Micronucleus Assay 1 Executive Summary of

3.0 In Vitro Micronucleus Assay
3.1 Executive Summary of In Vitro Comet Assay Features

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  Cell-based genotoxicity assay using Chinese Hamster Ovary (CHO) cell line.
  

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  The cell line has a rodent origin and has been widely used in genetic toxicology tests such as for chromosomal aberrations and mutations.
  

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  The Micronucleus assay demonstrated high concordance with the chromosomal aberrations, but can be executed more rapidly and requires less expertise.
  

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  Flow cytometric analysis enables automation of the scoring process and delivers superb sensitivity and throughput.
  

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  Microplate protocol testing up to 36 compounds per 96 well plate in duplicate.
  

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  Testing with or without exogenous metabolic activation using Aroclor 1254-induced, rat liver S9 fraction as a metabolic activation system.
  

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  Intra-microplate standard genotoxic (mitomycin C) and pro-genotoxic (cyclophosphamide) positive control compounds provide assay acceptability data.
  

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  Multiple cellular parameters -- cell cycle, relative survival, membrane integrity—measured currently with frequency of micronuclei
  

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  Detects mode of action (MOA) of test article: clastogen vs. aneugen.
  

3.2 Summary of In Vitro Micronucleus Assay

The in vitro micronucleus assay measures DNA damage by detecting extra-nuclear DNA fragments or entire chromosome following cell division (Figure 3.1). DNA fragments are associated with clastogenic mechanisms of action. Entire chromosomes lost during cell division are associated with aneugenic mechanisms of actions. Both endpoints identify potential human health hazard via DNA damaging mechanisms. Clastogenic mechanisms of action may act by direct interaction with DNA without thresholds. Aneugenic mechanisms are often attributed to threshold-based mechanisms of action and may have less impact on human health. For assays that measure micronuclei induction, the ability to also identify mechanism of action (clastogenic vs aneugenic) becomes important when making human risk evaluations. This type of nuanced genotoxicity data is also important when populating computational toxicology databases to identify alerting structures.
Standard microscopic scoring of micronucleus assays can differentiate clastogenic from aneugenic mechanisms of action. This can be achieved through the use of additional differential staining techniques that stain micronuclei for kinetochore proteins found only in the centromeres of intact chromosomes and missing in chromosome fragments. (Gudi, et al., 1992). Improved scoring methods using flow cytometric methods have been developed for scoring both in vitro and in vivo micronucleus assays.


Figure 3.1 Cell showing two nuclei in one cell with micronuclei

The in vitro micronucleus assay demonstrates high concordance with chromosome aberration analyses, but it is executed more rapidly and requires less technical expertise [Matsuoka et al., 1993; Miller et al., 1997]. These characteristics have led to its widespread use as an efficient and relatively simple method to screen drug candidates and other test articles for clastogenic and aneugenic potential. Draft OECD guidelines (487) have been written for the in vitro micronucleus assay. BioReliance has recently contributed in vitro micronucleus assay data using microscopic scoring methods in 2009 to Dr Mike Cimino, US EPA to help guide US approval of the draft guideline. Furthermore, it appears that the in vitro micronucleus assay will be added as one of the in vitro mammalian cell assays recommended by the next revision of the ICH S2 preclinical genotoxicity guideline. This guideline, titled “Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human” (FDA 2009) is in the ICH maintenance process and may possibly reach Step 4 of the ICH process at the next ICH meeting June 2009 in Yokohama, Japan

3.3 In Vitro Flow Cytometric Micronucleus Assay
Numerous efforts to automate the scoring phase of the assay have been described in the literature—methods based on image analysis, laser scanning cytometry, and flow cytometry have all been reported [Vral et al., 1994; Verhaegen et al., 1994; Bocker et al., 1995; Wessels and Nüsse, 1995; Viaggi et al., 1995; Roman et al., 1998; Diaz et al., 2007]. Most data have been reported for variations on the flow cytometric procedure described by Nüsse and colleagues [Nüsse and Kramer, 1984; Schreiber et al., 1992]. A commercially available product called the In Vitro MicroFlow™ kit has recently become available from Litron Laboratories, Rochester, NY. This kit is also based on a modification to the Nüsse method [Avlasevich et al., 2006; Bryce et al., 2007; Bryce et al., 2008].
In the past, the major limitation of flow cytometry-based techniques was their inability to distinguish true MN from apoptotic bodies [Nüsse and Marx, 1997]. Thus, an important goal of In Vitro MicroFlow^® kit development was differential staining of MN from the chromatin of dead and dying cells. The strategy that was developed to address this issue is labeling of necrotic and mid/late stage apoptotic cells with the fluorescent dye ethidium monoazide (EMA). After covalent binding of EMA to DNA via photoactivation, cells are washed, stripped of their cytoplasmic membranes, and incubated with RNase plus a pan-nucleic acid dye. This process provides a suspension of free nuclei and micronuclei that are differentially stained relative to chromatin associated with dead/dying cells (see Figure 3.2). By excluding EMA-positive events from analysis, more reliable MN measurements are acquired. Using this strategy, reliable data have been obtained in multiple cell lines.

Figure 3.2. Schematic showing the differential staining approach used for flow cytometric scoring of in vitro micronuclei.
Beyond providing rapid micronucleus frequency measurements (approximately 1 min per specimen), the flow cytometric analysis simultaneously acquires cell cycle information as well as two indices of cytotoxicity, relative survival and membrane integrity:
Cell Cycle Information: Test article-induced perturbations to the cell cycle are apparent by studying histograms of SYTOX Green fluorescence. For instance, expected G2/M blocks following treatment with alkylating agents are readily observed.
Relative Survival: 6 micron microspheres are added at either of several stages of the cell processing steps. By analyzing these “Counting Beads” on the flow cytometer, the number of healthy nuclei (i.e., EMA-negative) per bead can be determined. From these Nuclei-to-Bead ratios, relative survival values can be calculated. The advantage of these flow cytometry-derived relative survival measurements is that they represent a multi-parametric means of evaluating cell health. Indeed, this method has been found to reveal cytotoxicity that other cell scoring methods, such as Coulter counts, tend to underestimate.
Membrane Integrity: The health of treated cells can be inferred from the percentage of particles that are EMA-positive. Since each fragmented nucleus of apoptotic cells can form many such particles, this statistic is particularly sensitive to test article-induced apoptosis.
This comprehensive assessment of test article-induced toxicity has been found to be a valuable aid in micronucleus data interpretation, including the identification of test article concentrations that are too cytotoxic for reliable micronucleus readings [Bryce et al., 2008]. See Figure 3.2.

Figure 3.3. The high information content supplied by flow cytometric analyses is illustrated in these plots/graphs.
Figure 3.1 Left: Dexamethasone, a cytotoxic non-genotoxicant. MN frequency is low until excessively cytotoxic concentrations are reached. Excessive cytotoxicity for the top concentration is exhibited in two ways: low relative survival, and large increase in the incidence of EMA-positive events. At the top passing concentration, cell cycle is not perturbed.
Figure 3.2 Right: Vinblastine, a prototypical aneugen. MN frequency is increased, even at low non-cytotoxic concentrations. Excessive cytotoxicity for the top concentration is exhibited in two ways: low relative survival value, and large increase in EMA-positive events. At the top passing concentration, cell cycle is perturbed, as evidence by an accumulation of G2/M cells (grey = solvent control, white line = vinblastine).
3.4 Aneugenic Signature in the In Vitro Flow Cytometric Micronucleus Assay
In regards to CHO-K1 cells, other advantage characteristics of the methodology are realized. As an attachment cell line, all processing steps, including washing steps, occur without the need for centrifugation. This greatly improves the efficiency by which plates are processed. Additionally, owing to the large average size of CHO chromosomes, the assay is endowed with an ability to discriminate aneugenic from clastogenic modes of action. This “aneugenic signature” is apparent through the measurement of two endpoints. Firstly, aneugen-induced MN contains on average a greater DNA content than clastogen-induced MN. This assessment can be made concurrently with MN scoring on the flow cytometer by considering the SYTOX median fluorescent channel associated with these events. Secondly, a hallmark feature of aneugenic activity is non-disjunction. When chromosomes are not equally distributed to daughter nuclei, hyopdioploid nuclei are formed which can be detected and measured by the flow cytometer. As with median fluorescence channel information, this measurement is also captured simultaneously with MN scoring. Figure 3.3 illustrates this powerful capacity of flow cytometric micronucleus assay to discriminate aneugenic from clastogenic modes of action.

Figure 3.4a. “Clastogenic signature” of micronucleus flow cytometric data with a prototypical clastogen, mitomycin C (MMC) in CHO-K1 cells.

Figure 3.4b. “Aneugenic signature” of micronucleus flow cytometric data with a prototypical aneugen, Vinblastine in CHO-K1 cells.

While micronuclei are observed to increase in a concentration dependent manner with MMC, neither the micronucleus median channel fluorescence nor Hypodiploid nuclei increase appreciably. In the case of an aneugen, vinblastine, micronuclei are observed to increase in a concentration dependent manner as was seen with a classic clastogen. However, in the case with vinblastine, both the micronuclei median channel fluorescence and Hypodiploid nuclei are found to increase significantly.
Using the high throughput, miniaturized version of the assay, the above noted aneugenic signature has been verified with vinblastine, colchicine, carbendazim, and griseofulvine.
3.5 Assay Endpoints

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  Micronucleus(MN) frequency: this will be presented as a ratio of free MN vs. 5000 healthy nuclei collected per sample
  

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  Relative survival: nuclei vs beads ratio representing the relative number of viable cells in treated samples compared to the concurrent solvent control
  

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  % Hypodiploid: as a measurement for aneugenicity
  

3.6 High Throughput Modification of In Vitro Flow Micronucleus Assay

With the benefit of NIH-NCI funding, Litron scientists have miniaturized this basic methodology to 96 well plate format, thereby reducing test article requirements, and allowing for more automation through the use of liquid handlers. Importantly, it is possible to treat, stain, and analyze cells in the same 96 well plate, thereby greatly improving assay efficiency and throughput.
The assay protocols are described in greater detail in the assay SOPs. In summary:

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  A suspension of 1x10⁵ cells per well in the culture medium are plated into each 96-well flat-bottom plate 16-24 hours prior to treatment to allow cell attachment and incubated at 37ºC, 5% CO₂ and ≥85% humidity.
  

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  Test articles are prepared at the desired test concentration in a solution in dimethylsulfoxide (DMSO).
  

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  Test articles are diluted in medium 1:100 (v/v) to make the highest concentration. A dilution series of the test articles are made on a separate deep-well 96 well microplate in duplicate.
  

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  The culture medium is aspirated from the 96-well microplate where cells were seeded and the assay medium containing test articles are transferred into corresponding wells.
  

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  2 concentrations of a standard genotoxicant, mitomycin C and cyclophosphomide are tested in parallel as positive controls for without and with activation systems, respectively.
  

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  Replicate vehicle controls (1% DMSO) are included.
  

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  In the non-activated system, microplate are incubated continuously for 24 hours, at 37ºC, 5% CO₂ and ≥85% humidity. In the activated system, the cells are treated for 3-4 hours, washed and recovered in test article-free fresh medium for 20-21 hours at 37ºC, 5% CO₂ and ≥85% humidity.
  

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  The microplate is subject to staining procedures for flow cytometric analysis.
  

3.7 Test Compound Presentation

Compounds should be prepared for the assay in 100% DMSO. The stock solutions are diluted 1:100 to achieve the treatment doses. This is due to the 1% limit of tolerance of DMSO in the cellular assay. This limits the upper test concentration depending on the format of the supplied compound. For testing in Phase 1 of the EPA NCCT ToxCast program compounds were supplied as 20 mM solutions in DMSO. This would enable testing up to 200 µM in the in-vitro Micronucleus assay.
3.8 Technology Transfer and Assay Validation
BioReliance is one of the first companies to adapt the flow cytometric in vitro micronucleus technology from Litron Laboratories. The initial validation was conducted to test the 10 well-characterized chemicals in the Annex 3 of the Draft OECD Guidance Document 487 (Bryce, Shi, etc., in preparation). The work has been presented or accepted for presentation in the following international conferences:
Bryce, S., Shi, J.,Phonethepswath, S., Avlasevich1, S., Raja, S., Dertinger, S. “High content flow cytometry-based micronucleus scoring method is applicable to CHO-K1 cells” The 39th Environmental Mutagen Society Annual Meeting, 2008
Bryce, S., Shi, J.,Nicolette, J., Diehl, M., Sonder, P.,Phonethepswath, S., Avlasevich1, S., Raja, S., Bemis, J. and Dertinger, S. “High content flow cytometry-based micronucleus scoring method is applicable to attachment cell lines” The Society of Toxicology Annual Meeting, Baltimore, MD, Mar. 2009 (accepted)
Additionally, a total of 21 genotoxic or non-genotoxic chemicals were tested in CHO cells at BioReliance to thoroughly test the assay. Since it has been a concern that DNA fragments derived from the apoptotic process may interfere the results in the flow cytometric Micronucleus assay, a number of non-genotoxic apoptosis inducers were tested and satisfying results were observed in CHO cells. Moreover, a sensitivity of 83% and a specificity of 100% were observed (Shi, etc., in preparation). Therefore, this assay has been fully validated in CHO cells at BioReliance.