Genotoxicity tests should detect compounds that induce genetic damage. The induced changes are caused by different mechanisms and may require physiological activation of the chemical. Therefore the current regulatory measures to identify genotoxic properties comprise a test battery. The increasing demand of chemical safety assessment calls for alternative methods to reduce animal experiments. Indicator tests can provide additional useful information for initial classification.
Mammalian cell-based alternatives open up new opportunities for fast and reliable tests to screen and identify genotoxic potential of substances and possible modifications of their toxicity profile in substance mixtures.
The Fluorimetric Detection of Alkaline DNA Unwinding (FADU) is a valuable part of battery testing for genotoxicity. FADU assay is able to identify DNA strand breaks and also physiologically based alteration. From great importance is the capability to measure short exposition times (<5 min) and the corresponding DNA repair activity. The discrimination between those processes ensures reliable estimation of toxic potential. It could consequently reduce the number of false positive or false negative results and thus reduces the number of animals needed in scientific research or safety testing.
Principle of the FADU Assay
FADU assay detects DNA strand breaks and repair. The detection is based on progressive DNA unwinding under highly controlled conditions of alkaline pH, time and temperature. DNA “open sites” are the starting points for the unwinding process. They occur during cell circle, like replication forks or chromosome ends, but may also be induced by reactive oxygen species (ROS), irradiation or chemical compounds. A fluorescent dye is used as marker for double stranded DNA, and a decrease in the fluorescence intensity indicates an increase of DNA unwinding and consequently a greater number of DNA strand breaks.
Controls are to be run in parallel with the experimentally treated cells. Controls comprise samples with total DNA content (defined as 100 %), samples under physiological conditions, and completely unwound samples (background).
FADU Assay offers clear advantages
FADU assay is automated and the assay run time is less than 3h. Data concerning the classification of chemicals obtained using the automated FADU assay is very similar when compared with results coming from the comet assay. However, there are dramatic differences in the workflow and runtime of the two assays with the FADU assay being time and cost saving.
Due to the automation FADU assay is highly reproducible (inter-assay CV =9%) and robust. It is easy to handle. In addition, the reduced number of false positive or false negative results reduces the number of animals needed for subsequent safety testing.
FADU assay is able to test the genotoxic properties of chemicals, pharmaceuticals, and botanicals. Preliminary data of nanoparticle testing also indicate its feasibility and showed no interference with nanoparticles. Further validation for nanoparticles is currently done by BioTeSys as partner in the EU-founded project “Nanosolutions” for the assessment of nanotoxicology.
FADU and 3D skin models
Suitability of FADU assay for 3D skin models was proven in some first experiments.
EST-1000 skin models (CellSystems, Germany) were treated with different concentrations of methyl methanesulfonate (MMS). Skin models were dissociated and the cell suspension was analysed on DNA strand breaks by automated FADU. FADU assay revealed increasing DNA strand breaks with increasing MMS concentrations.
The results are very promising for adaptation of FADU for 3D skin models.
Contact:
Dr. Jürgen Bernhardt
Tel: +49-(0)711-31057150
info@biotesys.de
www.biotesys.de
Mammalian cell-based alternatives open up new opportunities for fast and reliable tests to screen and identify genotoxic potential of substances and possible modifications of their toxicity profile in substance mixtures.
The Fluorimetric Detection of Alkaline DNA Unwinding (FADU) is a valuable part of battery testing for genotoxicity. FADU assay is able to identify DNA strand breaks and also physiologically based alteration. From great importance is the capability to measure short exposition times (<5 min) and the corresponding DNA repair activity. The discrimination between those processes ensures reliable estimation of toxic potential. It could consequently reduce the number of false positive or false negative results and thus reduces the number of animals needed in scientific research or safety testing.
Principle of the FADU Assay
FADU assay detects DNA strand breaks and repair. The detection is based on progressive DNA unwinding under highly controlled conditions of alkaline pH, time and temperature. DNA “open sites” are the starting points for the unwinding process. They occur during cell circle, like replication forks or chromosome ends, but may also be induced by reactive oxygen species (ROS), irradiation or chemical compounds. A fluorescent dye is used as marker for double stranded DNA, and a decrease in the fluorescence intensity indicates an increase of DNA unwinding and consequently a greater number of DNA strand breaks.
Controls are to be run in parallel with the experimentally treated cells. Controls comprise samples with total DNA content (defined as 100 %), samples under physiological conditions, and completely unwound samples (background).
FADU Assay offers clear advantages
FADU assay is automated and the assay run time is less than 3h. Data concerning the classification of chemicals obtained using the automated FADU assay is very similar when compared with results coming from the comet assay. However, there are dramatic differences in the workflow and runtime of the two assays with the FADU assay being time and cost saving.
Due to the automation FADU assay is highly reproducible (inter-assay CV =9%) and robust. It is easy to handle. In addition, the reduced number of false positive or false negative results reduces the number of animals needed for subsequent safety testing.
FADU assay is able to test the genotoxic properties of chemicals, pharmaceuticals, and botanicals. Preliminary data of nanoparticle testing also indicate its feasibility and showed no interference with nanoparticles. Further validation for nanoparticles is currently done by BioTeSys as partner in the EU-founded project “Nanosolutions” for the assessment of nanotoxicology.
FADU and 3D skin models
Suitability of FADU assay for 3D skin models was proven in some first experiments.
EST-1000 skin models (CellSystems, Germany) were treated with different concentrations of methyl methanesulfonate (MMS). Skin models were dissociated and the cell suspension was analysed on DNA strand breaks by automated FADU. FADU assay revealed increasing DNA strand breaks with increasing MMS concentrations.
The results are very promising for adaptation of FADU for 3D skin models.
Contact:
Dr. Jürgen Bernhardt
Tel: +49-(0)711-31057150
info@biotesys.de
www.biotesys.de
