ABSTRACT
The suitability of the comet assay for quantifying DNA repair capacity at individual level was studied following the kinetics of nucleotide excision repair (NER) in human lymphocytes from four healthy donors, at various time steps after a single dose of UVC. A significant increase of DNA migration was seen as soon as 20 min after UV exposure, reaching the peak within 60-90 min. Afterwards, a rapid decline was observed, approaching the basal level at 180-240 min. The increase could be ascribed to excision activity, while the reduction to gap filling and rejoining, as demonstrated by the effects of phase-specific inhibitors, novobiocin and aphidicolin. Therefore, the comet assay should allow following the biphasic kinetics of NER. Wide inter-individual differences were observed, although repeated tests on the same donor cells revealed a large experimental variation. To quantitatively compare the individual patterns, a mathematical model was developed that adequately fitted the experimental results and estimated appropriate descriptors for each phase and for each donor. A second approach was also used to directly compare the distributions of damaged cells and to assess the differences between donors and between experiments visualizing them as reciprocal distances on a two-dimensional space computed with a principal component analysis (PCA). The results confirmed the inter-individual differences, but also the strong influence of experimental factors of the comet assay. The two approaches provided the means of accurately comparing DNA repair kinetics at individual level, taking also into account the experimental variability which poses serious doubts on the suitability of the comet assay. Nevertheless, since this methodology allows a detailed analysis of repair kinetics and it is potentially very useful for identifying individual with reduced repair capacity, further efforts have to be addressed to improve the reproducibility of the comet assay.
