Flow cytometric analysis of micronuclei in mammalian cell cultures: past, present and future

Mutagenesis vol. 26 no. 1 pp. 147 152, 2011 doi:10.1093/mutage/geq058 REVIEW Flow cytometric analysis of micronuclei in mammalian cell cultures: past, present and future Svetlana Avlasevich, Steven Bryce, Marlies De Boeck 1, Azeddine Elhajouji 2, Freddy Van Goethem 1, Anthony Lynch 3, John Nicolette 4, Jing Shi 5 and Stephen Dertinger* Litron Laboratories, 200 Canal View Boulevard, Rochester, NY 14623, USA, 1 Genetic & Exploratory Toxicology, Johnson and Johnson Pharmaceutical R&D, Janssen Pharmaceutica N.V., Turnhoutseweg 30, B-2340 Beerse, Belgium, 2 Preclinical Safety, Novartis Institutes for Biomedical Research, CH-4002 Basel, Switzerland, 3 GlaxoSmithKline R&D (3F02), Park Road, Ware, Hertfordshire, UK, 4 Abbott Laboratories, R45M/AP13A, 100 Abbott Park Raod, Abbott Park, IL 60064, USA and 5 BioReliance Corp., 14920 Broschart Raod, Rockville, MD 20850, USA *To whom correspondence should be addressed. Tel: þ585 442 0930; Fax: þ585 442 0934; Email: sdertinger@litronlabs.com Received on May 25, 2010; revised on July 15, 2010; accepted on August 23, 2010 The relative simplicity of the in vitro micronucleus (MNvit) endpoint has made it amenable to several automated scoring approaches. Flow cytometry is one such scoring platform that has been successfully employed. This review describes the origins of the MNvit assay, as well as the evolution and properties of flow cytometry-based scoring systems. While the current state-of-the-art methods acquire micronucleus (MN) frequency data very efficiently, it is becoming clear that they also endow the assay with high information content. For instance, simultaneous with MN frequency determinations, several additional endpoints are acquired that provide insights into cytotoxicity, cell cycle perturbations and, in the event of MN induction, information about genotoxic mode of action. This review concludes with a discussion regarding data gaps and also recommendations for additional work that is needed to more fully realise the potential of flow cytometric MNvit scoring. Significant achievements The in vitro micronucleus (MNvit) test is a simple cytogenetic assay based on the scoring of extranuclear chromatin-containing structures in actively dividing cell populations. These so-called micronuclei (MN) result from acentric chromatid or chromosome fragments (i.e. those lacking a centromere) or whole chromosomes that are unable to migrate to the spindle poles during mitosis and subsequently are not incorporated into either of the daughter nuclei. Therefore, MN can only arise in cells that have undergone cell division. The induction of these extranuclear bodies was first recorded in Vicia faba root cells by Thoday (1) and their use as a quantitative measure of cytogenetic damage was originally proposed by Evans et al. (2). Several years later, the idea of scoring MN as a biological dosimeter of radiation exposure in humans was proposed by Fliedner et al. (3). The MN test became a routine assay in environmental mutagenesis following the development of the rodent bone marrow MN test (4,5). A mammalian cell culture version of the MN test was originally proposed by Heddle (6) and later described by Countryman and Heddle (7) in human peripheral blood lymphocytes. The presence of whole chromosomes in MN can be ascertained using suitable molecular cytogenetic methods, e.g. immunochemical labelling of kinetochore proteins (8) or fluorescence in situ hybridisation (FISH) using a combination of centromeric and telomeric probes (9). This permits chromosome loss and/or non-disjunction (FISH only) to be ascertained and therefore the MNvit assay can provide evidence for the underlying mode of action (MOA) clastogenicity (structural chromosome aberrations) or aneugenicity (chromosome loss or gain). The predominant induction of either type of MN can be used to classify chemical activity and therefore the information is useful for risk assessment purposes. As many compounds that are positive in the MNvit test are mammalian genotoxic carcinogens, the endpoint is often used as a simple screen to identify agents that may be tumorigenic (for reviews see refs 10 12). The MNvit test has been used in a variety of established rodent cell lines (CHO, V79, CHL and L5178Y) and in primary human lymphocytes. Protocols have been described with and without the use of cytochalasin B, an agent that blocks cytokinesis (13). The cytokinesis-block method permits MN to be scored only in cells that have completed a single mitosis after genotoxicant exposure because such cells are easily recognised as binucleated cells (14,15). Limiting scoring of MN to once-divided binucleated cells guards against falsenegative results stemming from cytostatic effects of genotoxicants, suboptimal cell culture conditions and, in the case of primary lymphocytes, differences in mitogenic response (16). The MNvit test has been used in academia and industry for hazard identification as an alternative/replacement of the in vitro chromosome aberration test, a time-consuming method that requires considerable expertise to visually identify structural anomalies in metaphase chromosomes. MNvit is also a valuable follow-up test to elucidate MOA for risk assessment purposes. The test has already gained widespread international use as it offers significant advantages over the assessment of chromosome aberrations since analysis is more objective and quicker, resulting in a much higher throughput. Over the past decade, several studies have been performed to support the validity of the MNvit test for incorporation into the standard regulatory genotoxicity test battery as an alternative to the in vitro structural chromosome aberration test. These studies include, in part, the international validation efforts coordinated by the Société Francxaise de Toxicologie Génétique (17 21), the work performed by Homiski et al. (22) and studies by Miller et al. (23,24). Miller et al. reported.85% correlation between the in vitro structural chromosome aberration test and the MNvit test. More recently, formal validation for the MNvit Ó The Author 2010. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org. 147

S. Avlasevich et al. test has been carried out by the European Centre for the Validation of Alternative Methods (25). Based on this retrospective validation, it has been concluded that the MNvit test is reliable and a relevant genotoxicity assay that can be used as an alternative to the in vitro structural chromosome aberration test. Indeed, recent revisions of international regulatory guidances such as the new European Chemicals Regulation REACH (The Registration, Evaluation, Authorisation and Restriction of Chemicals) and International Conference on Harmonisation (ICH) S2 R guideline for pharmaceuticals have recommended the adoption of the MNvit test as an alternative to the structural chromosome aberration assay and/or mouse lymphoma assay. Furthermore, the Organisation for Economic Co-operation and Development (OECD) guideline for the conduct of the MNvit test (TG 487) for regulatory purposes was recently approved. It was realised early on that the simplicity of the MN endpoint should facilitate automation (26), and several different scoring platforms have been described. There were attempts at image analysis and DNA densitometry (27 30), as well as flow cytometry (31 36). The majority of the flow cytometric work was based on the approach of Nüsse and Kramer (31) that involved lysis of outer membranes. In conjunction with one or more nucleic acid dyes, it was possible to discriminate the liberated nuclei and MN based on their DNA-dye associated fluorescence intensities. Furthermore, beyond MN scoring, flow cytometry allowed sorting of MN to combine with FISH for centromeric probes or chromosomespecific paints to identify MN content and its genomic origin (37 39). While flow cytometry was clearly shown to be a highthroughput platform with great potential, the early methods experienced problems discriminating MN from cellular debris in the cell preparations. Later modifications to those of Nüsse and Kramer attempted to overcome these problems, taking advantage of the higher resolution of later machines and using improved gating based on a combination of light scatter and fluorescence signals to distinguish MN signals from debris (35,40). Although these modifications improved the reliability of the assay, it remained difficult to discriminate MN from cellular debris, especially chromatin associated with apoptotic cells. The reliability of flow cytometry-based MN scoring was improved when the procedures of Nüsse et al. were modified (41,42) and subsequently configured into a commercially available kit (In Vitro MicroFlowÒ; Litron Laboratories). The most significant of these modifications were (i) incorporation of a fluorescent dye to differentiate MN from chromatin associated with dead and dying cells and (ii) incorporation of concurrent assessments of cell/culture health that identify overly cyotoxic treatment conditions that tend to lead to unreliable MN measurements. The current state-of-the-art utilises a dual dye sequential staining procedure. More specifically, at the time of harvest, intact cells are incubated with ethidium monoazide bromide (EMA). EMA is a nucleic acid that enters the compromised membranes of necrotic and late-stage apoptotic cells (43). A unique characteristic of EMA is that it can be covalently bound to DNA following a photoactivation step. This makes it possible to label dead/dying cells with EMA without loss of signal as cells are further processed. This EMA labelling step is followed by exposure to a detergent-containing lysis solution that includes the nucleic acid dye SYTOXÒ Green, which 148 provides pan-dna labelling. In this manner, the sequential dye protocol results in differential staining of healthy cells chromatin (EMA /SYTOXþ) relative to necrotic and latestage apoptotic cells EMAþ/SYTOXþ profile. By excluding EMAþ chromatin from analysis, flow cytometric scoring of nuclei and MN is accomplished with significantly reduced interference from the presence of dead/dying cells (see Figure 1). This methodology has been shown to provide good agreement between flow- and microscopy-generated data (41,42), although as described below, to achieve adequate assay specificity, it is important to limit the highest concentrations of test article to those that are moderately, not overly, cytotoxic. Because cytotoxicity limits are an important element of mammalian cell culture genetic toxicology assays (44), one important advance to the flow-based methodology was the addition of fluorescent latex microspheres. The use of these socalled counting beads is common in flow cytometry applications that require absolute counts (45). The premise of counting beads is that when they are present at a known consistent density, they can supply information about the volume that has passed through the flow cytometer. Thus, by adding counting beads to the MN assay cultures (for instance to suspension cultures at time of seeding), it becomes possible to determine nuclei-to-bead ratios for each sample, and these values can be used to calculate relative survival. Efficiency remains high since these measurements are acquired concurrently with flow cytometric MN counts. As reported by Bryce et al. (45), the flow-based relative survival measurement often reveals cytotoxicity that alternate methods, including Coulter counter-based relative survival and population doubling times, as well as Annexin propidium iodide staining, often underrepresent. We hypothesise that flow cytometry-based assessment tends to be more sensitive because of the multiparametric nature of these measurements. For instance, to be included in a relative survival calculation, the flow-based analyses require liberated nuclei to exhibit forward and side scatter properties of healthy cells and also to exhibit characteristic EMA and SYTOX fluorescence profiles. By restricting MN scoring to test article concentrations that do not demonstrate excessive cytotoxic profiles based on this nuclei-to-bead ratio approach, assay specificity is improved (42,46). A second endpoint that reflects the health of a culture is the percentage of EMAþ events. As apoptotic cells contribute multiple EMAþ events, whereas necrotic nuclei contribute one, this statistic is particularly sensitive to programmed cell death. In some cell lines, especially those grown in suspension, this measurement can be useful for determining the validity of an assay (e.g. vehicle controls should have a characteristic low percentage of EMAþ events) or to identify test article concentrations that are too cytotoxic for reliable MN enumeration (41,46). Each of the authors has worked with the sequential labelling method described above for at least 3 years. Based on this collective experience, we have found that there are several compelling characteristics of flow cytometric scoring. Increased objectivity Automation is inherently a more objective means of acquiring data relative to visual inspection of slides. Furthermore, strategies for controlling intra- and inter-laboratory variation are readily incorporated when a flow cytometric platform is employed. For instance, in the case of MN scoring, the

Flow cytometric analysis of MNvit Fig. 1. Bivariate plots of nuclei, MN and other subcellular particles from bleomycin (genotoxicant)- or sodium dodecyl sulphate (SDS) (cytotoxicant)-treated TK6 cells as analysed by flow cytometry. Panels (A and D): these plots illustrate that a fraction of chromatin associated with apoptotic and necrotic cells is doublepositive, that is it exhibits both SYTOX-Green and EMA fluorescence. Through the use of software gating logic, it is possible to exclude events that exhibit an EMA-positive staining characteristic. Panels (B and E): these plots were used to score nuclei and MN values, and in these cases, gating logic was not configured to exclude EMA-positive events. By not considering EMA fluorescence characteristics, the cytotoxicant SDS exhibits artificially high MN frequencies. Panels (C and F): these plots were used to score nuclei and MN values, but in these cases, gating logic was configured so that only events that were SYTOX-Green-positive and EMA-negative were considered. By eliminating EMA-positive events from the analysis, the genotoxicant bleomycin but not the cytotoxicant SDS exhibits an elevated micronucleus frequency. standard practice is to adjust photomultiplier tube voltage settings so that G1 nuclei in vehicle control cultures appear in the same fluorescent channels each day analyses are performed. Furthermore, it is also possible to maintain very consistent definitions of the MN scoring region, for instance 1/100th to 1/10th of the G1 nuclei s fluorescence intensity. Number of cells scored As MN are relatively rare events, an accurate determination of their frequency clearly benefits from interrogating more cells per replicate. Based on practical considerations, microscopy and image analysis tend to limit analyses to 1000 2000 cells per replicate, whereas flow cytometry provides the opportunity to readily acquire MN frequency information based on 5000 cells, usually in timeframes of 2 3 min. This potential to increase the number of cells evaluated and thereby enhance statistical power may be especially important for work with weak genotoxicants or even potent MN inducers when one is interested in carefully defining the low end of the dose response curve. Efficiency and throughput Beyond the high rate of data acquisition, there are other features of the MN scoring application that makes conduct of the assay very efficient. For instance, 96-well plate versions of the assay have been described for both suspension and attachment cell lines (47,48). In both cases, all processing steps, from treatment, staining and lysis, occur in the same plate. Furthermore, for those flow cytometers that are equipped with a robotic sampler, it is also possible to acquire data from these same plates, a situation that clearly streamlines the conduct of the entire assay. When attachment cell lines are used, further efficiencies are realised because all washing steps can take place with simple aspirations without the need for centrifugation steps. Taken together, these characteristics can lead to new experimental designs that were not previously feasible with microscopic inspection. For example, a recent report described MNvit experiments that utilised 22 concentrations of test article studied in quadruplicate (49). Experiments of this type can be useful in several contexts, including screening programmes that wish to avoid the time and resources associated with preliminary dose-range finding assays. This type of design may also be valuable when one is interested in carefully defining the low end of a dose response curve, for instance to determine no observable effect levels and lowest observable effect levels. High information content As alluded to previously, flow cytometry provides a rich data set that conveys information about test article cytotoxicity, which is achieved concurrently with MN counts. This information can be extremely valuable when interpreting MN 149

S. Avlasevich et al. results. Thus, while nuclei-to-bead measurements can be readily converted to informative relative survival values, %EMAþ events are sensitive to necrosis/apoptosis, and the SYTOX fluorescence distribution of nuclei can detect test article-induced perturbations to the cell cycle. MOA signatures Related to the high information content nature of flow cytometric measurements, work with certain cell lines, most convincingly with CHO-K1 to date, suggest that clastogenic and aneugenic signatures are evident from flow cytometric data (48,50,51). For instance, while both clastogens and aneugens increase %MN, only aneugens are observed to markedly increase the frequency of hypodiploid nuclei and the median SYTOX fluorescence intensity of MN events (see Figure 2). Compatibility with diverse cell types The dual-staining technique and cell lysis procedures are compatible with several cell lines, a characteristic that provides investigators with a great deal of flexibility. Cell lines that have been studied and found to be compatible to date are L5178Y, TK6, WIL-2-NS, CHO-K1, V79 and HepG2 (41,42,50 52). While the advantageous characteristics of flow cytometrybased MN scoring described above have energised the genetic toxicology community to explore ways to incorporate this technology into their safety assessment work, it is also evident that the method imposes certain limitations. For instance, the use of cytochalasin B in microscopy-based methods restricts analysis to once-divided cells. Visual inspection is also able to detect effects such as nucleoplasmic bridges and nuclear buds, endpoints that are not apparent with flow cytometry. So as microscopy-based scoring continues to develop into a comprehensive cytome assay (12), progress with flow cytometric analysis of in vitro MN also continues, presenting considerable opportunities/challenges of its own. The authors perspectives on the future of MN scoring by flow cytometry are provided below. Knowledge gaps and recommendations for future research Flow cytometric analysis offers an opportunity for efficient MN scoring, as well as higher information content. That is, beyond the capacity to survey large numbers of cells, the multiparametric platform permits one to simultaneously collect cytotoxicity and cell cycle data, as well as clues as to the genotoxic MOA. Indeed, the promise of an automated mammalian cell assay that provides quantitative data and MOA information may allow the MNvit test to move from a simple hazard identification screen to one that may also supply weight of evidence information that is useful for risk assessment purposes. While enthusiasm for such a promising technology is warranted, it needs to be tempered by the fact that the latest protocols for flow cytometry analysis are still relatively new. Whereas it has been a relatively straight-forward process to compare automated MN scoring against microscopic counts, for other evaluations such as aneugenic versus clastogenic MOA signatures, validation strategies will be more complex. Assays for aneugenicity or non-disjunction events using antikinetochore probes or FISH have well-established historical data and criteria for judging a chemical aneugenic. The current flow cytometric state-of-the-art provides some characteristic MOA signatures, but to date, this has really only been demonstrated in two cell lines, CHO-K1 and V79 (47,49,50). Indeed, there is some evidence that these signatures are not as strong or specific in several commonly used cell lines L5178Y and TK6. More work is necessary to establish a better understanding of the cell lines that are capable of providing aneugenic versus clastogenic signatures, and there is a need to more definitively formulate quantitative criteria that are useful for making these MOA determinations. As described previously, a major challenge of flow cytometric systems has been the detection of bona fide MN resulting from a genotoxic effect as opposed to DNA fragments produced during apoptosis. While EMA labelling segregates out most DNA originating from dying cells, it is not effective at labelling those chromatin fragments generated before plasma membrane integrity has become compromised. For example, when Collins et al. (53) studied the cytotoxicant dexamethasone, they reported increased MN counts by flow cytometry that were not observed by microscopy, even though EMA was used to exclude chromatin from dead/dying cells. Only later when it became clear that induction of EMA-positive events and also nuclei-to-bead ratio measurements should be used to Fig. 2. Bivariate plots of nuclei, MN and other subcellular particles from bleomycin (clastogen)- or griseofulvin (aneugen)-treated CHO cells analysed by flow cytometry. This cell line exhibits flow cytometric profiles ( signatures ) that provide insights into genotoxic mode of action. Panel (A): solvent control, with a low (baseline) MN frequency. Panel (B): clastogenic signature, where a significant increase in MN frequency is not accompanied by marked induction of hypodiploid nuclei or an upward shift in MN fluorescence intensity (see histogram, insert, where counts are plotted versus SYTOX-Green fluorescence). Panel (C): aneugen signature, where a significant increase in MN frequency is accompanied by marked induction of hypodiploid nuclei and an upward shift in MN fluorescence intensity (see histogram, insert, where counts are plotted versus SYTOX-Green fluorescence). 150

set the top analysable concentration of test article was agreement between microscopy and flow cytometry observed (46). The message from work with this cytotoxicant and others is clear although EMA labelling markedly diminishes the influence that dead cells have on MN counts, it is not absolute. Rather, for reliable analyses and interpretation of flow cytometry-based data, a more holistic approach of evaluating test article-induced cytotoxicity is required. Gaining deeper insights into test article-induced toxicities can only benefit interpretation of cytogenetic endpoints such as MN. Thus, beyond scoring %EMA-positive events and performing sensitive flow-based relative survival measurements, a potentially productive line of investigation will be the evaluation of other indices of cytotoxicity that can be multiplexed onto flow cytometry-based MN scoring protocols. For instance, experiments are currently underway that are assessing the merits of collecting pretreatment and posttreatment cell-to-bead ratios (that is healthy cells based on light scatter properties prior to lysis/staining steps). Unlike nuclei-to-bead ratios at the time of harvest, these pretreatment and post-treatment cell-to-bead measurements can be used to calculate relative increased cell counts and population doublings. These cytotoxicity indices are considered useful because they are more responsive to cell death and cytostasis as compared to relative survival measurements (54). Other endpoints of cytotoxicity made in parallel or concurrently with MN scoring should be investigated for their ability to identify treatment conditions that lead to spurious MN counts or bona fide MN that are the result of secondary effect(s) stemming from excessive toxicity. Fluorescence-based assessment of mitochondrial membrane potential is one example. While the EMA/SYTOX sequential staining strategy works well with several mammalian cell lines, its compatibility with primary cells is less clear. Microscopically scored MN assays can include cytochalasin B to ensure interrogated cells have gone through one mitosis. This especially benefits assays that utilise primary cells such as human lymphocytes since only a relatively minor portion of the cell population undergoes division upon mitogen stimulation. Since the flow cytometric assay employs a lysing technique that does not distinguish between once-divided and quiescent cells, one would expect assay sensitivity to be negatively affected. To relate to this concern, two lines of investigation should be pursued as follows: (i) addition of an additional fluorescent reagent that labels chromatin from once-divided cells that is compatible with EMA and SYTOX Green and (ii) the use of parallel cultures that could be used to derive a cell division statistic. In either case, for instance, as described by Schreiber et al. (55), one can use this information to normalise flow-based MN counts to the proportion of cells that divided in culture. Success with either approach could have important implications, as it may allow one to extend the so-called in vitro MN scoring approach to human biomonitoring applications that are directed at assessing in vivo exposure situations. Finally, the bulk of work with flow cytometric MNvit assays has been in the areas of assay development, basic research and early hazard identification screening. With recent acceptance of OECD guidelines for the MNvit, there will be a natural desire to transition flow-based MN analyses from a basic research tool to one that can support the registration of new chemicals. This effort would obviously benefit from additional studies that further characterise sensitivity and specificity of flow-based approaches, as well as assessments of inter- and intralaboratory reproducibility. Data that addresses these concerns have been published with encouraging results (46,50), but wide adoption as a regulatory assay will require further evaluations with a broader variety of chemicals. Once assay validity, significant MN responses, preferred cytotoxicity assessments and MOA information are more clearly defined, we believe the assay could be used in a number of areas, including the regulatory environment. As with other assays being developed for broad dissemination and regulatory use, especially those that are endowed with high information content, a task for the developers and early adopters of the system will include education. This phase of work, in conjunction with continued assay validation efforts, is important so that the wealth of data generated by flow-based MN assays is interpreted appropriately. Acknowledgements The authors thank Michael Fenech for providing the opportunity to contribute to this special issue of Mutagenesis. 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