The creation of a double strand break (DSB) is accompanied by the phosphorylation of histone H2AX. The measurement of serine 139 phosphorylated histone H2AX (γH2AX) is reported to be a marker of interest to identify potential genotoxic activity. In order to evaluate the High Content Screening for γH2AX detection, 4 non genotoxic compounds and 9 genotoxic compounds from the ECVAM list I or II were selected to to be tested on HepG2 cell line. These cells offer the advantage to have H2AX expression data in the literature. In parallel, Human primary keratinocytes were included. Indeed these cells would be relevant for investigating skin adverse effects of topical applied xenobiotics with the advantage of High content imaging as valuable tool for screening in the early discovery phase.

1. Development of double stand break assessment assay with HCS by
using the HepG2 cell line and evaluation with primary skin cell (NHEK)
M. ROUDAUT1, 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
Abstract
The creation of a double strand break (DSB) is accompanied by the phosphorylation of histone H2AX. The measurement of serine 139
phosphorylated histone H2AX (γH2AX) is reported to be a marker of interest to identify potential genotoxic activity.
In order to evaluate the High Content Screening for γH2AX detection, 4 non genotoxic compounds and 9 genotoxic compounds from the
ECVAM list I or II were selected to to be tested on HepG2 cell line. These cells offer the advantage to have H2AX expression data in the
literature. In parallel, Human primary keratinocytes were included. Indeed these cells would be relevant for investigating skin adverse effects
of topical applied xenobiotics with the advantage of High content imaging as valuable tool for screening in the early discovery phase.
Methods
Conclusions & Perspectives
Cell culture: HepG2 were routinely maintained in MEM supplemented with 10% FBS, 1% non essential amino-acids, 1% peni/strepto and 1% L-glutamine. Neonatal human epidermal keratinocytes
(NHEK, pool of donors) were obtained and cultured following the recommendations from Lonza. Cells were seeded at 10 000 cells/well for HepG2 and 5 000 cells/well for NHEK in 96-well plates
(Greiner® advanced TC µClear). The cells were then incubated overnight at 37 °C in 5 % CO2 before treatment.
Genotoxic drugs exposure: 16 compounds were assessed for their DSBs inducing properties: Etoposide, azidothymidine (AZT), mitomycine C (MMC), vinblastin, taxol, colchicine, methylmethane
sulfonate (MMS), N-ethyl-N-nitrosourea (ENU), Cisplatin, 4-nitroquinoline-N-oxide (4NQO), Phenformin, cadmium chloride, hydroquinone, D-mannitol, ampicilline & n-butyl chloride. Exposure of the
cells was performed by replacement of the medium in each well with fresh medium with or without the different genotoxic agents at different concentrations ranging from 0.195 µM to 100 µM, except
for MMS and ENU (1.563 to 1000µM). Benzo-[a]-pyren (BaP, 10µM, genotoxic only if metabolized) and MMC (10µM) were used as positive controls. Incubations were done in duplicate.
Assessment of double strand breaks through γH2AX detection: Cells were paraformaldehyde fixed (4%) after 24h of exposure. After permeabilisation of the cells by PBS/Tween20, staining was done by
adding anti-γH2AX (phospho ser139) antibody (Abcam) followed by secondary antibody conjugated with Alexa-488. Nuclei were counterstained with Hoechst 33348. Imaging was done on an Operetta
(Perkin Elmer) with a 20x objective. Image analysis was performed with Columbus software (Perkin Elmer). 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 γH2AX intensity is significantly higher than the untreated cells.
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 Evaluation of γH2AX by HCS on HepG2 and Primary Human keratinocytes cells seems to be a promising technology.
 On Primary Human keratinocytes different technical aspect have to be improved (i.e. cell synchronization) to generate robust results and
more compounds have to be tested.
 The RCC cut off and the γH2AX positive threshold have to be accurately defined.
 Further work is needed on both cell types to fully validate the use of HCS for γH2AX assessment.
Results
Fig. 1: Inter and intra plate variability on HepG2 cells.
Fig. 2: Genotoxicity (γH2AX ) and cytotoxicity (RCC) assessment on HepG2 cells.
Example of data.
Fig. 3: Comparison of the sensitivity and specificity of HepG2 as a cellular
model to screen compounds for their DSBs inducing properties.
Fig. 4: Development of this assay on human primary keratinocytes.
Fig. 5: Nuclei of primary human epidermal
keratinocytes exposed to etoposide 6.25µM for 24h.
Blue : Hoechst staining. Green : γH2AX staining (a).
Red: 53BP1 staining (b). P53 binding protein 1 is
involved in the DSBs repair process, thus colocalising
with γH2AX (c : merged images).
a b c