Use of Automated High Content Analysis Applied To Assessment Of Primary DNA Damages With γH2AX and...
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Quantifying DNA damage is mandatory to assess potential adverse effects of candidate, drugs, molecules or extracts developed in dermo-cosmetic industry. Different assays can be performed to detect primary DNA damages, such as γH2AX or the single cell gel electrophoresis also known as the comet assay. • Various physical, chemical, and biological factors are involved in DSB induction. Cells respond to DNA damage by activating the so-called DNA damage response (DDR), a complex molecular mechanism developed to detect and repair DNA damage. The formation of DSBs triggers activation of many factors, including phosphorylation of the histone variant H2AX, producing γH2AX. Phosphorylation of H2AX plays a key role in DDR and is required for the assembly of DNA repair proteins in the sites containing damages. In general, analysis of γH2AX expression can be used to detect the genotoxic effect of different toxic substances. For γH2AX, 2 cell models were used: HepG2 and primary keratinocyte. HepG2 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. • The comet assay is a sensitive, well established technique for quantifying DNA damage in eukaryotic cells. Compatible with the detection of a wide range of DNA damaging agents, its principle consists in migration of fragmented DNA in an electrophoresis gel (damaged DNA forming the tail of the comet), while intact DNA moves at a slower rate (head of the comet). The percentage of fragmented DNA in the comet tail is a direct measure of DNA damage. Tk6 cell line was used to perform on automated comet assay in 96-well format. The High Content Imaging technology was used as a valuable tool for screening in early discovery phase. Four non genotoxic compounds and twelve genotoxic compounds from the ECVAM list I or II were selected to be tested on different cell models. Preliminary results on Mitomycine C, 4 Nitroquinoline-N-oxide and D- mannitol are shown here. Use of Automated High Content Analysis Applied To Assessment Of Primary DNA Damages With γH2AX and Comet Assays M. ROUDAUT 1 , S. ASTRI 2 , N. ORSINI 2 , A.-P. LUZY 2* , J. BURSZTYKA 1* & N. MAUBON 1 1 HCS Pharma, Biopôle, 6 rue Pierre Joseph Colin, 35000 Rennes 1* [email protected] 2 Galderma R&D, Les Templiers, 2400 route des Colles, 06410 Biot ²*[email protected] Abstract Methods Conclusions & Perspectives Cell culture: HepG2 were routinely maintained in MEM, TK6 in RPMI-1640, and both 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 % CO 2 before treatment. Genotoxic drugs exposure: 3 compounds were assessed for their DSBs inducing properties: mitomycine C (MMC), 4-nitroquinoline-N-oxide (4NQO), and D-mannitol. 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. Benzo-[a]-pyren (BaP, 10μM, genotoxic only if metabolized) and MMC (10μM) were used as positive controls. Incubations were done in duplicate for 3 hours or/and 24h. γH2AX detection: At the end of the exposure period, cells were fixed in paraformaldehyde 4%. After cell permeabilization by PBS/Tween20, staining was done by adding anti-γH2AX (phospho ser139) antibody (Abcam) followed by secondary antibody conjugated with Alexa-647. Nuclei were counterstained with Hoechst 33348. Comet assay: Cell embedding in agarose has been carried out with a Sciclone ALH3000 (Caliper), and the comet assay was done in alkaline conditions (Trevigen). Imaging was done using a Micro XLS (Molecular Devices) 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). γH2AX results are expressed as a fold change of the mean intensity in the nucleus. For the comet assay, the % of DNA in Tail is calculated as follows: (Sum of intensity in the tail/sum of intensity in the whole comet)*100 . To get this poster, please flash the QR- code You can use the I-NIGMA application from your store Evaluation of γH2AX by HCS on HepG2 and Primary Human keratinocytes cells seems to be a promising technology. On Primary Human keratinocytes different technical aspects have to be improved (i.e. cell synchronization) to generate robust results and more compound have to be tested. The RCC cut off and the γH2AX positive threshold have to be accurately defined to ensure the best performance (specificity and sensitivity) of the assay. Further work is needed on both cell types to fully validate the use of HCS for γH2AX assessment. Automation of the comet assay using 96-well slides allows medium throughput screening to assess potential adverse effects of new drug candidates on Tk6 cells. Assessment of cytotoxicity in parallel need to be developed. Encouraging preliminary results should be completed with additional list of genotoxic and non genotoxic compounds. Results Fig. 1: Genotoxicity (γH2AX) and cytotoxicity (RCC) assessment on NHEK and HepG2 cells after 3hours and 24h ours of exposure in duplicate . Fig. 3: Comet assay: %DNA in tail calculated after a 3 hours exposure of TK6 cells. Typical data sample. Mean of the duplicates, error bares show the two calculated values Fig. 2: Exemple of Picture extracted by HCS. Nuclei of HepG2 cells exposed to MMC 50μM for 3h. Blue : Hoechst staining. Red: γH2AX staining. Yellow: Nuclei border calculated with Columbus® software. NHEK HepG2 Fig. 4: Typical pictures generated during the comet assay. Left: TK6 cells exposed to 0,5%DMSO for 3 hours. Right: TK6 cells exposed to 3,125μM of 4-NQO for 3 hours. Grey : DNA after lysis and electrophoresis. Color: picture segmentation calculated with Columbus® software. TK6
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Transcript of Use of Automated High Content Analysis Applied To Assessment Of Primary DNA Damages With γH2AX and...
Quantifying DNA damage is mandatory to assess potential adverse effects of candidate, drugs, molecules or extracts developed in dermo-cosmetic industry. Different assays can be performed to detect primary DNA damages, such as γH2AX or the single cell gel electrophoresis also known as the comet assay. • Various physical, chemical, and biological factors are involved in DSB induction. Cells respond to DNA damage by activating the so-called DNA damage response (DDR), a complex molecular mechanism developed to
detect and repair DNA damage. The formation of DSBs triggers activation of many factors, including phosphorylation of the histone variant H2AX, producing γH2AX. Phosphorylation of H2AX plays a key role in DDR and is required for the assembly of DNA repair proteins in the sites containing damages. In general, analysis of γH2AX expression can be used to detect the genotoxic effect of different toxic substances. For γH2AX, 2 cell models were used: HepG2 and primary keratinocyte. HepG2 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.
• The comet assay is a sensitive, well established technique for quantifying DNA damage in eukaryotic cells. Compatible with the detection of a wide range of DNA damaging agents, its principle consists in migration of fragmented DNA in an electrophoresis gel (damaged DNA forming the tail of the comet), while intact DNA moves at a slower rate (head of the comet). The percentage of fragmented DNA in the comet tail is a direct measure of DNA damage. Tk6 cell line was used to perform on automated comet assay in 96-well format.
The High Content Imaging technology was used as a valuable tool for screening in early discovery phase. Four non genotoxic compounds and twelve genotoxic compounds from the ECVAM list I or II were selected to be tested on different cell models. Preliminary results on Mitomycine C, 4 Nitroquinoline-N-oxide and D-mannitol are shown here.
Use of Automated High Content Analysis Applied To Assessment Of Primary DNA Damages With γH2AX and Comet Assays
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* [email protected]
2 Galderma R&D, Les Templiers, 2400 route des Colles, 06410 Biot ²*[email protected]
Abstract
Methods
Conclusions & Perspectives
Cell culture: HepG2 were routinely maintained in MEM, TK6 in RPMI-1640, and both 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: 3 compounds were assessed for their DSBs inducing properties: mitomycine C (MMC), 4-nitroquinoline-N-oxide (4NQO), and D-mannitol. 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. Benzo-[a]-pyren (BaP, 10µM, genotoxic only if metabolized) and MMC (10µM) were used as positive controls. Incubations were done in duplicate for 3 hours or/and 24h.
γH2AX detection: At the end of the exposure period, cells were fixed in paraformaldehyde 4%. After cell permeabilization by PBS/Tween20, staining was done by adding anti-γH2AX (phospho ser139) antibody (Abcam) followed by secondary antibody conjugated with Alexa-647. Nuclei were counterstained with Hoechst 33348.
Comet assay: Cell embedding in agarose has been carried out with a Sciclone ALH3000 (Caliper), and the comet assay was done in alkaline conditions (Trevigen).
Imaging was done using a Micro XLS (Molecular Devices) 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). γH2AX results are expressed as a fold change of the mean intensity in the nucleus. For the comet assay, the % of DNA in Tail is calculated as follows: (Sum of intensity in the tail/sum of intensity in the whole comet)*100 .
To get this poster, please flash the QR-
code
You can use the I-NIGMA application
from your store
Evaluation of γH2AX by HCS on HepG2 and Primary Human keratinocytes cells seems to be a promising technology. On Primary Human keratinocytes different technical aspects have to be improved (i.e. cell synchronization) to generate robust results and more compound have to be tested. The RCC cut off and the γH2AX positive threshold have to be accurately defined to ensure the best performance (specificity and sensitivity) of the assay. Further work is needed on both cell types to fully validate the use of HCS for γH2AX assessment.
Automation of the comet assay using 96-well slides allows medium throughput screening to assess potential adverse effects of new drug
candidates on Tk6 cells. Assessment of cytotoxicity in parallel need to be developed. Encouraging preliminary results should be completed with additional list of genotoxic and non genotoxic compounds.
Results
Fig. 1: Genotoxicity (γH2AX) and cytotoxicity (RCC) assessment on NHEK and HepG2 cells after 3hours and 24h ours of exposure in duplicate .
Fig. 3: Comet assay: %DNA in tail calculated after a 3 hours exposure of TK6 cells. Typical data sample. Mean of the duplicates, error bares show the two calculated values
Fig. 2: Exemple of Picture extracted by HCS. Nuclei of HepG2 cells exposed to MMC 50µM for 3h. Blue : Hoechst staining. Red: γH2AX staining. Yellow: Nuclei border calculated with Columbus® software.
NHEK HepG2
Fig. 4: Typical pictures generated during the comet assay. Left: TK6 cells exposed to 0,5%DMSO for 3 hours. Right: TK6 cells exposed to 3,125µM of 4-NQO for 3 hours. Grey : DNA after lysis and electrophoresis. Color: picture segmentation calculated with Columbus® software.
TK6
