Comet assay: Better test methods - fewer animals
All chemicals must now be tested for genotoxicity. This applies to both new and existing substances. Researchers at the Norwegian Institute of Public Health have adapted the popular Comet assay to test chemicals on a large scale. This means quick and effective testing with a significant reduction in the number of laboratory animals needed.
There are more than 100 000 chemicals available today, with 3000 new substances being added every year.
According to new rules from the European Commission (REACH Directive), both existing and new substances - if used at more than one tonne per year - must be tested for genotoxicity i.e. their ability to damage DNA. Substances that are genotoxic can lead to cancer after considerable exposure. They can also damage germ cells which may have implications for descendants.
As well as new requirements for chemical testing, in both Norway and the EU there is a strong desire to reduce the number of animals used in experiments.
Researchers at the NIPH have participated in an EC project (COMICS) where the goal has been to develop methods for efficient and accurate genotoxicity testing. As part of this work, the Norwegian researchers have adapted the Comet assay (single cell gel electrophoresis) for large-scale testing.
800 samples simultaneously
”We can run up to 800 samples in one experiment which is a great improvement. Originally, the method could be used to examine only 12 to 20 samples at the same time,” said Kristine Bjerve Gutzkow at the Division of Environmental Medicine.
Gutzkow and Gunnar Brunborg of the NIPH, together with Professor Andrew Collins from the University of Oslo have developed a system that is both efficient and economical and which reduces the need for animal testing.
The improved method was presented and discussed at two recent conferences on environment and health that were recently held in Oslo, jointly organised by the NIPH and University of Oslo.
“There is a great need for sensitive, reliable methods that simultaneously have a high capacity. Chemical manufacturers, test laboratories and the monitoring authorities are enthusiastic about these improved methods," added Gutzkow.
She believes that interest in the method will increase further as the new and stricter legislation in the EU is implemented.
Testing cells in gel
Cell samples are suspended and encapsulated in agarose gel. The cells are then lysed to remove proteins and other unwanted materials to leave the DNA available for analysis. The gels with DNA are placed in a bath with high pH before a low electric field is applied that causes the denatured DNA to migrate as a round "ball" in the gel.
If there is any DNA damage, the "DNA-ball" will develop a tail of migrated DNA in the gel – resembling a comet in the sky, hence the name of the assay. When the gel is fixed, dried and studied under the microscope, one can see if the DNA is round or comet-shaped. The brighter and longer the tail, the higher the level of damage. Microscopic analysis will soon be fully automated, which will make the assay even more efficient.
The Comet assay is also known as single cell gel electrophoresis because the cells can be studied individually. In the traditional COMET assay that was developed 25 years ago, cells are encapsulated into large gels layered on a small glass lens. The NIPH researchers have contributed adjustments, primarily by casting 96 micro-gels on a polyester film. In addition, the method has been simplified and has become faster and more robust.
For health monitoring
Can the method be used for anything other than to test new chemicals?
”It can also be used in health monitoring and in various research projects. For example, blood samples from population groups or individuals can be studied to see if they have an increased level of DNA damage. Such investigations may be relevant for accidents, long-term impact from possible environmental toxins or to study the lifestyle effects. Since the method has a large capacity, it is possible to examine a large number of people in a short time. We can also add specific enzymes to distinguish between different types of DNA damage” added Gutzkow.
Both fresh and frozen material can be used, such as whole blood, white blood cells, sperm, or organ samples. Tissues and cells from animals, insects and humans can be examined. The method has been successfully tested on fish blood. Other applications are in vitro fertilised eggs from mice, earthworms, starfish and springtails.