Stephen D. Dertinger

In vivo rodent erythrocyte micronucleus assay. II. Some aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring.

An expert working group on the in vivo micronucleus assay, formed as part of the International Workshop on Genotoxicity Test Procedures (IWGTP), discussed protocols for the conduct of established and proposed micronucleus assays at a meeting held March 25–26, 1999 in Washington, DC, in conjunction with the annual meeting of the Environmental Mutagen Society. The working group reached consensus on a number issues, including: (1) protocols using repeated dosing in mice and rats; (2) integration of the (rodent erythrocyte) micronucleus assay into general toxicology studies; (3) the possible omission of concurrently‐treated positive control animals from the assay; (4) automation of micronucleus scoring by flow cytometry or image analysis; (5) criteria for regulatory acceptance; (6) detection of aneuploidy induction in the micronucleus assay; and (7) micronucleus assays in tissues (germ cells, other organs, neonatal tissue) other than bone marrow. This report summarizes the discussions and recommendations of this working group. In the classic rodent erythrocyte assay, treatment schedules using repeated dosing of mice or rats, and integration of assays using such schedules into short‐term toxicology studies, were considered acceptable as long as certain study criteria were met. When the micronucleus assay is integrated into ongoing toxicology studies, relatively short‐term repeated‐dose studies should be used preferentially because there is not yet sufficient data to demonstrate that conservative dose selection in longer term studies (longer than 1 month) does not reduce the sensitivity of the assay. Additional validation data are needed to resolve this point. In studies with mice, either bone marrow or blood was considered acceptable as the tissue for assessing micronucleus induction, provided that the absence of spleen function has been verified in the animal strains used. In studies with rats, the principal endpoint should be the frequency of micronucleated immature erythrocytes in bone marrow, although scoring of peripheral blood samples gives important supplementary data about the time course of micronucleus induction. When dose concentration and stability are verified appropriately, concurrent treatment with a positive control agent is not necessary. Control of staining and scoring procedures can be obtained by including appropriate reference samples that have been obtained from a separate experiment. For studies in rats or mice, treatment/sampling regimens should include treatment at intervals of no more than 24 hr (unless the test article has a half‐life of more than 24 hr) with sampling of bone marrow or blood, respectively, within 24 or 40 hr after the last treatment. The use of a DNA specific stain is recommended for the identification of micronuclei, especially for studies in the rat. In the case of a negative assay result with a non‐toxic test article, it is desirable that systemic exposure to the test article is demonstrated. The group concluded that successful application of automated scoring by both flow cytometry and image analysis had been achieved, and defined criteria that should be met if automated scoring is employed. It was not felt appropriate to attempt to define specific recommended protocols for automated scoring at the present time. Other issues reviewed and discussed by the working group included micronucleus assays that have been developed in a number of tissues other than bone marrow. The group felt that these assays were useful research tools that could also be used to elucidate mechanisms in certain regulatory situations, but that these assays had not yet been standardized and validated for routine regulatory application. Environ. Mol. Mutagen. 35:234–252, 2000

In vivo mutation assay based on the endogenous Pig-a locus

The product of the X‐chromosomes Pig‐a gene acts in the first step of glycosylphosphatidylinositol (GPI) anchor biosynthesis, and is thereby essential for attaching certain proteins to the cell surface. The experiments described herein were designed to evaluate whether lack of GPI‐anchored proteins could form the basis of an in vivo mutation assay. Specifically, we used a CD59‐negative cell surface phenotype to denote Pig‐a mutation. Besides anti‐CD59‐PE, two other fluorescent reagents were used: thiazole orange to differentiate mature erythrocytes, reticulocytes (RETs), and leukocytes; and anti‐CD61 to resolve platelets. These experiments were performed with Sprague Dawley rats, and focused on two cell populations, total erythrocytes and RETs. The ability of the analytical method to enumerate CD59‐negative erythrocytes was initially assessed with reconstruction experiments whereby mutant‐mimicking cells were added to control bloods. Subsequently, female rats were treated on three occasions with the model mutagens ENU (100 mg/kg/day) or DMBA (40 mg/kg/day). Blood specimens were harvested at various intervals, as late as 6 weeks post‐exposure. Considering all week 4–6 data, we found that CD59‐negative cells ranged from 239 to 855 × 10−6 and 82 to 405 × 10−6 for ENU and DMBA, respectively. These values were consistently greater than those observed for negative control rats (18 ± 19 × 10−6). The elevated frequencies observed for the genotoxicant‐exposed animals were usually higher for RETs compared to total erythrocytes. These data support the hypothesis that an efficient in vivo mutation assay can be developed around flow cytometric enumeration of erythrocytes and/or RETs that exhibit aberrant GPI‐anchored protein expression. Environ. Mol. Mutagen., 2008.

Simple and reliable enumeration of micronucleated reticulocytes with a single-laser flow cytometer

A flow cytometric procedure for scoring micronuclei in mouse peripheral blood erythrocytes, especially reticulocytes, is described. The methods reported herein were developed in an effort to simplify the techniques and to reduce the equipment requirements associated with automated micronucleus analyses. With this procedure, fluorescein-conjugated monoclonal antibodies which bind to the CD71-defined antigen (the transferrin receptor) are used to label reticulocytes. The nucleic acid dye propidium iodide is used to identify cells with micronuclei. Given 488 nm excitation, four populations of erythrocytes are clearly resolved: normochromatic erythrocytes with and without micronuclei, and reticulocytes with and without micronuclei. Since the method is capable of simultaneously providing the incidence of micronuclei in both mature and immature erythrocyte populations, it is compatible with either chronic or acute treatment regimens. To demonstrate cell handling and flow cytometric procedures for quantitatively analyzing peripheral blood micronuclei, an experiment with the model clastogen methyl methanesulfonate is described. Additionally, a reconstruction experiment was performed whereby three mouse blood samples were spiked with successively greater volumes of blood from a clastogen-treated animal so each preparation differed slightly, but definitely, in micronucleus content. Each sample was scored six times by conventional microscopy and by flow cytometry so that the two methods could be directly compared. Collectively, the results from the methyl methanesulfonate experiment and the reconstruction study demonstrate the accuracy and reliability of the flow cytometric method. Furthermore, advantages associated with objective, high throughout scoring methodology are clearly indicated.

The in vivo pig‐a gene mutation assay, a potential tool for regulatory safety assessment

The Pig‐a (phosphatidylinositol glycan, Class A) gene codes for a catalytic subunit of the N‐acetylglucosamine transferase complex involved in an early step of glycosylphosphatidyl inositol (GPI) cell surface anchor synthesis. Pig‐a is the only gene involved in GPI anchor synthesis that is on the X‐chromosome, and research into the origins of an acquired genetic disease involving GPI anchor deficiency (paroxysmal nocturnal hemoglobinuria) indicates that cells lacking GPI anchors, or GPI‐anchored cell surface proteins, almost always have mutations in the Pig‐a gene. These properties of the Pig‐a gene and the GPI anchor system have been exploited in a series of assays for measuring in vivo gene mutation in blood cells from humans, rats, mice, and monkeys. In rats, flow cytometric measurement of Pig‐a mutation in red blood cells requires microliter volumes of blood and data can be generated in hours. Spontaneous mutant frequencies are relatively low (<5 × 10−6) and rats treated with multiple doses of the potent mutagen, N‐ethyl‐N‐nitrosourea, display Pig‐a mutant frequencies that are close to the sum of the frequencies produced by the individual exposures. A general observation is that induced mutant frequencies are manifested earlier in reticulocytes (about 2 weeks after treatment) than in total red blood cells (about 2 months after exposure). Based on data from a limited number of test agents, the assay shows promise for regulatory applications, including integration of gene mutation measurement into repeat‐dose toxicology studies. Environ. Mol. Mutagen., 2010. Published 2010 Wiley‐Liss, Inc.

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.

Reduction of use of animals in regulatory genotoxicity testing : Identification and implementation opportunities-Report from an ECVAM workshop

In vivo genetic toxicology tests measure direct DNA damage or the formation of gene or chromosomal mutations, and are used to predict the mutagenic and carcinogenic potential of compounds for regulatory purposes and/or to follow-up positive results from in vitro testing. These tests are widely used and consume large numbers of animals, with a foreseeable marked increase as a result of the EU chemicals legislation (REACH), which may require follow-up of any positive outcome in the in vitro standard battery with appropriate in vivo tests, regardless of the tonnage level of the chemical. A 2-day workshop with genotoxicity experts from academia, regulatory agencies and industry was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) in Ranco, Italy from 24 to 25 June 2008. The objectives of the workshop were to discuss how to reduce the number of animals in standard genotoxicity tests, whether the application of smarter test strategies can lead to lower animal numbers, and how the possibilities for reduction can be promoted and implemented. The workshop agreed that there are many reduction options available that are scientifically credible and therefore ready for use. Most of these are compliant with regulatory guidelines, i.e. the use of one sex only, one administration and two sampling times versus two or three administrations and one sampling time for micronucleus (MN), chromosomal aberration (CA) and Comet assays; and the integration of the MN endpoint into repeat-dose toxicity studies. The omission of a concurrent positive control in routine CA and MN tests has been proven to be scientifically acceptable, although the OECD guidelines still require this; also the combination of acute MN and Comet assay studies are compliant with guidelines, except for sampling times. Based on the data presented at the workshop, the participants concluded that these options have not been sufficiently utilized to date. Key factors for this seem to be the uncertainty regarding regulatory compliance/acceptance, lack of awareness, and an in many cases unjustified uncertainty regarding the scientific acceptance of reduction options. The workshop therefore encourages the use and promotion of these options as well as the dissemination of data related to reduction opportunities by the scientific community in order to boost the acceptance level of these approaches. Furthermore, experimental proof is needed and under way to demonstrate the credibility of additional options for reduction of the number of animals, such as the integration of the Comet assay into repeat-dose toxicity studies.

The in vivo Pig-a assay: A report of the International Workshop On Genotoxicity Testing (IWGT) Workgroup.

The in vivo Pig-a assay uses flow cytometry to measure phenotypic variants for antibody binding to cell surface glycosylphosphatidylinositol (GPI)-anchored proteins. There is good evidence suggesting that the absence of antibody binding is the result of a mutation in the endogenous X-linked Pig-a gene, which forms the rationale for the assay. Although the assay has been performed with several types of hematopoietic cells and in a variety of mammalian species, including humans, currently it is optimized only for measuring CD59-deficient (presumed Pig-a mutant) erythrocytes in the peripheral blood of rats. An expert workgroup formed by the International Workshop on Genotoxicity Testing considered the state of assay development and the potential of the assay for regulatory use. Consensus was reached on what is known about the Pig-a assay and how it should be conducted, and recommendations were made on additional data and refinements that would help to further enhance the assay for use in hazard identification and risk assessment.

Analysis of micronucleated cells by flow cytometry. 1. Achieving high resolution with a malaria model

Micronucleated cells (MN cells) are present in the blood as rare events (i.e. about 2 MN cells/1000 total). Scoring MN cells by hand is both time-consuming and tedious, which is the primary reason why only 1000-2000 total cells (PCEs) are routinely scored for each sample. It is generally recognized that scoring larger numbers of cells would improve assay statistics and is desirable, but impractical with hand-scoring. In contrast, automated scoring methods can process large numbers of cells, thus improving statistical analysis. In order to accurately and quickly evaluate clastogenic activity, we have developed a flow cytometry based method of scoring micronucleated cells. One of the first steps in developing an automated assay is to demonstrate the ability of the method to resolve the cells of interest. In this case, micronucleated cells must be resolved from DNA-deficient red blood cells (RBCs). Since micronuclei are heterogeneous rare events which vary in both size and DNA content, we chose to use a more enriched and homogeneous biological model for optimizing the experimental variables of this assay, leading to high resolution of the rare cells. Experiments are described in which the murine malaria parasite, P. berghei, served as a micronucleus model and facilitated the development of an accurate flow cytometry based scoring method. This parasite resides in the red blood cell population and endows the cells with a homogeneous (genetically determined) DNA component in the micronucleus size range. The conditions developed with the malaria parasite are readily applied to the analysis of micronucleus events in blood samples.(ABSTRACT TRUNCATED AT 250 WORDS)