Investigations of DNA damage in living cells: Application of the comet assay and the development of a novel time-resolved fluorescence measurement technique.

Abstract

Non-steroidal anti-inflammatory drug (NSAID) photo-induced DNA damage in human peripheral blood mononuclear cells measured using the alkaline comet assay is presented. Whereas Tiaprofenic Acid photo-induced DNA damage is promptly produced (i.e., observed at low radiation doses), Ketoprofen (KP) photo-induced DNA damage is delayed (observed at relatively higher radiation doses). This prompt and delayed effect is observed with UVA (320--400 nm), UVB (290--320 nm) and solar simulated radiation and is attributed to the NSAIDs' different photochemical properties. The results from these experiments mark the first account of NSAID photo-induced DNA damage in living cells. The neutral version of the comet assay, carried out using HL60 cells, revealed that KP photo-induced DNA damage did not lead to apoptosis, indicating that HL60 cells can sustain and possibly recover from this damage. A new technique for measuring DNA damage based on the time-resolved fluorescence decay measurements of PicoGreenRTM-DNA complexes is also presented. PicoGreenRTM exhibits a longer fluorescence lifetime when complexed to double-stranded DNA compared single-stranded DNA. This discovery allows for the quantification of single-stranded DNA in a given sample. This technique, which also incorporates key concepts such as alkaline unwinding buffers and higher unwinding rates of damaged DNA compared to non-damaged DNA, was able to reproducibly measure and differentiate DNA damage from 0--100 Gray of gamma radiation. The results presented include experiments carried out using CT-DNA as well as DNA isolated from sheep white blood cells, suggesting its potential use with isolated DNA from any eukaryotic cell.