Response of Bromodeoxyuridine-Substituted Chinese Hamster Cells to UVA Light Exposure in the Presence of Hoechst Dye #33258: Survival and DNA Repair Studies

Previous work has established that DNA double-strand breaks (DSBs) are formed when Chinese hamster cells are substituted with 5-bromo-2′-deoxyuridine (BrdU) and exposed to UVA light in the presence of Hoechst dye #33258. Double-strand breaks produced by this treatment ($5.1\times 10^{-6}\ {\rm DSBs}/{\rm BrdU}\ \text{residue}/{\rm kJ}\ {\rm m}^{-2}$) were found to depend linearly on the level of BrdU substitution, Hoechst dye and fluence of UVA light. To examine the biological consequences of these novel DSBs, clonogenic assays were used to score cell survival, and elution assays were used to measure strand break levels at various times after photolysis. Using this system, marked cell killing was observed; photosensitivity could be increased by four orders of magnitude compared to cells without BrdU and dye. Decreases in the F₀ value and the shoulder of survival curves followed increasing levels of BrdU substitution. In addition, the results indicate that DSBs produced by this photolysis protocol are two to three times more effective in causing cell killing than the DSBs produced by the action of ionizing radiation. To investigate the cause of the toxicity, repair of DSBs after photolysis was measured. Unexpectedly, DSB levels increased two- to threefold over 1 h at 37°C, then decreased to initial damage levels over the next 2 h. The implications of this break induction are discussed in terms of mechanism and cell killing.