The use of silver-stained "comets" to visualize DNA damage and repair in normal and Xeroderma pigmentosum fibroblasts after exposure to simulated solar radiation

The alkaline and neutral comet assays have been widely used to assess DNA d amage and repair in individual cells after itt vivo or in vitro exposure to chemical or physical genotoxins. Cells processed under neutral conditions generate comets primarily from DNA double strand breaks, whereas under alka line conditions, comets arise from DNA single and double strand breaks and alkali-labile lesions. A modified version of the. alkaline comet assay, as described here, used silver stain to visualize the comets and a (TM)Gelbond base to facilitate the manipulation and processing of samples, To demonstr ate how these modifications improve the assay, fibroblasts derived from bot h normal and Xeroderma pigmentosum (Xp) individuals were exposed to simulat ed solar radiation and the resulting DNA damage and repair evaluated and co mpared with results from the relevant literature. Comets from normal fibrob lasts reached their maximum length at about an hour after irradiation. Dose -dependent increases in comet length were observed up to at least 360 mJ/cm (2). In contrast, comet lengths from repair deficient Xp fibroblasts were s horter than normal cells reflecting their reduced capacity to generate sing le strand breaks by the excision of DNA dimers. For incubation times of mor e than 1 h, comet lengths from normal fibroblasts underwent a time-dependen t decrease, supporting the contention that this change was related to the l igation step in the DNA repair process. These changes were compatible with the model of DNA damage and repair established by others for ultraviolet ra diation.