Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

An international, multi-lab trial was conducted to evaluate a flow cytometry-based method for scoring micronuclei in mouse lymphoma L5178Y cells [S.L. Avlasevich, S.M. Bryce, S.E. Cairns, S.D. Dertinger, In vitro micronucleus scoring by flow cytometry: differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability, Environ. Mol. Mutagen. 47 (2006) 56-66]. A reference laboratory investigated the potential of six chemicals to induce micronuclei -- the genotoxicants mitomycin C (MMC), etoposide (ETOPO), and vinblastine (VB), and the non-genotoxicants sucrose (SUC), staurosporine (STS), and dexamethasone (DEX). The latter two non-genotoxicants were selected as extreme challenges to the assay because of their potent apoptogenic activity. Three collaborating laboratories were supplied with prototype In Vitro MicroFlow kits, and each was assigned one genotoxicant and one non-genotoxicant. Cells were treated continuously for 24h over a range of concentrations up to 5 mg/ml, or overtly cytotoxic concentrations. Micronuclei were scored via standard microscopy and flow cytometry. In addition to enumerating micronucleus frequencies, a cytotoxicity measurement that is simultaneously acquired with the flow cytometric micronucleus scoring procedure was evaluated (Flow-NBR). With this method, latex particles served as counting beads, and facilitated relative survival measurements that exclude the presence of dead/dying cells. For comparison purposes, additional cytotoxicity endpoints were measured, including several that are based on cell number, and others that reflect compromised membrane integrity, including dye permeability and/or phospholipid distribution. Key findings for this set of compounds include the following: (1) significant discrepancies in top concentration selection were found when cytotoxicity measurements were based on different methods, with the Flow-NBR approach tending to be the most sensitive, (2) both microscopy- and flow cytometry-based scoring methods detected concentration-dependent micronucleus formation for the three genotoxic agents studied, with good agreement between the reference laboratory and the collaborating laboratories, and (3) whereas flow cytometric analyses showed no significant increases for the non-genotoxicants when top concentration selection was based on Flow-NBR, significantly elevated micronucleus frequencies were observed for concentrations that were chosen based on less-sensitive cytotoxicity assays. Collectively, these results indicate that rapid assessment of genotoxicity can be accomplished with a relatively simple flow cytometric technique, and that the scoring system is transferable across laboratories. Furthermore, a concurrent assessment of cytotoxicity, Flow-NBR, may help reduce the occurrence of irrelevant positive results, as it may represent a more appropriate means for choosing top concentration levels. Finally, the data presented herein reinforce concerns about the manner in which cytotoxicity limits are described in guidance documents, since these recommendations tend to cite fixed cut-off values without reference to methodology.     

In vitro micronucleus assay scored by flow cytometry provides a comprehensive evaluation of cytogenetic damage and cytotoxicity

This laboratory has previously reported on the development of a flow cytometry-based method for scoring in vitro micronuclei in mouse lymphoma (L5178Y) cells [S.L. Avlasevich, S.M. Bryce, S.E. Cairns, S.D. Dertinger, In vitro micronucleus scoring by flow cytometry: differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability, Environ. Molec. Mutagen. 47 (2006) 56–66]. With this method, necrotic and mid/late stage apoptotic cells are labeled with the fluorescent dye ethidium monoazide. Cells are then washed, stripped of their cytoplasmic membranes, and incubated with RNase plus a pan-nucleic acid dye (SYTOX Green). This process provides a suspension of free nuclei and micronuclei that are differentially stained relative to chromatin associated with dead/dying cells. The current report extends this line of investigation to include the human cell line TK6. Additionally, methods are described that facilitate simultaneous quantitative analysis of cytotoxicity, perturbations to the cell cycle, and what we hypothesize is aneuploidization. This comprehensive cytogenetic damage assay was evaluated with the following diverse agents: etoposide, ionizing radiation, methyl methanesulfonate, vinblastine, ethanol, and staurosporine. Cells were harvested after 30 h of continuous treatment (in the case of chemicals), or following graded doses of radiation up to 1 Gy. Key findings include the following: (1) Significant discrepancies in top dose selection were found for five of the six agents studied when relative survival measurements were based on Coulter counting versus flow cytometry. (2) Both microscopy- and flow cytometry-based scoring methods detected dose-dependent micronucleus formation for the four genotoxic agents studied, whereas no significant increases were observed for the presumed non-genotoxicants ethanol and staurosporine when top dose selection was based on flow cytometric indices of cytotoxicity. (3) SYTOX and ethidium monoazide fluorescence signals conveyed cell cycle and cell death information, respectively, and appear to represent valuable aids for interpreting micronucleus data. (4) The frequency of hypodiploid nuclei increased in response to each of the genotoxic agents studied, but not following exposure to ethanol or staurosporine. Collectively, these results indicate that a comprehensive assessment of genotoxicity and other test article-induced toxicities can be acquired simultaneously using a simple two-color flow cytometry-based technique.     

In vitro micronucleus assay scored by flow cytometry provides a comprehensive evaluation of cytogenetic damage and cytotoxicity

This laboratory has previously reported on the development of a flow cytometry-based method for scoring in vitro micronuclei in mouse lymphoma (L5178Y) cells [S.L. Avlasevich, S.M. Bryce, S.E. Cairns, S.D. Dertinger, In vitro micronucleus scoring by flow cytometry: differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability, Environ. Molec. Mutagen. 47 (2006) 56-66]. With this method, necrotic and mid/late stage apoptotic cells are labeled with the fluorescent dye ethidium monoazide. Cells are then washed, stripped of their cytoplasmic membranes, and incubated with RNase plus a pan-nucleic acid dye (SYTOX Green). This process provides a suspension of free nuclei and micronuclei that are differentially stained relative to chromatin associated with dead/dying cells. The current report extends this line of investigation to include the human cell line TK6. Additionally, methods are described that facilitate simultaneous quantitative analysis of cytotoxicity, perturbations to the cell cycle, and what we hypothesize is aneuploidization. This comprehensive cytogenetic damage assay was evaluated with the following diverse agents: etoposide, ionizing radiation, methyl methanesulfonate, vinblastine, ethanol, and staurosporine. Cells were harvested after 30h of continuous treatment (in the case of chemicals), or following graded doses of radiation up to 1Gy. Key findings include the following: (1) Significant discrepancies in top dose selection were found for five of the six agents studied when relative survival measurements were based on Coulter counting versus flow cytometry. (2) Both microscopy- and flow cytometry-based scoring methods detected dose-dependent micronucleus formation for the four genotoxic agents studied, whereas no significant increases were observed for the presumed non-genotoxicants ethanol and staurosporine when top dose selection was based on flow cytometric indices of cytotoxicity. (3) SYTOX and ethidium monoazide fluorescence signals conveyed cell cycle and cell death information, respectively, and appear to represent valuable aids for interpreting micronucleus data. (4) The frequency of hypodiploid nuclei increased in response to each of the genotoxic agents studied, but not following exposure to ethanol or staurosporine. Collectively, these results indicate that a comprehensive assessment of genotoxicity and other test article-induced toxicities can be acquired simultaneously using a simple two-color flow cytometry-based technique.     

Sensitivity of the erythrocyte micronucleus assay: Dependence on number of cells scored and inter-animal variability

Until recently, the in vivo erythrocyte micronucleus assay has been scored using microscopy. Because the frequency of micronucleated cells is typically low, cell counts are subject to substantial binomial counting error. Counting error, along with inter-animal variability, limit the sensitivity of this assay. Recently, flow cytometric methods have been developed for scoring micronucleated erythrocytes and these methods enable many more cells to be evaluated than is possible with microscopic scoring. Using typical spontaneous micronucleus frequencies reported in mice, rats, and dogs we calculate the counting error associated with the frequency of micronucleated reticulocytes as a function of the number of reticulocytes scored. We compare this counting error with the inter-animal variability determined by flow cytometric scoring of sufficient numbers of cells to assure that the counting error is less than the inter-animal variability, and calculate the minimum increases in micronucleus frequency that can be detected as a function of the number of cells scored. The data show that current regulatory guidelines allow low power of the test when spontaneous frequencies are low (e.g., ≤0.1%). Tables and formulas are presented that provide the necessary numbers of cells that must be scored to meet the recommendation of the International Working Group on Genotoxicity Testing that sufficient cells be scored to reduce counting error to less than the inter-animal variability, thereby maintaining a more uniform power of detection of increased micronucleus frequencies across laboratories and species.     

HUMN project: detailed description of the scoring criteria for the cytokinesis-block micronucleus assay using isolated human lymphocyte cultures

Criteria for scoring micronuclei and nucleoplasmic bridges in binucleated cells in the cytokinesis-block micronucleus assay for isolated human lymphocyte cultures are described in detail. Morphological characteristics of mononucleated cells, binucleated cells, and multinucleated cells as well as necrotic and apoptotic cells and nuclear buds are also described. These criteria are illustrated by a series of schematic diagrams as well as a comprehensive set of colour photographs that are of practical assistance during the scoring of slides. These scoring criteria, diagrams and photographs have been used in a HUman MicronNucleus (HUMN) project inter-laboratory slide-scoring exercise to evaluate the extent of variability that can be attributable to individual scorers and individual laboratories when measuring the frequency of micronuclei and nucleoplasmic bridges in binucleated cells as well as the nuclear division index. The results of the latter study are described in an accompanying paper. It is expected that these scoring criteria will assist in the development of a procedure for calibrating scorers and laboratories so that results from different laboratories for the cytokinesis-block micronucleus assay may be more comparable in the future.     

Validation of a flow cytometric acridine orange micronuclei methodology in rats

Our laboratory has previously reported a flow cytometric acridine orange method for detection of micronucleus (MN) in the rat using cyclophosphamide as a test compound. To replace the manual method of scoring and satisfy Good Laboratory Practice (GLP) requirements, an extensive validation of the flow method was required. Therefore, manual scoring and flow cytometric determination of MN were compared using vincristine, chlorambucil, methotrexate, and doxorubicin compounds known to induce MN formation with various mechanisms of action. 1,2-Dimethylhydrazine (1,2-DH), a compound with negative or equivocal MN findings also was evaluated. The flow method consistently demonstrated dose- and time-dependent responses for MN production at all concentrations of vincristine, methotrexate, clorambucil, and doxorubicin. In contrast, manual scoring of slides failed to detect an increase in MN at the lowest doses of doxorubicin (1mg/kg) at 24 or 48 h, and methotrexate at 48 h, or any dose of methotrexate (50, 100, or 250 mg/kg) at 24h. Additionally, a dose-response for methotrexate at 48 h, and chlorambucil at 24 h were missed using manual scoring. For 1,2-DH, the flow method showed a low level (< 1.4-fold) increase in MN at all doses and times. In contrast, the manual method showed five-seven-fold increases at 24 h, but a < two-fold increase at 48 h in the highest dose only. These data may suggest that the flow method has a greater sensitivity and possibly accuracy than manual scoring. Significant decreases in polychromatic erythrocytes (PCE) were seen using both methods at approximately the same dose for all compounds. However, absolute flow cytometric PCE values were consistently higher than manual. Additional cytotoxicity parameters obtained by the flow method allowed a more complete assessment of cytotoxicity than PCE alone. Furthermore, data reported here combined with improved throughput, shortened data turnaround and reporting times, and possibly better precision due to evaluation of much larger numbers of cells clearly demonstrate the usefulness of flow cytometry method in the routine micronucleus evaluation.     

Flow cytometric analysis of micronucleated reticulocytes in mouse bone marrow

This laboratory has previously reported a flow cytometric procedure for quantitatively analyzing mouse peripheral blood reticulocytes for micronucleus content. The current study extends this line of investigation by evaluating whether these same flow cytometric scoring procedures can be applied to the analysis of mouse bone marrow samples. To validate the method, three groups of male BALB/c mice were treated with 100 mg/kg b.wt. methyl methanesulfonate. Bone marrow samples were collected 20, 40 or 60 h after administration. A set of 5 untreated animals was included to provide an indication of spontaneous micronucleus frequencies. The cells were fixed with ultracold methanol, treated with ribonuclease, and labeled with anti-CD71 antibody (FITC conjugate) and propidium iodide. This fixing and labeling procedure resulted in the resolution of the micronucleated reticulocyte population and facilitated high-speed acquisition and enumeration via flow cytometry. The number of micronucleated reticulocytes was determined flow cytometrically by the analysis of 10?000 total reticulocytes per bone marrow sample. In addition to these automated measurements, slides stained with acridine orange were prepared and the number of micronuclei per 1000 reticulocytes was determined microscopically for each sample. The resulting data demonstrate that flow cytometry can effectively enumerate micronucleated reticulocytes in mouse bone marrow. The advantages associated with an objective, high throughput scoring methodology are also clearly indicated.     

Sensitivity of the erythrocyte micronucleus assay: dependence on number of cells scored and inter-animal variability

Until recently, the in vivo erythrocyte micronucleus assay has been scored using microscopy. Because the frequency of micronucleated cells is typically low, cell counts are subject to substantial binomial counting error. Counting error, along with inter-animal variability, limit the sensitivity of this assay. Recently, flow cytometric methods have been developed for scoring micronucleated erythrocytes and these methods enable many more cells to be evaluated than is possible with microscopic scoring. Using typical spontaneous micronucleus frequencies reported in mice, rats, and dogs we calculate the counting error associated with the frequency of micronucleated reticulocytes as a function of the number of reticulocytes scored. We compare this counting error with the inter-animal variability determined by flow cytometric scoring of sufficient numbers of cells to assure that the counting error is less than the inter-animal variability, and calculate the minimum increases in micronucleus frequency that can be detected as a function of the number of cells scored. The data show that current regulatory guidelines allow low power of the test when spontaneous frequencies are low (e.g., < or =0.1%). Tables and formulas are presented that provide the necessary numbers of cells that must be scored to meet the recommendation of the International Working Group on Genotoxicity Testing that sufficient cells be scored to reduce counting error to less than the inter-animal variability, thereby maintaining a more uniform power of detection of increased micronucleus frequencies across laboratories and species.     

Cyclophosphamide and etoposide canine studies demonstrate the cross-species potential of the flow cytometric peripheral blood micronucleated reticulocyte endpoint

Erythrocyte-based micronucleus tests have traditionally been performed with bone marrow specimens, since, in most preclinical animal models, the spleen can efficiently remove aberrant erythrocytes from the circulation. Even so, evidence is mounting that by examining tens of thousands of young (CD71-positive) circulating reticulocytes for the presence of micronuclei via flow cytometry, a sensitive assay of cytogenetic damage is realized. The work described herein was designed to test this hypothesis further, using an important preclinical toxicology model, the beagle dog. In these experiments, purebred male beagles were treated for five consecutive days with cyclophosphamide (0, 6.25, 12.5 or 25mg/m(2)/day) or for two consecutive days with etoposide (0, 1.56, 6.25 or 12.5mg/m(2)/day). Before treatment, and on each day of administration, blood specimens were collected and processed for flow cytometric scoring of micronucleated reticulocyte (MN-RET) frequency. Twenty-four hours after the final administration, blood MN-RET frequencies were determined via flow cytometry, and frequencies of micronucleated bone marrow polychromatic erythrocytes (MN-PCE) were determined using acridine orange and May-Grunwald Giemsa staining. In the case of cyclophosphamide, elevated blood MN-RET frequencies were observed 2 days after treatment began, and the maximal frequency was achieved 1 day later. Similarly, etoposide-induced blood MN-RET were not evident 1 day after administration began, but a robust effect was apparent 2 days after treatments were initiated. Twenty-four hours after the final administrations, dose-related micronucleus responses were evident for both agents and in both blood and bone marrow compartments. Good overall agreement between MN-RET and MN-PCE frequencies was evidenced by high Spearman's correlation coefficients-0.89 for blood flow cytometry versus bone marrow acridine orange staining and 0.83 for blood flow cytometry versus bone marrow May-Grunwald Giemsa staining. Taken together, these results provide further support for the cross-species utility of flow cytometry-based blood MN-RET measurements.     

Technical aspects of automatic micronucleus analysis in rodent bone marrow assays

The mouse bone marrow micronucleus assay is anin vivo test commonly used in the pharmaceutical industry to evaluate the genotoxic potential of new compounds. The test detects agent-induced chromosomal damage or damage of the mitotic spindle apparatus. In this paper the state-of-the-art in automated rodent micronucleus evaluation using computerized image analyis in combination with high-quality slides obtained by the cellulose column fractionation technique is reviewed. The latter allows the effective removal of nucleated cells from rodent bone marrow. It has been found that automatic micronucleus scoring with the Leitz MIAC image analyzer is substantially faster than labor-intensive manual analysis. Automatic scoring can be performed overnight for up to 16 slides. We have been successfully using automatic micronucleus analysis for the testing of new pharmaceutical drugs for more than 3 years.Abbreviations MNE NCE containing micronuclei - MPE PCE containing micronuclei - NCE normochromatic erythrocyte - PCE polychromatic erythrocyte deceased on 25 May 1994     

The micronucleus assay in human buccal cells as a tool for biomonitoring DNA damage: the HUMN project perspective on current status and knowledge gaps

The micronucleus (MN) assay in exfoliated buccal cells is a useful and minimally invasive method for monitoring genetic damage in humans. This overview has concluded that although MN assay in buccal cells has been used since the 1980s to demonstrate cytogenetic effects of environmental and occupational exposures, lifestyle factors, dietary deficiencies, and different diseases, important knowledge gaps remain about the characteristics of micronuclei and other nuclear abnormalities, the basic biology explaining the appearance of various cell types in buccal mucosa samples and effects of diverse staining procedures and scoring criteria in laboratories around the world. To address these uncertainties, the human micronucleus project (HUMN; see http://www.humn.org) has initiated a new international validation project for the buccal cell MN assay similar to that previously performed using human lymphocytes. Future research should explore sources of variability in the assay (e.g. between laboratories and scorers, as well as inter- and intra-individual differences in subjects), and resolve key technical issues, such as the method of buccal cell staining, optimal criteria for classification of normal and degenerated cells and for scoring micronuclei and other abnormalities. The harmonization and standardization of the buccal MN assay will allow more reliable comparison of the data among human populations and laboratories, evaluation of the assay's performance, and consolidation of its world-wide use for biomonitoring of DNA damage.     

Use of the cytokinesis-block method for the analysis of micronuclei in V79 Chinese hamster lung cells: results with mitomycin C and cyclophosphamide

The cytochalasin B (CYB)-blocked binucleated cell assay has been explored to analyze micronuclei and cell cycle kinetics using 2 known mutagenic carcinogens in V79 Chinese hamster lung cells. To determine the optimum time to obtain the maximum number of binucleated cells for micronucleus analysis, duplicate cultures of exponentially growing cells were treated with 3 micrograms/ml CYB for varying durations (8-48 h). A peak appearance of binucleated cells at 16 h in the presence of CYB suggested this as an optimum time for micronucleus analysis in binucleated V79 cells. To evaluate the capacity for induction of micronuclei in V79 cells, 2 mutagenic carcinogens, mitomycin C (0.125-1.0 micrograms/ml) and cyclophosphamide (2-12 micrograms/ml) were tested in duplicate cultures. Mitomycin C, a direct-acting alkylating agent, caused approximately an 18-fold increase in micronucleus frequency over controls at the highest concentration tested (1.0 micrograms/ml), and this increase occurred in a dose-related manner (r = 0.92). The concentrations of mitomycin C tested also caused a significant dose-related cell cycle delay, thus suggesting cytotoxicity to V79 cells. Cyclophosphamide, an indirect-acting alkylating agent, requiring the presence of S9 mix, caused approximately a 17-fold increase in micronucleus frequency over controls at the highest tested concentration (12 micrograms/ml), with a clear dose response (r = 0.99). The various concentrations of cyclophosphamide also caused cytotoxicity in a dose-related fashion. Thus, this study demonstrates the usefulness of the cytokinesis-block method in V79 cells as a possible screen to analyze micronucleus induction and cytotoxicity. Because this approach is much less labor intensive than conducting a structural chromosomal analysis, this assay has great potential both as an initial screen for clastogenic activity and as a tool for investigating the underlying mechanisms for clastogenicity.     

Multiparametric flow cytometric analysis of radiation-induced micronuclei in mammalian cell cultures.

A new flow cytometric method is presented that quantifies the frequency of radiation-induced micronuclei in mammalian cell cultures with high precision. After preparing a suspension of main nuclei and micronuclei stained with ethidium bromide and Hoechst 33258, both types of particles are measured simultaneously in a flow cytometer using forward light scatter and three fluorescence emission intensities excited by UV, 488 nm, and by energy transfer from Hoechst 33258 to ethidium bromide. Nonspecific debris overlapping the micronucleus distribution especially in the low fluorescence intensity region was discriminated from micronuclei by calculating ratios of the different fluorescences. The frequencies of radiation-induced micronuclei measured with this new technique agreed well with results obtained by conventional microscopy. The lower limit of the DNA content of micronuclei identified by this technique was found to be about 0.5%-0.75% of the DNA content of G1-phase nuclei. Dose effect curves and the time-dependent induction of micronuclei were measured for two different mouse cell lines.     

Enumeration of micronucleated reticulocytes in rat peripheral blood: a flow cytometric study

Micronuclei (MN) are routinely enumerated in mouse peripheral blood to index genotoxicity. Recent data from the Collaborative Study Group for the Micronucleus Test (CSGMT) [CSGMT (The Collaborative Study Group for the Micronucleus Test), Evaluation of the rat micronucleus test with bone marrow and peripheral blood: summary of the 9th collaborative study by CSGMT/JEMS MMS, Environ. Mol. Mutagen. 32 (1998) 84-100] suggest that rat peripheral blood may also be appropriate for the enumeration of MN, if scoring is limited to the youngest fraction of reticulocytes. The experiments described herein were designed to test whether modifications to a flow cytometric scoring procedure for measuring micronucleated reticulocytes (MN-RET) in mouse peripheral blood could be extended to accurately enumerate MN in rat peripheral blood. Rats were treated with saline or one of three genotoxic agents (6-mercaptopurine, ethyl methanesulfonate or propane sultone) in an acute dosing protocol. Peripheral blood samples were subsequently collected for both microscopic and flow cytometric analysis. Micronucleus frequencies were scored in the youngest fraction of reticulocytes: scoring by microscopy was restricted to the types I and II reticulocytes based on RNA content utilizing acridine orange supravital staining; flow cytometric measurements were restricted to the youngest fraction of reticulocytes based on transferrin receptor (CD71) staining. A statistically significant dose-related increase in the incidence of MN was observed, irrespective of scoring method. A higher level of statistical discrimination between control and genotoxin-treated groups was observed for the flow cytometric data and can most likely be explained by the increased number of cells scored (10x more than microscopy) and the lower scoring variability. Together, these data suggest that (i) rat peripheral blood represents an appropriate compartment for evaluating genotoxin-induced MN when the analysis is restricted to young reticulocytes, and (ii) the measurement of MN in rat peripheral blood reticulocytes benefits from the high throughput methodology of flow cytometry.     

Evaluation of an automated in vitro micronucleus assay in CHO-K1 cells

In this paper, we describe the evaluation of an automated in vitro micronucleus assay using CHO-K1 cells in 96-well plates. CHO-K1 cells were pre-loaded with a cell dye that stains the cytoplasm, after which the cells were treated with the test compounds for either 3h (for the +S9 condition) or 24h (for the -S9 condition). A total of 10 concentrations were tested, of which the top five concentrations were scored (limited by either cytotoxicity or solubility). At the end of the incubation period the cells were fixed and their DNA was stained with Hoechst. The visualization and scoring of the cells was done using an automated fluorescent microscope coupled with proprietary automated image analysis software provided by Cellomics (Pittsburg, PA). A total of 46 compounds were used in this evaluation, including 8 aneugens and 25 clastogens with varied mechanisms of action. Thirteen non-genotoxic compounds were also included. The automated scoring had a sensitivity of 88% and a specificity of 100%, with a predictive value positive of 100% and a predictive value negative of 76%, compared to data from the literature that was obtained with manual scoring. We also describe the incorporation of a metabolic activation system using rat liver S9 homogenates, and the use of cell number counts as a cytotoxicity index which is complementary to the CBPI- (cytokinesis-block proliferation index) based index. Finally, we also discuss the potential for artefactual findings due to fluorescent precipitate, which should be carefully monitored to prevent false positive results. In conclusion, the automated in vitro micronucleus scoring is a valid alternative to the manual scoring of slides, and it has the advantage of generating data in a rapid and consistent manner, and with low compound requirements, which makes it well suited as a screening assay in the early stages of compound development.     

A comparison of two methods for evaluating drug-induced chromosome alterations.

Evaluating the technique and procedure for mutagenicity testing in mammals is a prerequisite to the development of a broad spectrum mutagenic assessment program. Two techniques, chromosome examination and micronucleus scoring, show promise but their applicability for mass screening is uncertain. We determined the slide observation time for these two techniques in mice treated orally, subcutaneously, intravenously, and intraperitoneally with cyclophosphamide (CY). In each instance, we detected a dose-response in less observation time by counting micronuclei in polychromatophilic erythrocytes. The simplicity of the scoring method, the ease of micronucleus identification and the rapidity of scoring all suggest the micronucleus test may be favorably integrated into a mutagenicity screening program.     

H2AX phosphorylation as a genotoxicity endpoint

The γH2AX focus assay, based on phosphorylation of the variant histone protein H2AX, was evaluated as a genotoxicity test in immortalised wild-type mouse embryonic fibroblasts (MEFs) treated for 4 h with a panel of reference compounds routinely used in genotoxicity testing. The topoisomerase II poison etoposide (0.006–60 μg/ml), the alkylating agent methyl methanesulfonate (1.3–65 μg/ml) and the direct DNA-damaging agent bleomycin (0.1–10 μg/ml) all produced a positive concentration–response relationship. The non-genotoxic compounds ampicillin (0.035–3500 μg/ml) and sodium chloride (0.058–580 μg/ml) showed no such response with increased concentrations. The H2AX phosphorylation results were compared with the outcome of two standard in vitro genotoxicity tests, namely the micronucleus and comet assays. Compounds that produced measurable DNA damage in the focus assay generated micronuclei at comparable concentrations. In this study, the focus assay identified genotoxic agents with the same specificity as the comet assay.These results were substantiated when H2AX phosphorylation was analysed using flow cytometry in the murine cell line L5178Y, growing in suspension. The data were in concordance with the manual scoring focus assay. To further this investigation, the γH2AX flow cytometry was compared to the in vitro micronucleus flow cytometry and mouse lymphoma assay using the same cell population after MMS treatment. The median γH2AX value increased significantly above the control at all four MMS concentrations tested. The percentage of micronucleus events in the in vitro micronucleus flow test and the mutation frequency in the mouse lymphoma assay were also significantly increased at each MMS concentration. The current data indicate that H2AX phosphorylation could be used as a biomarker of genotoxicity, which could predict the outcome of in vitro mammalian cell genotoxicity assays.     

FISH in analysis of gamma ray-induced micronuclei formation in barley

A micronucleus test in combination with fluorescent in situ hybridization (FISH) using telomere-, centromere-specific probes and 5S and 25S rDNA was used for a detailed analysis of the effects of gamma ray irradiation on the root tip meristem cells of barley, Hordeum vulgare (2n = 14). FISH with four DNA probes was used to examine the involvement of specific chromosomes or chromosome fragments in gamma ray-induced micronuclei formation and then to explain their origin. Additionally, a comparison of the possible origin of the micronuclei induced by physical and chemical treatment: maleic hydrazide (MH) and N-nitroso-N-methylurea (MNU) was done. The micronuclei induced by gamma ray could originate from acentric fragments after chromosome breakage or from whole lagging chromosomes as a result of a dysfunction of the mitotic apparatus. No micronuclei containing only centromeric signals were found. An application of rDNA as probes allowed it to be stated that 5S rDNA–bearing chromosomes are involved in micronuclei formation more often than NOR chromosomes. This work allowed the origin of physically- and chemically-induced micronuclei in barley cells to be compared: the origin of micronuclei was most often from terminal fragments. FISH confirmed its usefulness in the characterization of micronuclei content, as well as in understanding and comparing the mechanisms of the actions of mutagens applied in plant genotoxicity.     

Enumeration of micronucleated CD71-positive human reticulocytes with a single-laser flow cytometer

The extreme rarity of micronucleated reticulocytes (RETs) in the peripheral blood of non-splenectomized humans has precluded facile enumeration of these cells, as well as evaluation of this endpoint as an index of cytogenetic damage. In this report, we describe a high-throughput, single-laser flow cytometric system for scoring the incidence of micronuclei (MN) in newly formed human RETs. The procedure is based on an immunochemical reagent that differentially labels the most immature fraction of RETs from mature erythrocytes based on the expression level of the transferrin receptor (also known as CD71). The resolution of four erythrocyte populations (young RETs and mature erythrocytes, with and without MN) was achieved for human blood cells treated with phycoerythrin-conjugated anti-CD71, RNase, and either SYTOX Green or SYBR Green I nucleic acid dyes. Anti-glycophorin A labeling of erythroid cells (CyChrome conjugate) was also incorporated into the staining procedure to ensure that debris or other potential artifacts did not adversely impact the analyses. Instrument calibration procedures utilizing malaria-infected rodent erythrocytes were also developed, and are described. Using this analytical system, blood samples from 10 healthy non-splenectomized human volunteers were analyzed for micronucleus frequencies with a single-laser flow cytometer. Average micronucleus frequencies in the mature and most immature fraction of RETs were 0.016 and 0.19%, respectively. Blood samples from three healthy splenectomized volunteers were also evaluated. As expected, these samples exhibited higher micronucleus frequencies in the mature subset of erythrocytes (range 0.03-0.18%). The resulting data suggest that MN can be quantified in human erythrocyte populations with a single-laser flow cytometer, and that the frequency of MN cells in the youngest reticulocyte population approaches values expected in the absence of splenic selection against MN-erythrocytes. This high throughput system is potentially important for evaluating the value of the micronucleated reticulocyte endpoint as an index of chromosome breakage and/or chromosome segregational abnormalities in human populations.     

Indispensable function of the germ nucleus for postconjugational stomatogenesis in Paramecium caudatum: Evidence against its genic function shown by hypohaploid micronuclei

It is known that the germinal micronucleus at the stages of gametogenesis and/or fertilization has an indispensable function for the postconjugational development of oral apparatus (stomatogenesis) in Paramecium caudatum. To determine whether this function is due to some specific genes in the micronucleus, postconjugational stomatogenesis was examined in the conjugation of haploid and hypohaploid cells. Haploid clones were obtained by conjugation between amicronucleate cells and diploid micronucleate cells. After conjugation between these haploid clones or between the haploid clones and amicronucleate clones, we succeeded in obtaining hypohaploid clones that have various types of nullisomic micronuclei. If a few genes in the micronucleus control postconjugational stomatogenesis, some hypohaploid micronuclei should undergo stomatogenesis normally, but others should not. In the present work, however, almost all the hypohaploid micronuclei developed the oral apparatus and formed food vacuoles. We can apparently rule out the possibility that a few specific genes of the micronucleus are required for postconjugational stomatogenesis in Paramecium caudatum, unless selection operates to retain the chromosomes with the essential gene(s). Dev. Genet. 23:142–150, 1998. © 1998 Wiley-Liss, Inc.