Protocol to evaluate DNA damage in whole nematodes using the comet assay

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• Created: Monday, 08 August 2016 09:32

The scientists at King’s College, London, and their collaborators have been working to establish a protocol for cell dissociation from the nematode Caenorhabditis elegans.  Ultimately, these cells will be used to assess the genotoxicity of the environmental pollutant benzo[a]pyrene (BaP) using the alkaline comet assay.  For full background and experimental information, please refer to the original publication (details below).

C. elegans is a useful model organism for studying toxicogenomic responses to environmental pollutants at the molecular level as well as at the level of the organism.  Due to the availability of the whole genome sequence, C. elegans has been subjected to gene expression studies.

Polycyclic aromatic hydrocarbons (PAHs) are products of incomplete combustion of fossil fuels and are present in the particulate phase of polluted air, diesel exhaust, and tobacco smoke. BaP is often studied as a model PAH and is listed as Group 1 human carcinogen by the International Agency for Research on Cancer (IARC).

BaP genotoxicity was assessed in C. elegans after 48 h exposure (0–40 μM).

The alkaline comet assay was conducted and comets were analysed using a Leica fluorescence microscope. DNA damage was scored using the Comet Assay IV capture system (version 4.11; Perceptive Instruments, UK). Fifty cell nucleoids were assessed per slide, and each sample was analysed in duplicate. All samples were measured blind. The tail intensity (% tail DNA), defined as the percentage of DNA migrated from the head of the comet into the tail, was used as a measure of DNA damage induced, which is a meaningful end-point to assess genotoxicity.

The investigators reported that induction of comets by BaP was concentration-dependent up to 20 μM; comet% tail DNA was ∼30% at 20 μM BaP and ∼10% in controls. Similarly, BaP-induced DNA damage was evaluated in C. elegans mutant strains deficient in DNA repair. In xpa-1 and apn-1 mutants BaP-induced comet formation was diminished to wild-type background levels.  This led the investigators to conclude that the DNA damage formed by BaP that is detected in the comet assay is not recognised in cells deficient in nucleotide and base excision repair, respectively.

In conclusion, the scientists were able to establish a cell dissociation protocol of the whole nematode C. elegans that allowed the assessment of genotoxicity of environmental pollutants using the comet assay. They found that the ubiquitous environmental pollutant BaP induces comets in wild-type C. elegans in a dose-dependent manner and that transgenic C. elegans strains can be used to further characterise BaP-induced DNA damage pathways. The scientists believe that the results suggest both nucleotide and base excision repair-related single strand breaks contribute to comet formation induced by BaP.

The investigators reported that future applications of the established protocol may include the modified comet assay using repair-specific endonucleases to detect oxidative damage to DNA.

For full details of the methods and other articles used for reference in this article, please refer to the original publication:  The application of the comet assay to assess the genotoxicity of environmental pollutants in the nematode Caenorhabditis elegans.  Soudabeh Imanikia, Francesca Galea, Eszter Nagy, David H. Phillips, Stephen R. Stürzenbaum, and Volker M. Arlt. Environ Toxicol Pharmacol. 2016 Jul; 45: 356–361.