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ADME PK

In vitro Comet assay

  • Cyprotex have partnered with Gentronix, specialists in genotoxicity screening, to offer the in vitro Comet assay.
  • The Comet assay, also called the single cell gel electrophoresis (SCGE) assay is a sensitive, rapid and relatively simple method for detecting DNA damage at the individual eukaryotic cell level1.
  • Electrophoresis is performed on exposed cells that have been embedded in agarose microgels. Damage resulting in DNA fragmentation is detectable by migration away from the nucleus giving a comet-like appearance with the head consisting of intact DNA and the tail consisting of damaged DNA.
  • The assay delivers rapid and sensitive DNA damage assessment with the benefit of low compound requirements.
  • The Comet assay complements other genotoxicity assays to provide a mechanistic insight into genotoxicity.
In addition to the capability of the Comet assay to identify DNA damage at the single cell level, other significant advantages include its sensitivity for detecting low levels of DNA damage, the requirement for only small numbers of cells per sample, its ease of application, and the short time needed to perform the assay.

2Liao W, McNutt MA and Zhu W-G (2009) Methods 48; 46-53

Protocol

In vitro Comet genotoxicity protocol

Test System Alkaline Comet (single cell gel electrophoresis)
Cell Line TK6 cells
Test Article Concentration 5 serial dilutions, typically 2-fold; e.g. 1000, 500, 250, 125, 62.5 µg/mL
Incubation Time 3 hr exposure time
Quality Controls 1% DMSO (negative control)
Etoposide (positive genotoxic control)
Cell Viability Assesment Propidium iodide exclusion using flow cytometry; 80% relative survival required for Comet processing
Test Article Requirements 10 mg solid - Other compound preparations are acceptable
Data Analysis 50 comets per compound dose acquired from two agarose gels (25 from each gel)
Data Delivery Written report presenting overall results
Excel worksheets with full graphical dose response data for genotoxicity including % tail intensity

Data

Data from the in vitro Comet genotoxicity assay

The in vitro Comet assay from Gentronix has been validated against a number of different genotoxic and non-genotoxic compounds.

 
in vitro comet Cycloheximide data

in vitro comet Etoposide data

in vitro comet Hydrogen peroxide data

in vitro comet 4-Nitroquinoline-1-oxide data
Figure 1
Graphs illustrating mean tail intensity data from the Gentronix Comet assay for non-genotoxic (cycloheximide) and genotoxic (etoposide, hydrogen peroxide and 4-nitroquinoline-1-oxide) compounds (mean ± sd; n=3).

References

1 Singh NP et al. (1988) Exp Cell Res 175; 184-191
2 Liao W et al. (2009) Methods 48; 46-53

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