Mechanistic studies of photosensitised DNA strand breakage by iodinated DNA ligands

Full text: Halogenated analogues of Hoechst 33258, which bind in the minor groove of DNA, are being investigated as potential radiosensitisers and photosensitisers. A proposed model of sensitisation involves radiation-induced dehalogenation of the ligand, generating a carbon-centered radical, which abstracts a deoxyribosyl H-atom, leading to a DNA strand break. There is more than ten fold difference in sensitisation activity between the isomeric ligands meta- and ortho-iodo Hoechst as measured by a single strand breakage assay using pBR322, with the ortho-compound being the more active. The basis for this difference was investigated by comparing the relative yields of dehalogenation and strand breakage for each isomer. Dehalogenation was monitored by HPLC, and DNA damage was assayed by agarose gel electrophoresis. Under aqueous conditions and in the presence of salt, which favours minor groove binding of DNA, both compounds were approximately equally efficient at inducing a strand break for a given number of dehalogenation events. By contrast, the quantum yields for dehalogenation were 0.56 and 0.049 for the ortho- and meta-isomers respectively. Therefore, the difference between the two isomers as photosensitisers of DNA cleavage can be attributed to the difference in the UV-induced dehalogenation efficiency rather than a difference in the efficiency in the corresponding ligand radical species. Aqueous ethanol reduces the DNA strand breakage efficiency of both compounds, due to the action of ethanol as a radical scavenger. However, the meta-compound is more markedly affected, with the ligand radical species having a DNA strand breakage efficiency (dehalogenation events per ssb) of 139, compared with a value of 43 for ortho-iodo Hoechst. This indicates that the radical generated from the ortho-ligand might be more deeply embedded in the minor groove, which is less accessible to scavenger activity than the meta- counterpart. Sensitisation of DNA strand breakage by ionizing radiation could only be demonstrated for ortho-iodo Hoechst, and only in a buffered solvent system which contained 20% ethanol as an hydroxyl radical scavenger