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.

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.

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.

Flow cytometric analysis of Pig-a gene mutation and chromosomal damage induced by procarbazine hydrochloride in CD-1 mice.

Procarbazine is a genotoxic carcinogen whose DNA-damaging activities are not reliably detected in vitro. We evaluated the in vivo genotoxic effects of procarbazine on hematopoietic cells of male CD-1 mice using a multi-endpoint study design that scored micronucleated reticulocyte (MN-RET) frequency and gene mutation at the Pig-a locus. CD-1 mice were treated for 3 days with procarbazine, up to 150 mg/kg/day.

Efficient monitoring of in vivo pig-a gene mutation and chromosomal damage: summary of 7 published studies and results from 11 new reference compounds.

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.

In vivo flow cytometric Pig-a and micronucleus assays: highly sensitive discrimination of the carcinogen/noncarcinogen pair benzo(a)pyrene and pyrene using acute and repeated-dose designs.

Combining multiple genetic toxicology endpoints into a single in vivo study, and/or integrating one or more genotoxicity assays into general toxicology studies, is attractive because it reduces animal use and enables comprehensive comparative analysis using toxicity, metabolism, and pharmacokinetic information from the same animal. This laboratory has developed flow cytometric scoring techniques for monitoring two blood-based genotoxicity endpoints-micronucleated reticulocyte frequency and gene mutation at the Pig-a locus-thereby making combination and integration studies practical. The ability to effectively monitor these endpoints in short-term and repeated dosing schedules was investigated with the carcinogen/noncarcinogen pair benzo(a)pyrene (BP) and pyrene (Pyr).

Interlaboratory Pig-a gene mutation assay trial: Studies of 1,3-propane sultone with immunomagnetic enrichment of mutant erythrocytes.

An international collaborative trial was established to systematically investigate the merits and limitations of a rat in vivo Pig-a gene mutation assay. The product of this gene is essential for anchoring CD59 to the plasma membrane, and mutations in this gene are identified by flow cytometric quantification of circulating erythrocytes without cell surface CD59 expression. Initial interlaboratory data from rats treated with several potent mutagens have been informative, but the time required for those flow cytometric analyses (∼20 min per sample) limited the number of cells that could be interrogated for the mutant phenotype.

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.

International Pig-a gene mutation assay trial: evaluation of transferability across 14 laboratories.

A collaborative international trial was conducted 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, SYTO 13 dye, and flow cytometry were used to determine the frequency of CD59-negative erythrocytes (RBC(CD59-)) and CD59-negative reticulocytes (RET(CD59-)). To provide samples with a range of mutant phenotype cell frequencies, male rats were exposed to N-ethyl-N-nitrosourea (ENU) via oral gavage for three consecutive days (Days 1-3).

When pigs fly: immunomagnetic separation facilitates rapid determination of Pig-a mutant frequency by flow cytometric analysis.

In vivo mutation assays based on the Pig-a null phenotype, that is, the absence of cell surface glycosylphosphatidylinositol (GPI) anchored proteins such as CD59, have been described. This work has been accomplished with hematopoietic cells, most often rat peripheral blood erythrocytes (RBCs) and reticulocytes (RETs). The current report describes new sample processing procedures that dramatically increase the rate at which cells can be evaluated for GPI anchor deficiency.

Miniaturized flow cytometric in vitro micronucleus assay represents an efficient tool for comprehensively characterizing genotoxicity dose-response relationships.

This laboratory has developed a flow cytometric approach for scoring in vitro micronuclei (In Vitro MicroFlow(®)) whose characteristics are expected to benefit studies designed to comprehensively investigate genotoxicity dose-response relationships. In particular, new experimental designs become possible when automated scoring is combined with treatment, processing and sampling that all occur in microtiter plates. To test this premise, experiments described herein investigated micronucleus (MN) formation in TK6 cells treated with genotoxic agents applied at 22 closely spaced concentrations in quadruplicate, with 10,000 cells analyzed per replicate.

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.

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.

Reticulocyte and micronucleated reticulocyte responses to gamma irradiation: effect of age.

The effect of age on the formation of radiation-induced micronucleated reticulocytes (MN-RETs) and reticulocytes (RETs) was investigated by exposing female C57BL/6J mice to graded doses of gamma rays from a (137)Cs source. Age at time of irradiation was 6, 16, or 32 weeks, and doses ranged from 0.5 to 3 Gy.

Erythrocyte-based Pig-a gene mutation assay: demonstration of cross-species potential.

Glycosylphosphatidylinositol (GPI) anchors attach specific proteins to the cell surface of hematopoietic cells. Of the genes required to form GPI anchors, only Pig-a is located on the X-chromosome. Prior work with rats suggests that the GPI anchor deficient phenotype is a reliable indicator of Pig-a mutation [Bryce et al.