivity of flow cytometry-based micronucleus and Pig-a gene mutation assays at and below tumorigenic dose rate 50 (TD50) levels. The incidence of micronucleated reticulocytes (MN-RET) was used to evaluate chromosomal damage, and the frequency of CD59-negative reticulocytes (RET(CD59-) ) and erythrocytes (RBC(CD59-) ) served as phenotypic reporters of mutation at the X-linked Pig-a gene. Several leukemogenic agents with a presumed genotoxic MoA were studied. Specifically, male Sprague Dawley rats were treated via oral gavage for 28 days with chlorambucil, thiotepa, melphalan, and 1,3-propane sultone at doses corresponding to 0.33x, 1x, and 3x TD50, as well as at the maximum tolerated dose. Frequencies of MN-RET were determined at Days 4 and 29, and RET(CD59-) and RBC(CD59-) data were collected pretreatment as well as Days 15/16, 29, and 56/57. Dose-related increases were observed for each endpoint, and time to maximal effect was consistently: MN-RET < RET(CD59-) < RBC(CD59-) . For each of the chemicals studied, the genotoxic events occurred long before tumors or preneoplastic lesions would be expected. Furthermore, in the case of Pig-a gene mutation, the responses were observed at or below the TD50 dose for three out of the four chemicals studied. These data illustrate the potential for quantitative blood-based analyses to provide dose-response and temporality information that relates genetic damage to cancer induction.