Souk Phonethepswath

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. This new method was applied to blood specimens from vehicle, 1,3-propane sultone, melphalan, and N-ethyl-N-nitrosourea treated Sprague Dawley rats. Leukocyte- and platelet-depleted blood samples were incubated with anti-CD59-phycoerythrin (PE) and anti-CD61-PE, and then mixed with anti-PE paramagnetic particles and Counting Beads (i.e., fluorescent microspheres). An aliquot of each specimen was stained with SYTO 13 and flow cytometric analysis was performed to determine RET percentage, RET:Counting Bead ratio, and RBC:Counting Bead ratio. The major portion of these specimens were passed through ferromagnetic columns that were suspended in a magnetic field, thereby depleting each specimen of wild-type RBCs (and platelets) based on their association with anti-PE paramagnetic particles. The eluates were concentrated via centrifugation and the resulting suspensions were stained with SYTO 13 and analyzed on the flow cytometer to determine mutant phenotype RET:Counting Bead and mutant phenotype RBC:Counting Bead ratios. The ratios obtained from pre- and post-column analyses were used to derive mutant phenotype RET and mutant phenotype RBC frequencies. Results from vehicle control and genotoxicant-treated rats are presented that indicate the scoring system is capable of returning reliable mutant phenotype cell frequencies. Using this wild-type cell depletion strategy, it was possible to interrogate ≥ 3 million RETs and ≥ 100 million RBCs per rat in approximately 7 min. Beyond considerably enhancing the throughput capacity of the analytical platform, these blood-processing procedures were also shown to enhance the precision of the measurements.

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.

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., Environ. Mol. Mutagen., 49 (2008) 256-264]. The current report extends this line of investigation by describing simplified blood handling procedures, and by testing the assay principle in a second species, Mus musculus. With this method, erythrocytes are isolated, incubated with anti-CD24-PE, and stained with SYTO 13. Flow cytometric analyses quantify GPI anchor-deficient erythrocytes and reticulocytes. After reconstruction experiments with mutant-mimicking cells demonstrated that the analytical performance of the method is high, CD-1 mice were treated on three occasions with 7,12-dimethyl-1,2-benz[a]anthracene (DMBA, 75 mg/kg/day) or ethyl-N-nitrosourea (ENU, 40 mg/kg/day). Two weeks after the final treatment, DMBA-treated mice were found to exhibit markedly elevated frequencies of GPI anchor deficient erythrocytes and reticulocytes. For the ENU experiment, blood specimens were collected at weekly intervals over a 5-week period. Whereas the frequencies of mutant reticulocytes were significantly elevated 1 week after the last administration, the erythrocyte population was unchanged until the second week. Thereafter, both populations exhibited persistently elevated frequencies for the duration of the experiment (mean frequency at termination=310x10(-6) and 523x10(-6) for erythrocyte and reticulocyte populations, respectively). These data provide evidence that Pig-a mutation does not convey an appreciable positive or negative cell survival advantage to affected erythroid progenitors, although they do suggest that affected erythrocytes have a reduced lifespan in circulation. Collectively, accumulated data support the hypothesis that flow cytometric enumeration of GPI anchor deficient erythrocytes and/or reticulocytes represents an effective in vivo mutation assay that is applicable across species of toxicological interest.

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. 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.

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 (RBCCD59−) and CD59‐negative reticulocytes (RETCD59−). 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). 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: predosing and on Days 15 and 30. Blood samples were processed according to a standardized sample processing and data acquisition protocol, and three endpoints were measured: %reticulocytes, frequency of RETCD59−, and frequency of RBCCD59−. The methodology was found to be reproducible, as the analysis of technical replicates resulted in experimental coefficients of variation that approached theoretical values. Good transferability was evident from the similar kinetics and magnitude of the dose‐related responses that were observed among different laboratories. Concordance correlation coefficients showed a high level of agreement between the reference site and the test sites (range: 0.87–0.99). 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. Environ. Mol. Mutagen. 2011.

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.

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. The genotoxicants colchicine, vinblastine sulfate, ethyl methanesulfonate, methyl methanesulfonate, ethyl nitrosourea, methyl nitrosourea, and bleomycin were applied continuously for 24-30 h. Following treatment, all cell processing, sampling and data acquisition steps were accomplished in the same 96-well plate. Data acquisition occurred in a walk-away mode via the use of a high throughput sampling device. The resulting flow cytometric MN values were evaluated with a statistical model that indicated non-linear relationships describe the data better than linear fits. The one exception was bleomycin, where MN induction was consistently best described by a linear dose-response relationship. Collectively, these results suggest that flow cytometry represents a practical and efficient approach for thoroughly examining the dose-response relationship, and clearly benefits studies that seek to characterize no observable genotoxic effect levels, lowest observable genotoxic effect levels, and/or benchmark doses.

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. Thus, it was desirable to establish a new higher throughput scoring approach before expanding the trial to include weak mutagens or nongenotoxicants. An immunomagnetic column separation method that dramatically increases analysis rates was therefore developed (Dertinger et al. [ 2011 ]: Mutat Res 721:163‐170). To evaluate this new method for use in the international collaborative trial, studies were conducted to determine the mutagenic response of male Sprague Dawley rats treated for 3 or 28 consecutive days with several doses of 1,3‐propane sultone (1,3‐PS). Pig‐a mutant frequencies were measured over a period of several weeks and were supplemented with another indicator of genetic toxicity, peripheral blood micronucleated reticulocyte (MN‐RET) counts. 1,3‐PS was found to increase Pig‐a mutation and MN‐RET frequencies in both 3‐ and 28‐day study designs. While the greatest induction of MN‐RETs was observed in the 3‐day study, the highest Pig‐a responses were found with 28‐days of treatment. Pig‐a measurements were acquired in approximately one‐third the time required in the original method, while the number of erythrocyte and reticulocyte equivalents analyzed per sample were increased by factors of 100 and 10, respectively. The data strongly support the value of using the immunomagnetic separation technique for enumerating Pig‐a mutation frequencies. These results also demonstrate that the ongoing international trial will benefit from the inclusion of studies that are based on both acute and protracted repeat dosing schedules in conjunction with the acquisition of longitudinal data, at least until more data have been accumulated. Environ. Mol. Mutagen. 2011.

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 (RETCD59−) and erythrocytes (RBCCD59−) 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 RETCD59− and RBCCD59− 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‐RETu2009<u2009RETCD59−u2009<u2009RBCCD59−. 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. Environ. Mol. Mutagen. 55:299–308, 2014.

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 cisplatins 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.