The future of the comet assay

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• Created: Wednesday, 31 May 2017 09:51

The International Comet Assay Workshops are a series of scientific conferences dealing with practical and theoretical aspects of the comet assay.  The workshops take place every two years; the next will be August 2017, Pamploma, Spain.  For more information visit the ICAW website. 

The first paper describing such an assay was published over 30 years ago in 1984 by Swedish researchers O. Ostling and K. J. Johanson. Appropriately, the theme for the 2015 meeting was looking to the future: ‘The Next 3 Decades of the Comet Assay’. The programme included 25 oral and 43 poster presentations depicting the latest advances in technical developments as well as applications of the comet assay in genotoxicity testing (in vitro and in vivo) and biomonitoring of both humans and the environment. Open discussion sessions based on questions from the participants allowed exchange of practical details on current comet assay protocols. 

For a full report of the meeting, please refer to the publication (The next three decades of the comet assay: a report of the 11th International Comet Assay Workshop Mutagenesis (2017) 32 (3): 397-408).  The aim of the workshop and subsequent publication is to provide information on ways to improve the assay performance, by testing for cytotoxicity, by using reference samples to reduce or allow for inter-experimental variation, and by standardising quantification of the damage, including replicates and scoring enough comets to ensure statistical validity.

Here, some of the important technical issues which were discussed during the sessions are briefly summarised.  However for full analysis and interpretation, please refer to the original publication.  After 30 years of experimentation with the comet assay, the authors of the article believe that important experimental parameters can be controlled and the comet assay is a truly reliable method with a wealth of possible applications.

Current and future applications

A questionnaire was given to the ICAW 2015 attendees.  It was completed by 97 participants and provides a good overview of the various application fields. As expected, the majority used the standard alkaline comet assay for in vitro and in vivo genotoxicity testing as well as human biomonitoring.  Other applications included genotoxicity testing of nanoparticles, FISH (fluorescent in situ hybridisation) comet assay, and ecogenotoxicity.

Innovations in the comet assay used for human biomonitoring

The comet assay has been extensively used in environmental and occupational human biomonitoring studies.  The review looks at recent trends and innovations within this field.  Highlights include non-invasively collected samples and the novel modification of the comet assay for the detection of global DNA methylation levels.  For more information on this subject, please refer to the original publication. 

Current and new developments for genotoxicity testing

The use of the comet assay in the genotoxicity testing of chemicals and nanomaterials was discussed at ICAW 2015 and some of the key points discussed were cell lines, 3D skin models, the OECD guideline, sample collection and storage. 

In vitro comet assay and in vitro skin model

While the in vitro comet assay is not yet formally recognised in regulatory toxicology, it is recommended as an appropriate test for use in the technical guidance documents of REACH. During the workshop, the development and standardisation of the use of the 3D skin models for in vitro genotoxicity testing was discussed. 

Plant comet assay

At ICAW 2015, the last 20 years plant comet assay research was reviewed and promising results were shown in the development of the plant comet assay scoring method. 

Technical aspects and critical parameters

ICAW 2015 participants were asked to indicate any technical problems they were experiencing and the technical aspects they considered important when performing the comet assay.  For a full breakdown of the responses, please refer to the original publication. 

Viability testing

Some ICAW 2015 participants were worried about the use of non-viable cells in the comet assay.  The publication discusses this matter in more detail, however, when using the comet assay for in vitro as well as in vivo genotoxicity testing, it is crucial to assess whether cells are viable until comet analysis.

Lysis time

Approximately a quarter of ICAW 2015 participants had questions regarding the importance of the duration of the lysis step.  Again, the publication discusses this matter in more detail, but the lysis time is not believed to be critical (although there are exceptions to this).

Detection of specific DNA lesions

According to the survey of ICAW delegates, a substantial amount of attendees were interested in ways to detect genotoxic lesions other than strand breaks and alkali-labile sites (ALS).  The usefulness of DNA repair enzymes (with endonuclease or lyase activity) to detect specific DNA lesions was discussed at the workshop, acknowledging that some base modifications are highly mutagenic, whereas others are not.

Electrophoresis

Previously, several ring trials have been conducted with the in vitro comet assay to study the effects of electrophoresis conditions.  Some areas of particular interest during the workshop were: “Adjusting sensitivity by altering electrophoresis conditions,” “Influence of pH on strand breaks detected” and “The voltage makes the difference”.  Again, for full interpretation please refer to the original publication. 

Scoring, reproducibility and robust results

The publication ends with a section on viewing comets and scoring them.  Themes include: anomalous comets (e.g. hedgehogs, ghosts and clouds) and comets which are found at the edge of the slide.  The publication also talks about quality control measures in the comet assay (e.g. reference cells) and how many cells should be scored.  

This article is a summary of some of the topics discussed in the publication.  We hope you enjoyed this round-up and we look forward to meeting you at ICAW 2017 at University of Navarra, Pamplona, in northern Spain, on August 28–31, 2017

For full information, please refer to the original publication: 

Gudrun Koppen, Amaya Azqueta, Bertrand Pourrut, Gunnar Brunborg, Andrew R. Collins, Sabine A. S. Langie; The next three decades of the comet assay: a report of the 11th International Comet Assay Workshop. Mutagenesis 2017; 32 (3): 397-408. doi: 10.1093/mutage/gex002