C

Conclusions

Use of Automated High Content Analysis or Imaging Applied To Assessment Of Primary DNA Damage With

γH2AX and Comet Assays on different Cell types

Introduction

M. ROUDAUT1, K. JARNOUEN1, S. ASTRI2, N. ORSINI2, A.-P. LUZY2*, J. BURSZTYKA1* & N. MAUBON1

1 HCS Pharma, Biopôle, 6 rue Pierre Joseph Colin, 35000 Rennes 1* julian.bursztyka@hcs-pharma.com

2 Galderma R&D, Les Templiers, 2400 route des Colles, 06410 Biot

²*anne-pascale.luzy@galderma.com

Materials and methods

This experiment was

conducted on Tk6 cells (). C

γH2AX phosphorylation summary results

Nine compounds were tested on the Comet and γH2AX assays in a 96 wells format and analysis was done by high content imaging. Three non genotoxic compounds (Ampicilin trihydrate, Mannitol and

Phenformin HCl), three clastogens (Etoposide, MMC and MMS) and three aneugens ( Paclitaxel, Vinblastine and Colchicine) were dissolved in DMSO and tested at different concentrations ranging from

0.2 µM to 100 µM, except for MMS (1.6 to 1000 µM). Incubations were done in duplicate. Imaging was done on an ImageXpress Micro XLS (Molecular Devices) with a 20X objective. Image analysis was

performed with MetaXpress software (Molecular Devices).

Comet assay

This experiment was conducted on human lymphoblastoid cell line (TK6). Compounds and

cells were incubated for 3h, and embedded in agarose with a Sciclone ALH3000 (Caliper).

The comet assay was done in alkaline conditions (Trevigen). The % of DNA in Tail is

calculated as follows: (Sum of intensity in the tail/sum of intensity in the whole comet)*100 .

The cytotoxicity was evaluated by XTT measurement.

γH2AX assay The experiment was conducted on the HepG2 human hepatic cell line (ATCC) and neonatal human epidermal

keratinocytes (nHEK, pool of donors, Lonza). Compounds, caspase 3/7 staining kit and cells were incubated for

3h and 24h and paraformaldehyde fixed (4%). After permeabilisation of the cells by PBS/Tween 20, staining was

done by adding anti-γH2AX (phospho ser139) and 53BP1 antibodies (Abcam) followed by secondary antibodies

conjugated with Alexa-647 and Alexa-555 respectively. Nuclei were counterstained with Hoechst 33348.

Relative cell count (RCC) was calculated as a percentage of the cell count in the control (0.5% DMSO). For

γH2AX, results are expressed as a fold change of the mean intensity in the nucleus after removing the

caspase3/7 positive cells.

Compound Genotoxicity (MoA) Results on TK6

Mannitol Non Genotoxic Negative

Ampiciline Trihydrate Non Genotoxic Negative

Phenformin Hcl Non Genotoxic Negative

Etoposide Clastogen Positive

Methylmethane sulfonate

(MMS) Clastogen Positive

Mitomicyne C (MMC) Clastogen Negative

Vinblastine sulfate Aneugen Negative

Colchicine Aneugen Negative

Paclitaxel (taxol) Aneugen Negative

C

Genotoxicity potential assessment of pharmaceuticals is mandatory for registration. In addition to the standard in vitro/ in vivo test battery, other assays can be of interest because of their high

throughput in the drug discovery stage but also for mechanistic understanding.

Different other assays can be performed to detect DNA damage, such as γH2AX and the single cell gel electrophoresis (or comet assay).

The γH2AX assay was suggested as a new in vitro assay for detecting genotoxic properties of chemicals. The phosphorylation of γH2AX has been demonstrated to be a sensitive marker for a wide

range of DNA damage. In this study, primary human keratinocytes were evaluated to provide a more relevant testing system for genotoxicity assessment in the skin for topical dermatological/cosmetic

substances. Primary human keratinocytes were compared to the well known HepG2 cell line for which data have already been published.

The comet assay is a sensitive, well established technique for quantifying DNA damage in eukaryotic cells and enabling the detection of a wide range of DNA damaging agents. The assay can be

adapted in a 96 wells format allowing higher throughput.

The high content imaging (HCI) analysis applied to genotoxicity assays is a valuable tool allowing higher throughput and multi-parametric analysis in order to improve predictability of these assays.

gH2AX phosphorylation on HepG2 cells for the 9 compounds at 3h and 24h

Green (negative compound), red (clastogen compound) and orange (aneugen compound)

Media Etoposide 10µM

The objective of this study was to assess the feasibility of using HCI to

analyze γH2AX and comet results. Nine known compounds were tested on

different cell types.

• Comet assay performed on TK6 cells gave expected results (published).

MMC was misclassified as a negative compound due to cross link of DNA

preventing DNA migration and/or inappropriate experimental conditions.

• HCI adapted to γH2AX assay allows the detection of the genotoxic

response on HepG2 cells (already published) and on primary human

keratinocytes. Measurement of RCC and caspase 3/7 help to discriminate

phosphorylation due to apoptosis, from those due to DNA damage.

Multiplexing staining : γH2AX, apoptosis and 53BP1 colocation help to

discriminate more accurately genotoxic from non genotoxic compounds

and to discriminate clastogens from aneugens. The RCC cut off and the

γH2AX positive threshold are yet to be accurately defined.

Further work is needed on both cell types to fully validate the use of high

Content Imaging for γH2AX assessment.

Alkaline Comet Assay summary results

A compound was considered as positive when the Relative DNA tail increase was >2 fold in at

least two concentrations with a mitochondrial activity >80% versus vehicle control C

Based on literature, we defined as positive a response if RCC*> 30% and ɣH2AX fold induction > 1.2-1.5.

The 3 clastogens compounds were detected at 3h and 24h and the 3 non genotoxic compounds never induced gH2AX. Aneugen compounds showed

an equivocal induction of ɣH2AX with a decrease of RCC probably due to their mechanism of action. [*RCC = relative cell survival]

gH2AX phosphorylation on primary human keratinocytes for the 9 compounds at 3h and 24h

C The same criteria of positivity were applied.

The primary human keratinocytes were less sensitive than HepG2 after 3h. At 24h, the results obtained were similar on both cell lines : clastogen

compounds induced ɣH2AX phosphorylation, non genotoxic compounds not and aneugen compounds gave equivocal results (1/3 positive).

An example of high content imaging analysis results obtained with MMC at 6,25 µM on primary human keratinocytes

Hoescht γH2AX Caspase 3/7 53BP1 Merge Hoescht γH2AX Caspase 3/7 Merge 53BP1

Colocalisation 53BP1/ gH2AX at 24h on HepG2 and primary human keratinocytes

C Clastogen compounds induced a recruitment of 53BP1 at the nuclear foci gH2AX on both cell types

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