Abstract
This study aimed to establish a protocol for cell dissociation from the nematode Caenorhabditis elegans (C. elegans) to assess the genotoxicity of the environmental pollutant benzo[a]pyrene (BaP) using the alkaline version of the single cell electrophoresis assay (comet assay). BaP genotoxicity was assessed in C. elegans (wild-type [WT]; N2, Bristol) after 48h exposure (0-40μM). Induction of comets by BaP was concentration-dependent up to 20μM; comet% tail DNA was ∼30% at 20μM 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 WT background levels suggesting that the 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 summary, our study provides a protocol to evaluate DNA damage of environmental pollutants in whole nematodes using the comet assay.
Highlights
The single-cell gel electrophoresis assay is widely used for the detection of DNA damage and repair in a variety of cells in vitro and in vivo (Olive and Banath, 2006)
Whereas both these studies aimed to investigate DNA damage in a particular population of cells in C. elegans our approach focused to establish a protocol for cell dissociation from the whole nematode C. elegans to assess genotoxicity of environmental pollutants using the comet assay, thereby providing a measure of genotoxicity in the whole organism
Because C. elegans are fed on E. coli OP50 it is essential to analyse the possible effects of BaP on bacterial growth before introducing the carcinogen-infused OP50 to the nematodes
Summary
The single-cell gel electrophoresis assay (comet assay) is widely used for the detection of DNA damage and repair in a variety of cells in vitro and in vivo (Olive and Banath, 2006). The assay has the advantage of being a rapid, sensitive and relatively inexpensive method. It is commonly used in genotoxicity testing but it has widespread applications in environmental biomonitoring and human population monitoring (Amaeze et al, 2015; Forchhammer et al, 2012). Due to the availability of the whole genome sequence, C. elegans has been subjected to gene expression studies (Steinberg et al, 2008). The mutational signatures of different chemotherapeutic drugs and environmental carcinogens in C. elegans were studied by whole genome sequencing (Meier et al, 2014)
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