Quantifying DNA damage is mandatory to assess potential adverse effects of candidate drugs or molecules or extracts developed in the
dermo-cosmetic industry, but also to assess the efficacy of therapeutic approaches with the aim of producing tumor cell genotoxicity in cancer treatment. Genotoxic potential effect can be analysed by different assays as for example: gamma-H2AX, comet assay and micronuclei analysis.
In HCS Pharma, We have developed in an automated fashion this diferent assay.
To learn more on these assays, see also our posters presented in different congress:
- Genotoxicity assay
- Genotoxicity on HepG2
- Genotoxicity by γH2AX detection on HepG2 & keratinocytes
- γH2AX and Comet Assays
- Comet assay on TK6
H2AX becomes phosphorylated on serine 139, then called gamma-H2AX, as a reaction to DNA Double-strand breaks (DSB). Thus, gamma-H2AX is a sensitive target for measuring DSBs in cells.
After double stranded DNA breaks, H2AX is rapidly phosphorylated. Depending on the penetration of the compound in the cell, kinetics has to be performed. Below are two examples of phosphorylation of H2AX in SH-SY5Y cells exposed to etoposide or camptothecin for one hour in a dose effect:
Columbus software allows us to analyze these pictures by counting the number of nuclei marked by an antibody against gamma-H2AX. In these two examples, after 1 hour of incubation, 80% of the neuronal cells have their H2AX phoshorylated in the nucleus with etoposide 100 µM and around 60% with camptothecin 100 µM.
In-house, we have already performed this assay on different cell lines: SH-SY5Y, HepG2, Raw264.7, Hela, HT29 … and on primary cells as keratynocytes as other example with etoposide on HT29 cells :
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 the 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.
The comets are evaluated via fluorescence staining and microscopy. Two drawbacks are often mentioned about the comet assay: preparing the comet slides with cells in agarose to perform electrophoresis is time consuming and reduce the throughput of the method, and, if manual scoring of the comets are done, there can be a high inter-operator variability.
In HCS Pharma, We have combined process to perform an automated comet assay, from the cell and agarose handling, to comet assessment and scoring, by using automated instrumentation and a 96-well format comet slide.
Micronucleus analysis is a well known assay for genotoxicity analysis of compounds which can be performed in an automated fashion by High content Screening.
Here is an example of a micronucleus induced by etoposide 100 µM after a 24H incubation on HepG2 cells:
or by camptothecin 100 µM:
These two assays can be performed in the same experiment and in a screening mode. For more details, please contact us.