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. Author manuscript; available in PMC: 2023 Apr 22.
Published in final edited form as: Environ Mol Mutagen. 2022 Apr 22;63(3):151–161. doi: 10.1002/em.22480

In vitro Human Cell-Based Aneugen Molecular Mechanism Assay

Nikki E Hall 1,*, Kyle Tichenor 1,*, Steven M Bryce 1, Jeffrey C Bemis 1, Stephen D Dertinger 1
PMCID: PMC9106857  NIHMSID: NIHMS1798652  PMID: 35426156

Abstract

This laboratory previously described an in vitro human cell-based assay and data analysis scheme that discriminates common molecular targets responsible for chemical-induced in vitro aneugenicity: tubulin destabilization, tubulin stabilization, and inhibition of Aurora kinases [Bernacki et al., Toxicol. Sci. 170 (2019) 382–393]. The current report describes updated procedures that simplify benchtop processing and data analysis methods. For these experiments, human lymphoblastoid TK6 cells were exposed to each of 25 aneugens over a range of concentrations in the presence of fluorescent paclitaxel (488 Taxol). After a 4 hr treatment period, cells were lysed and nuclei were stained with a nucleic acid dye and labeled with fluorescent antibodies against phospho-histone H3 (p-H3). Flow cytometric analyses revealed several unique signatures: tubulin stabilizers caused increased frequencies of p-H3-positive events with concentration-dependent increases in 488 Taxol-associated fluorescence; tubulin destabilizers caused increased frequencies of p-H3-positive events with concomitant decreases in 488 Taxol-associated fluorescence; and Aurora kinase B inhibitors caused reduced frequencies of p-H3-positive events and lower median fluorescent intensities of p-H3-positive events. These results demonstrate a simple rubric based on 488 Taxol- and p-H3-associated metrics can reliably discriminate between several commonly encountered aneugenic molecular mechanisms.

Keywords: TK6 cells, flow cytometry, aneugen, microtubules, aurora kinase inhibitors

INTRODUCTION

Due to the consequential health effects associated with aneugenicity, safety assessment programs routinely investigate chemicals’ potential to cause chromosome malsegregation [ICH, 2011; Tweats et al., 2019]. The most commonly used approaches are based on micronucleus formation—both in mammalian cell culture and in laboratory rodent models. However, it is important to recognize that micronucleus formation is responsive to clastogenesis as well an aneugenicity. Therefore, unlesss CREST antibodies or FISH probes are used [Eastmond and Tucker, 1989; Kirsch-Volders et al., 2003], micronucleus formation does not provide genotoxic mode of action (MoA) information.

It is not suprising then that many genotoxicity assays have been developed to deliver greater insights into MoA, especially in regard to clatogenicity versus aneugenicity. Examples include tubulin polymerization assays [Mirigian et al., 2013], a combination γH2AX/phospho-histone H3 assay [Khoury et al., 2016], the MultiFlow® DNA Damage Assay [Bryce et al., 2016, 2017, 2018], ToxTracker-ACE [Brandsma et al., 2020], MEGA-Screen [Wilson et al., 2021], iScreen [Sun et al., 2021], and TubulinTracker [Geijer et al., 2022].

This laboratory previously described a follow-up test to the MultiFlow DNA Damage Assay that elucides common molecular mechanisms responsible for aneugenicity [Bernacki et al., 2019]. This so-called MultiFlow Aneugen Molecular Mechanism (AMM) Assay utilized fluorescent taxol (488 Taxol) to provide information about tubulin polymerization dynamics. Two fluorescent antibodies were used in this test system—anti-Ki-67 and anti-p-H3. Anti-Ki-67 represented a pan-mitotic cell signal, and anti-p-H3 measured the fraction of mitotic cells that exhibited the p-H3 epitope. Since the phosphorylation of histone H3 at Ser10 is dependent on Aurora kinase B activity [de Groot et al., 2015], the ratio of p-H3-positive events to Ki-67-positive events represented a useful reporter of Aurora kinase B inhibition. In addition to treating cells with a suspected aneugen, a low concentration of non-fluorescent taxol was used to increase the number of mitotic cells available for analysis.

While the previous incarnation of the AMM Assay showed promise, over time, two issues become apparent. First, we found that the degree to which the Ki-67 antibody was able to differentiate mitotic and non-mitotic chromatin was too variable. Second, each lot of non-fluorescent taxol required precise re-titering in order to increase the fraction of mitotic cells without obscuring effects induced by the suspected aneugenic test article. To simplify the assay, both cell treatment and the labeling strategy were revised. While the new method shares many features of the original assay, it omits the two reagents that proved problematic.

This report describes experiments based on the simplified AMM Assay. As described below, we tested the assay with TK6 cells exposed to each of 25 reference aneugens over a range of concentrations. The treatments occurred in the presence of 488 Taxol (without non-fluorescent taxol), and the labeling protocol no longer included Ki-67 antibody. In addition to the streamlined cell processing procedure, we also describe a simplified data analysis approach.

MATERIALS AND METHODS

Chemicals, Cells, Culture Conditions

The identity and source of 25 reference chemicals, as well as the molecular targets said to explain their aneugenicity, are provided in Table I. The majority of these chemicals interfere with tubulin polymerization or inhibit Aurora kinase B. Note that from this point on, we use AKB(+) to abbreviate Aurora kinase B inhibitors, with the plus sign to express the idea that most of these agents affect Aurora B in a promiscuous, off-target manner, and generally inhibit more than one Aurora kinase, not just the B isoform. That being said, we did include two agents that very specifically inhibit the B or A isoforms (Barasertib and Aurora A Inhibitor I, respectively; see Table I).

Table I.

Aneugens and Presumed Molecular Target.

Chemical CAS No. Source Presumed Aneugenic Molecular Target Notes; References
Carbendazim 2068-78-2 Millipore Sigma Tubulin destablizer Yenjeria et al., 2009
Colchicine 1120-71-4 Millipore Sigma Tubulin destablizer Kirkland et al., 2016
Diethylstilbestrol 56-57-5 Millipore Sigma Tubulin destablizer Parry et al., 2002
Flubendazole 51-21-8 Millipore Sigma Tubulin destablizer Tweats et al., 2016
Griseofulvin 38966-21-1 Millipore Sigma Tubulin destablizer Oliver et al., 2006
Mebendazole 446-86-6 Millipore Sigma Tubulin destablizer Van Hummelen et al., 1995
Nocodazole 30516-87-1 Millipore Sigma Tubulin destablizer Verdoodt et al., 1999
Noscapine 9041-93-4 Millipore Sigma Tubulin destablizer Schuler et al., 1999
Rigosertib 15663-27-1 Selleckchem Tubulin destablizer Tubulin destabilization attributed to an impurity found in commercial products, not the clinical-grade drug; Baker et al., 2020
Vinblastine sulfate 7689-03-4 Millipore Sigma Tubulin destablizer Kirkland et al., 2016
Vincristine sulfate 305-03-3 Millipore Sigma Tubulin destablizer Kirkland et al., 2016
17β-Estradiol 143-67-9 Millipore Sigma Tubulin destablizer Hernández et al., 2013
Epothilone A 147-94-4 Selleckchem Tubulin stablizer Bollag et al., 1995
Ixabepilone 23214-92-8 Selleckchem Tubulin stablizer Lee et al., 2001
Paclitaxel 518-82-1 Millipore Sigma Tubulin stablizer Kirkland et al., 2016
Alisertib 50-28-2 Selleckchem Aurora kinase(s) Aurora kinase A > B; Sehdev et al., 2012
AMG-900 877399-52-5 Selleckchem Aurora kinase(s) Payton et al., 2010
Barasertib 56-53-1 Selleckchem Aurora kinase B Highly specific for Aurora kinase B; Yang et al., 2007
Crizotinib 31430-15-6 Selleckchem Aurora kinase(s) c-Met, ALK, however Aurora kinase B implicated; Zou et al., 2007; Kong et al., 2018
Danusertib 126-07-8 Selleckchem Aurora kinase(s) Potent activity against Auora kinases with cross-reactivity against some cancer-relevant tyrosine kinases; Carpinelli et al., 2007
Hesperadin 31431-39-7 Selleckchem Aurora kinase(s) Hauf et al., 2003
PF-03814735 31430-18-9 Selleckchem Aurora kinase(s) Jani et al., 2010
VX-680 (Tozasertib) 128-62-1 Selleckchem Aurora kinase(s) Harrington et al., 2004
ZM447439 33069-62-4 Selleckchem Aurora kinase(s) Ditchfield et al., 2003
Aurora A Inhibitor I 945595-80-2 Selleckchem Aurora kinase A Highly specific for Aurora kinase A; Aliagas-Martin et al., 2009
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TK6 cells were purchased from ATCC® (cat. no. CRL-8015). Cells were grown in a humidified atmosphere at 37°C with 5% CO2, and maintained at or below 1 × 106 cells/mL. The culture medium consisted of RPMI 1640 with 200 μg/mL sodium pyruvate (both from Sigma-Aldrich, St. Louis, MO), 200 μM L-glutamine, 50 units/mL penicillin and 50 μg/mL streptomycin (from Mediatech Inc., Manassas, VA), and 10% v/v heat-inactivated horse serum (Gibco®, a Thermo Fisher Scientific Company, Waltham, MA).

Cell Treatments

Prototype MultiFlow Aneugen Molecular Mechanism Kit (Litron Laboratories, Rochester, NY) supplied 488 Taxol was added to TK6 cells for a final concentration of 0.5 μM. The cells were seeded into wells of a U-bottom 96 well plate (6 × 105/mL; 198 μL/well). Test chemicals were then added over a range of concentrations in a volume of 2 μL. The top concentrations were the same as the highest acceptable concentration from the initial MultiFlow—DNA Damage Assay reported previously [Bernacki et al., 2019]. A square root 2 dilution scheme was used, meaning each successively lower concentration was 70.71% of the preceding one. At least four solvent control (DMSO) wells were included on each 96 well plate. For these experiments, cells were exposed to test chemical in a humid incubator for 4 hr, typically at 20 concentrations in single wells.

Sample Processing

After 4 hr of co-treatment (488 Taxol plus test article), cells were resuspended with pipetting, and 35 μL from each well were added to a new 96-well plate containing 70 μL/well of pre-aliquoted lysis/labeling solution from a prototype MultiFlow Aneugen Molecular Mechanism Kit. The proprietary lysis/labeling solution was used to simultaneously digest cytoplasmic membranes, stain chromatin with a fluorescent nucleic acid dye, and label the p-H3 epitopes with a fluorescent antibody (anti-phospho-histone H3-PE). RNase was used to digest RNA and propidium iodide was used to stain chromatin, and a known concentration of latex microspheres (SPHERO™ Multiple Fluorophore Fluorescent Particles, cat. no. FP-3057-2; Spherotech, Inc., Lake Forest, IL) provided a means to calculate nuclei density. One non-kit-supplied reagent was added to the working lysis/labeling solution immediately before use—PhosSTOP™ (cat. no. 4906845001, MilliporeSigma, Burlington, MA). We found that inhibiting phosphatase activities greatly improved the stability of p-H3-associated fluorescence in the p-H3-positive cell population over time. PhosSTOP was incorporated by solubilizing one tablet in 1 mL phosphate buffered saline, and adding 154 μL of this stock solution to 7.93 mL lysis/labeling solution. Note that the remainder of the PhosSTOP solution was stored frozen for later use; aliquots limited to one freeze-thaw cycle. Cells and lysis/labeling solution were allowed to incubated at ambient temperature for 30 min in the dark. At that point, samples were analyzed via flow cytometry as described below.

Flow Cytometric Analysis

Flow cytometric analysis was performed using Miltenyi Biotec MACSQuant® Analyzer 10 flow cytometers with integrated 96-well MiniSampler devices. Kit-supplied SPHERO™ Multiple Fluorophore Fluorescent Particles were used to set and monitor PMT voltages, and electronic compensation was used to eliminate fluorescence spillover. The user-defined mixing and fluidics parameters for the MACSQuant were as follows: 80 μL of sample were mixed, and then 30 μL were analyzed at a flow rate of 50 μL/min until the 30 μL volume was exhausted. Note that each well was sampled/analyzed twice to provide two pseudo-replicates. Flow cytometry data were analyzed using FlowJo software v10.5.0.

p-H3-positive events were defined by propidium iodide-associated fluorescence (4n and greater DNA content), and their high PE signals. The frequency of positive events was expressed as a percentage relative to total events that exhibited 2n and greater DNA content. For all graphical representations and statistical analyses presented herein the p-H3 data were first converted to fold-change relative to a plate-specific solvent control arithmetic mean value. The median fluorescence channel of p-H3-positive events were also recorded. For all graphical representation and statistical analyses these values were first converted to fold-change relative to a plate-specific solvent control arithmetic mean.

The 488 Taxol biomarker was based on median channel fluorescence, and for all graphical representation and statistical analyses these values were first converted to fold-change relative to a plate-specific solvent control arithmetic mean. Gating logic required these events to exhibit propidium iodide-associated fluorescence corresponding to 2n - 4n DNA content. p-H3-positive events were excluded from the median channel fluorescence calculation.

Data Analysis

The reference chemicals were organized into four dichotomous groups: Tubulin binders vs. Non-tubulin binders; Tubulin stabilizers vs. Non-tubulin stabilizers; Tubulin destabilizers vs. Non-tubulin destabilizers; and AKB(+) vs. Non- AKB(+). In order to generate cutoff values that discriminate between each dichotomous group, aggregate fold-change response data for each chemical were applied to JMP software’s partition platform (JMP v12.0.1, SAS Institute, Cary, NC). The partitional platform considered the following biomarkers: %p-H3-positive events, p-H3-median fluorescence, and Taxol 488-median fluorescence. The resulting cutoff values are shown below, and are expressed as fold-change compared to concurrent mean solvent control values:

  • Tubulin binders vs. Non-tubulin binders: increase in %p-H3-positive events ≥ 2.56-fold;

  • Tubulin stabilizers vs. non-tubulin stabilizers: increase in 488 Taxol median fluorescence ≥ 1.44-fold;

  • Tubulin destabilizers vs. non-tubulin destabilizers: decrease in 488 Taxol median fluorescence < 0.86-fold;

  • AKB(+) inhibitors vs Non-AKB(+) inhibitors: decrease in %p-H3-positive events < 0.68-fold; and

  • AKB(+) inhibitors vs Non- AKB(+) AKB(+) inhibitors: decrease in p-H3 median fluorescence < 0.38-fold.

These cutoff values were used in a rubric to group chemicals according to their most likely aneugenic mechanism. Figure 1 illustrates our a priori expectations for several aneugenic mechanisms and associated phenotypes.

Figure 1.

Figure 1.

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Several mechanistic classes of aneugens are described, along with expected phenotypes. Green and red arrows signify elevations and decreases, respectively. Grey arrows indicate no substantial changes are expected.

In addition to the decision tree-like rubric described above, we also evaluated the biomarker response data using ToxPi software [Reif et al., 2010; Marvel et al., 2018]. For these analyses, an Excel file with 25 rows was created, each row corresponding to a different reference test chemical. One column contained chemical name, and three others recorded the largest mean fold-change value observed relative to mean solvent control value. These nadir or zenith fold-change values were entered for three biomarkers: %p-H3 positive events, p-H3 median associated fluorescence, and 488 Taxol median fluorescence. The Excel spreadsheet was converted to a csv file, and analyzed with ToxPi software, v2.3. A model comprised of three slices was created, each corresponding to the three biomarkers described above. The fold-change values were transformed (−log10), and the resulting ToxPi profiles were grouped using the program’s hierarchical clustering function (clustering options: complete; circular format).

RESULTS AND DISCUSSION

Tubulin destabilizers

Twelve tubulin-binding destabilizers were evaluated in the AMM assay at concentrations that showed evidence of aneugenic activity in the standard MultiFlow DNA Damage Assay [Bernacki et al., 2019]. As shown by Figure 2, these agents all induced remarkable increases in the percentage of p-H3-positive events, generally in excess of 4-fold. This signature was shared by tubulin stabilizers. Conversely, the AKB(+) and Aurora kinase A inhibitors did not increase %p-H3-positive events, or as was the case for Alisertib, did so to a lower extent (maximum increase: 2.54-fold).

Figure 2.

Figure 2.

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Percentage of phospho-histone H3-positive events (expressed as fold change relative to mean solvent control) is graphed against test chemical μM concentration (log10 transformed; solvent/zero concentration arbitrarily set to −6 log10). Note that these are aggregate data for all 25 test chemicals described in Table I, and they are split into the dichotomous groups: Non-tubulin binder (left) and Tubulin binder (right). Additional subgrouping is provided by the color-coded key, inset. The horizontal dashed line (2.56-fold) represents a useful cutoff value for distinguishing between the two groups: Non-tubulin binder versus Tubulin binder.

As a class, tubulin-binding stabilizers did not greatly affect p-H3-associated fluorescence in the p-H3 positive cell population (Figure 3).

Figure 3.

Figure 3.

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Median phospho-histone H3-associated fluorescence in the p-H3 positive cell population (expressed as fold change relative to mean solvent control) is graphed against test chemical μM concentration (log10 transformed; solvent/zero concentration arbitrarily set to −6 log10). Note that these are aggregate data for all 25 test chemicals described in Table I, and they are split into the dichotomous groups: Aurora kinase B(+) inhibitors (left) and Non-Aurora kinase B(+) inhibitors (right). Additional subgrouping is provided by the color-coded key, inset. The horizontal dashed line (0.38-fold) represents a useful cutoff value for distinguishing between the two groups: Aurora kinase B(+) versus Non-Aurora kinase B(+).

488 Taxol-associated fluorescence was reduced by each of the destabilizers (see Figure 4). This characteristic was specific to this class of tubulin binder, whereas stabilizers and kinase inhibitors increased these values, dramatically in the case of the former.

Figure 4.

Figure 4.

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Median 488 Taxol-associated fluorescence (expressed as fold change relative to mean solvent control) is graphed against test chemical μM concentration (log10 transformed; solvent/zero concentration arbitrarily set to −6 log10). Note that these are aggregate data for all 25 test chemicals described in Table I, and they are split into the dichotomous groups: Tubulin destabilizers (left) and Non-tubulin destabilizers (right). Additional subgrouping is provided by the color-coded key, inset. The horizontal dashed line (0.86-fold) represents a useful cutoff value for distinguishing between the two groups: Tubulin destabilizers versus Non-tubulin destabilizers.

A summary profile is provided in Table II. This phenotypic signature is consistent with our a priori expectations (Figure 1).

Table II.

Summary Results.

Chemical Aneugenic Mechanism Biomarker
Increase in % p-H3 (≥ 2.56-fold) Decrease in % p-H3 (< 0.68-fold) Decrease in p-H3 Median Channel Fluourescence (< 0.38-fold) Increase in 488 Taxol Median Channel Fluorescence (≥ 1.44-fold) Decrease in 488 Taxol Median Channel Fluorescence (< 0.86-fold)
Carbendazim Tublin Binder: Destablizer Y N N N Y
Colchicine Y N N N Y
Diethylstilbestrol Y N N N Y
Flubendazole Y N N N Y
Griseofulvin Y N N N Y
Mebendazole Y N N N Y
Nocodazole Y N N N Y
Noscapine Y N N N Y
Rigosertib (impurity) Y N N N Y
Vinblastine Y N N N Y
Vincristine Y N N N Y
β-estradiol Y N N N Y
Epothilone A Tublin Binder: Stablizer Y N N Y N
Ixabepilone Y N N Y N
Paclitaxel Y N N Y N
Alisertib Aurora Kinase B(+) Inhibitors N Y Y N N
AMG-900 N Y Y N N
Barasertib N Y Y N N
Crizotinib N Y Y N N
Danusertib N Y Y N N
Hesperadin N Y Y N N
PF-03814735 N Y Y N N
Tozasertib N Y Y N N
ZM447439 N Y Y N N
Aurora A Inhibitor Other N N N N N
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Tubulin stabilizers

Three tubulin-binding stabilizers were evaluated in the AMM assay at concentrations that showed evidence of aneugenic activity in the standard MultiFlow DNA Damage Assay. As was the case for the destabilizers, each of these agents caused remarkable increases in the percentage of p-H3-positive events (Figure 2).

Tubulin-binding stabilizers did not affect p-H3-associated fluorescence in the p-H3 positive cell population (Figure 3). On the other hand, 488 Taxol-associated fluorescence was enhanced by each of the stabilizers (see Figure 5). Interestingly, the Epothilone A response exhibited a downturn at the highest concentrations tested, a finding that has implications for proper experimental design that considers sufficient concentrations that are not overtly cytotoxic. Importantly, none of the destabilizers or kinase inhibitors cause increased 488 Taxol-associated fluorescence of similar magnitudes.

Figure 5.

Figure 5.

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Median 488 Taxol-associated fluorescence (expressed as fold change relative to mean solvent control) is graphed against test chemical μM concentration (log10 transformed; solvent/zero concentration arbitrarily set to −6 log10). Note that these are aggregate data for all 25 test chemicals described in Table I, and they are split into the dichotomous groups: Non-tubulin stabilizers (left) and Tubulin stabilizers (right). Additional subgrouping is provided by the color-coded key, inset. The horizontal dashed line (1.44-fold) represents a useful cutoff value for distinguishing between the two groups: Non-tubulin stabilizers versus Tubulin stabilizers.

A summary profile is provided in Table II. This phenotypic signature is consistent with our a priori expectations (Figure 1).

Aurora kinase B(+) inhibitors

Nine AKB(+) inhibitors were evaluated in the AMM assay. Each had previously been identified as exhibiting aneugenic activity in TK6 cells as assessed by the base MultiFlow DNA Damage Assay [Bernacki et al., 2019]. For some of these agents, the lowest concentrations tested caused modest increases in %p-H3-positive events. However, in all nine cases, p-H3-positive frequencies were dramatically decreased at the highest concentrations, often close to zero (see Figure 6).

Figure 6.

Figure 6.

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Percentage of phospho-histone H3-positive events (expressed as fold change relative to mean solvent control) is graphed against test chemical μM concentration (log10 transformed; solvent/zero concentration arbitrarily set to −6 log10). Note that these are aggregate data for all 25 test chemicals described in Table I, and they are split into the dichotomous groups: Aurora kinase B(+) inhibitors (left) and Non-Aurora kinase B(+) inhibitors (right). Additional subgrouping is provided by the color-coded key, inset. The horizontal dashed line (0.68-fold) represents a useful cutoff value for distinguishing between the two groups: Aurora kinase B(+) inhibitors versus Non-Aurora kinase B(+) inhibitors.

The AKB(+) inhibitors were also observed to cause concentration-related reductions to p-H3-associated fluorescence in the p-H3 positive cell population (Figure 3). This was a clear signature of this class of aneugens, as none of the other test articles caused this effect to the same degree.

488 Taxol-associated fluorescence was not greatly affected by AKB(+) agents, certainly not to the same extent as tubulin destabilizers (Figure 4) or stabilizers (Figure 5).

A summary profile is provided in Table II. This phenotypic signature is consistent with our a priori expectations (Figure 1).

Other

We included one specific Aurora kinase A inhibitor in these studies—Aurora A kinase Inhibitor I, and it appears as “O” for “other” in Figures 2 - 6. As with the other agents, it had also been previously identified as an aneugen when evaluated by the base MultiFlow assay [Bernacki et al., 2019].

Whereas Aurora A kinase Inhibitor I increased %p-H3-positive events and 488 Taxol-associated fluorescence (Figures 2 and 4), these were modest effects that were well below the cutoff values used to categorize aneugens according to their tubulin-binding or AKB(+) inhibition properties (Table II). Thus, we considered this test article “inactive” in the AMM assay, or in other words, its aneugenic molecular mechanism is not classifiable with this methodology.

Hierarchical clustering

In addition to aggregate graphs (Figures 2 - 6) and summary rubric (Table II), we considered the magnitude of the biomarker responses using ToxPi software. This provides a view into chemical-specific responses, and represented another opportunity to evaluate the assay’s ability to differentiate among several important aneugenic mechanisms. The results of a hierarchical clustering algorithm are provided in Figure 7. Here, as with the decision tree-like rubric, we find that destabilizers, stabilizers, and AKB(+) inhibitors are clearly separated. As expected, the Aurora A kinase Inhibitor I falls into a subclade owing to its lack of effect on this suite of biomarkers.

Figure 7.

Figure 7.

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ToxPi profiles for 25 chemicals are shown following hierarchical clustering (complete, circular format). Red slices = greatest fold-change value observed for %p-H3; yellow slices = greatest fold-change value observed for p-H3-associated mean fluorescence; and blue slices = greatest fold-change value observed for 488 Taxol-associated fluorescence. Based on the response profiles of these three biomarkers, tubulin stabilizers, tubulin destabilizers, and AKB(+) inhibitors are found to cluster together.

Conclusions

In general, in vitro assays currently used to study aneugenicity have concentrated on hazard identification, or to distinguish between micronucleus induction occurring via clastogenicity versus aneugenicity [Eastmond and Tucker, 1989; Kirsch-Volders et al., 2003; Khoury et al., 2016; Bryce et al., 2016, 2017, 2018; Brandsma et al., 2020]. The simplified AMM assay and data analysis rubric described herein is capable of elucidating common genotoxic mechanisms responsible for in vitro aneugenicity. This methodology is therefore well-aligned with initiatives that are seeking to develop adverse outcome pathways [Sasaki et al., 2020].

In addition to the decision tree-type rubric, we found it useful to evaluate AMM assay data with ToxPi software. First, it represented an efficient tool for conveying multiple biomarker results for many chemicals (twenty-five). Second, the hierarchical clustering results provided additional evidence that the three biomarker signatures described herein are capable of discriminating aneugenic mechanisms of action.

We anticipate that among the potential use cases of the AMM assay is the attribution of drug candidates’ in vitro aneugenicity to off-target, promiscuous inhibition AKB(+). Such a finding could lead to margin of exposure-type analyses [Dearfield et al., 2017]. That is, one may be able to demonstrate sufficiently large differences between therapeutically-relevant dose levels versus considerably higher concentrations required to inhibit AKB(+) and cause aneugenic effects.

Additional research is needed to expand the number and types of chemicals tested through these assays, especially aneugens that affect other mitotic kinases aside from AKB(+). Other work aimed at evaluating interlaboratory transferability of benchtop protocols and data analysis strategies are planned.

ACKNOWLEDGMENTS

This work was funded in part by a grant from the National Institute of Health/National Institute of Environmental Health Sciences (NIEHS; grant no. R44ES029014). The contents are solely the responsibility of the authors, and do not necessarily represent the official views of the NIEHS.

The authors are indebted to colleagues that have been articulating the need for molecular initiating event-centric assays, and others that provided specific advice and shared personal experiences with reagents similar to those described herein. The authors are especially grateful to scientists at Pfizer, Groton, including Maik Schuler, Richard Spellman, and Maria Engel. An early prototype AMM Kit was evaluated by several collaborating laboratories, and their important work stimulated the development of the simpler cell processing protocol described herein.

Footnotes

CONFLICT OF INTEREST STATEMENT

The authors are employed by Litron Laboratories. Litron currently sells a collection of MultiFlow® Kits, and intends to sell the MultiFlow Aneugen Molecular Mechanism Kit and offer chemical testing services based on the procedures described herein.

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