Frontiers in Genetics (May 2015)
Comparing the alkaline comet with 53BP1 immunocytochemistry assay: sensitivity and cost/time effectiveness
Abstract
Both the comet and p53-binding protein 1 (53BP1) Immunocytochemistry (ICC) assays are well known, sensitive techniques to determine DNA damage, albeit with different perspectives. Whilst the comet assay determines DNA strand breaks (SSB/DSB/ alkali labile sites), the 53 BP1 assay reflects the cellular response to DSBs that promotes the end-joining of distal DNA ends. Both assays however require fluorescence image analysis at the cellular level under a microscope (i.e. different comet parameters using commercially available software, and counting of foci per cell). Whilst these techniques are considered to be rapid compared to many other established methodologies, their relative sensitivity and effectiveness have however not been compared effectively. In this study we compare the two techniques examining their sensitivity as well as cost/time effectiveness. SH-SY5Y dopaminergic neuronal cell line, a commonly used neuronal cell line for neurotoxicity testing, was exposed for 6 and 24h to a range of concentrations (150, 300 and 900 µM) of copper (Cu), a known toxicant which exerts its toxicity mainly via free radical production. Exposure of the cells to Cu showed a concentration dependent increase in DNA damage. The Student’s T-test was used to analyse the data from the comet and ICC assays showing that there was a significant difference (p<0.001) at 150µM (data normalised to cell protein content). This analysis indicated that ICC was more sensitive compared to the comet assay at this concentration. At mid concentration (i.e. 300 µM), no significant difference was observed between the two assays. In contrast, the comet assay was able to measure DNA damage at higher concentration (900 µM).This was mainly due to overlapping foci for ICC, which made it difficult to measure the damage quantitatively. The cost of running the experiments was compared, taking into account the costs of reagents, slides, fluorochromes and other consumables from the suppliers (i.e. Sigma, Fisher and ABCAM). The total cost was obtained and then the amount of materials used per assay (defined as 16 slides), with cost per sample (2 slides). The cost analysis indicated that the overall cost of the comet assay per sample is £5.66 while the 53BP1 ICC per sample is £1.80. The time taken per assay was measured from end of exposure to start of analysis. Here the comet assay and ICC require a similar amount of time to complete (6h). However the analysis for each sample is significantly longer with the ICC (30min) while the comet assay takes 10min. Overall although the biological damage measured by the two assays are mechanistically different (one reflecting SSB/DSB and another only DSB) the alkaline comet assay appears to be more suitable for larger sample sizes that have high levels of DNA damage, while the ICC due to its higher sensitivity and cost effectiveness is more suitable to smaller studies which aim to determine DNA damage at lower levels of exposure to genotoxicants.
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