Benchtop flow cytometry facilitates discrimination between induction of DNA damage, oxidative stress and protein misfolding using a novel multicell GFP reporter assay

Giel Hendriks1, Remco S. Derr1, Harry Vrieling2, Robin T. Clark3

1Toxys B.V., Robert Boyleweg 4, 2333 CG, Leiden, The Netherlands

2Department of Human Genetics, Leiden University Medical Center, PO Box 9600, 2300 RC, Leiden, The Netherlands

3MilliporeSigma, a business of Merck KGaA, Darmstadt, Germany

Introduction

With increasing demand for the production of novel compounds for a wide range of pharmaceutical, chemical, and cosmetics applications comes a need for effective approaches that can reliably assess their adverse and potentially carcinogenic properties during the early phases of development.  Exposure of cells to novel chemicals, xenobiotics and biologicals may induce damage at the organelle or biomolecular level that can jeopardize homeostasis and lead to induction of carcinogenicity.  Current approaches to in vitro genotoxicity assessment that include the classical Ames bacterial mutation test followed by mammalian mutation assays lack specificity and provide limited information about mechanisms of genotoxicity (Walmsley and Billinton, 2011). Cells have evolved defense mechanisms wherein specific programs are activated to restore healthy cell function in response to the detection of subcellular damage. Quantitative assessment of damageassociated signal transduction activity may therefore serve as a more sensitive reporter for induction of structural, organelle, or DNA damage (Hendriks et al., 2013).

The ToxTracker™ assay was developed by Toxys B.V. and is a mammalian stem cell-based genotoxicity assay that uses fluorescent reporter genes specific to cell damage pathways capable of tracking DNA replication, oxidative stress induction and protein misfolding.  Validation of this assay was achieved using compounds recommended by the European Centre for the Validation of Alternative Methods (ECVAM) for the specific effects of toxicity (Kirkland et al., 2016).  The additional development of the dedicated software tool Toxplot for automated data analysis and graphical interpretation of test results enhances rapid and reliable identification by the ToxTracker™ assay of specific biological processes that improve in vitro assessment of chemicals that are potentially hazardous to humans. The ToxTracker™ assay uses GFP reporters of six key damage pathway activation genes to provide a sensitive and specific assay for elucidating mechanisms of cellular toxicity. Although microscopy can detect fluorescent reporters, a quantitative method is required for assessment of cellular response on a population scale. ToxTracker™ was validated on and is optimized for the Guava® easyCyte HT (high-throughput) benchtop flow cytometer. Available 96-well automated sample acquisition facilitates the screening of multiple compounds at a range of concentrations. Measurement of toxicity indicators using the Guava® easyCyte platform augments the sensitivity and specificity of the ToxTracker™ assay with the kind of quantitative data from significant cell populations that is characteristic of flow cytometry.

Materials & Methods

Cell culture and treatments

C57/Bl6 B4418 wild type mouse embryonic stem (mES) reporter cells were cultured in mES knockout medium (Gibco) containing 10% Fetal Calf serum (FCS), 2 mM glutamax, 1 mM sodium pyruvate, 100 μM β-mercaptoethanol and leukemia inhibitory factor (LIF), and were propagated on irradiated primary mouse embryonic fibroblasts as feeders according to established protocols (Hendriks et al., 2012). For chemical exposure, cells were seeded 24 hours prior to exposure on gelatincoated plates in BRL-conditioned mES cell medium in the absence of feeder cells, and subsequently exposed to the test compounds for 24 hours. For analysis of compounds that require metabolic activation, cells were exposed for 3 hours in the presence of 1% S9 rat liver extract in 3.2 mM KCl, 0.8 mM MgCl2, 0.5 mM glucose-6-phosphate and 0.4 mM NADP. After 3 hours, cells were washed with phosphate-buffered saline (PBS) and cultured for 24 hours in Buffalo rat liver (BRL) cells -conditioned medium minus the tested compounds and S9.

Detection of GFP expression

GFP reporter expression was determined by flow cytometry on the Guava® easyCyte 5HT benchtop cytometer, equipped with a 488 nm blue laser and 96-well plate reader. Cells were incubated with the test substances in gelatin-coated wells of 96-well plates. All tested compounds were dissolved in DMSO, MilliQ® water, or PBS and diluted in fresh BRL-conditioned mES cell medium just before incubation with the cells. Following 24 hours of exposure, cells were washed with PBS, detached with trypsin and resuspended into PBS supplemented with 2% FCS, immediately followed by flow cytometry analysis. Reporter activity was determined by the mean fluorescence intensity (MFI) of 5000 intact cells, which can be further assessed for relative induction using the MFI heat-mapping feature of the Guava® InCyte software (Figure 1).

Workflow for ToxTracker™ assay on the Guava® easyCyte 5HT.

Figure 1. Workflow for ToxTracker™ assay on the Guava® easyCyte 5HT. (A) mES GFP reporter cell lines were cultured on gelatincoated wells for 24h exposure to putative toxic compounds, then acquired on the Guava® easyCyte 5HT flow cytometer. (B) The Guava® InCyte software features a heatmapping display that provides rapid visual and quantitative assessment for compound screening approaches such as ToxTracker™. Color intensity of wells on the map corresponds to mean fluorescence intensity from the selected channel. In the ToxTracker™ assay, the green channel intensity is mapped to assess relative induction of GFP reporter genes.

Test criteria

Activation of a reporter cell line was considered positive when, at any applied dose, exposure to a compound resulted in >1.5 fold induction of GFP expression being at least 5 times higher than the standard deviation of background fluorescence in mock-exposed cells. Concentrations that induced >75% cytotoxicity were not considered for data analysis. Application of the 1.5 fold induction cut-off threshold provides positive test results with a confidence of >99.9%. In a representative experiment, reporter cells were exposed to five different concentrations of a compound, typically at 2-fold dilutions, starting with a concentration that induces no detectable cytotoxicity, up to a concentration that resulted in less than 50% cell survival. The relative cell survival after 24 hours of treatment was calculated as the ratio in concentration of intact cells for treated versus untreated samples as determined by the Guava® InCyte software. All data presented are the summary of at least three independent biological replicates.

Validation of GFP reporter cell lines

GFP reporter cells were exposed to 50 genotoxic and non-genotoxic compounds at five or more concentrations. The selection of compounds was largely based on the ECVAM-suggested list of chemicals for validation of in vitro genotoxicity test assays (Kirkland et al., 2008). Compound concentration ranges were based on the degree of cytotoxicity they induced.  Samples exhibiting >25% cell death following 24 hours of treatment were not included in results.

Results

Biomarker identification

Two highly specific biomarker genes for DNA damage (BSCL2) and oxidative stress (SRXN1) were used to generate fluorescent reporter cell lines when the original ToxTracker™ assay was developed (Hendriks et al., 2012). Utilizing extensive transcription profiling data, the panel of biomarker genes was expanded to six biomarkers to allow monitoring of a broader spectrum of signal transduction routes following exposure (Hendriks et al., 2015).

Whole genome transcription data of mES cells after exposure to various genotoxic and non-genotoxic carcinogens clearly demonstrate the differential responsiveness of the selected biomarker genes (Figure 2B) (Hendriks et al., 2011, Schaap et al., 2014). BSCL2 and RTKN are preferentially induced by DNA damaging agents, while SRXN1 and BLVRB are induced upon oxidative stress. Upregulated BTG2 expression reflects activation of the P53 response and is therefore induced upon DNA damage and oxidative stress. Lastly, DDIT3 upregulation indicates activation of the unfolded protein response.

Principles and design of the ToxTracker™ assay.

Figure 2. Principles and design of the ToxTracker™ assay. A. Previous toxicogenomic studies revealed genes upregulated following exposure of mES cells to genotoxic and nongenotoxic carcinogens. Genes unique to specific damage pathways were selected to generate six GFP-reporter cell lines (B).

Flow cytometry detects damage-associated gene upregulation following exposure to DNA-damaging and stress-inducing compounds.

Figure 3. Flow cytometry detects damage-associated gene upregulation following exposure to DNA-damaging and stress-inducing compounds. Following exposure to 10 µM cisplatin (CisPt), a DNA-damaging chemotherapeutic (middle row of each panel) SRXN1-GFP cells (right panel) demonstrate moderate fluorescence when compared with untreated mES cells (top row). Treatment with the oxidative stress-inducing agent diethyl maleate (DEM) induced robust fluorescence, consistent with the anticipated response from the SRXN1 gene. In the RTKN-GFP reporter line (left panel), cisplatin induced brighter fluorescence than did DEM, as predicted for the role of RTKN in NFκB signaling in the context of DNA damage. Graphs shown are mapped to their corresponding wells in the plate map, where the InCyte™ heat map feature provides a graphical representation of relative GFP fluorescence—as shown here—or of relative fluorescence from any channel, from up to six plates simultaneously.

Metabolic activation

Occasionally, compounds are not directly reactive but can become genotoxic following metabolic activation during detoxification reactions in the liver, kidney and lung (Nebert and Dalton, 2006). The major enzymes that are involved in the metabolic activation of pro-genotoxins, e.g. cytochrome P450s and epoxide hydrolyses, are scarcely or not expressed in mES cells. To test whether the ToxTracker™ assay could detect genotoxic properties of compounds that require metabolic activation, reporter cell lines were exposed for three hours to six reference pro-genotoxins in the presence of S9 rat liver extract. After removal of S9 and compounds, fresh medium was added and after 24 hours recovery, GFP reporter induction was measured by flow cytometry.  All compounds tested induced both DNA damage reporters BSCL2 and RTKN. In addition to activation of the DNA damage reporters, exposure to the pro-genotoxins in the presence of S9 extract frequently led to concurrent activation of the oxidative stress reporters. Only after treatment with Benzo(a)pyrene  was induction of the unfolded protein response detected (Figure 4), as observed  in previous  studies (Boysen and Hecht 2003).

Selective activation of the ToxTracker™ reporter cell lines.

Figure 4. Selective activation of the ToxTracker™ reporter cell lines. (A) GFP reporter cells were exposed to increasing concentrations of the DNA damaging agent cisplatin (CisPt), the oxidative stress-inducing agent diethyl maleate (DEM) and the UPR-activating compound tunicamycin. GFP induction levels in intact cells were determined by flow cytometry at 24h. after initiation of the exposure. (B) Cell survival was determined by the Guava® easyCyte 5HT after 24h. exposure as the relative decrease in cell concentration compared to untreated controls.

Conclusions and discussion

The current standard battery of in vitro genotoxicity assays — that generally includes the bacterial Ames gene mutation test and a micronucleus test — is profoundly limited by an inability to provide insight into the genetic programs of toxicity response that are induced by a compound. Understanding the mechanisms of toxicity induction helps to eliminate false positive genotoxicity findings, and to better estimate the relevance of test results for assessment of human health risk.

An attractive approach to mechanistic insight into toxicity is to monitor and measure the specific cellular stress response pathways that are activated following exposure.  The ToxTracker™ assay addresses this need with GFP reporter cell lines that provide specific and sensitive indicators of the activation of genes involved in cell-damage responses.

A means to collect this specific signaling induction information from large numbers of cells is clearly required to ensure quantitative data that provides confidence in reporter cell tracking.   Because of the need to screen numerous compound formulations at a relevant range of concentrations, systems that can automatically acquire samples in a single experimental session expedite toxicity screening and eliminate the introduction of interassay variation.

Guava® easyCyte HT (high-throughput) flow cytometers are equipped with an integrated multiwell plate carrier to permit single setup and walkaway acquisition for up to 96 samples.  The unique microcapillary fluidics design of Guava® cytometers enables direct sampling, conferring the distinct advantage of absolute cell counts, and permitting direct correlation between cellular responses and cytotoxicity.  Together, these features provide solutions for the need for convenient and consistent data collection compatible with toxicity and drug discovery assays characterized by multisample testing.

Materials

     

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