PHOTOLYSIS OF N-HYDROXYPYRIDINETHIONES - A NEW SOURCE OF HYDROXYL RADICALS FOR THE DIRECT DAMAGE OF CELL-FREE AND CELLULAR DNA

N-Hydroxypyridine-2-thione (2-HPT), known to release hydroxyl radicals on irradiation with visible light, and two related compounds, viz. N- hydroxypyridine-4-thione (4-HPT) and N-hydroxyacridine-9-thione (HAT), were tested for their potency to induce DNA damage in L1210 mouse leu kemia cells and in isolated DNA from bacteriophage PM2. DNA single-str and breaks and modifications sensitive to various repair endonucleases (Fpg protein, endonuclease III, exonuclease III, T4 endonuclease V) w ere quantified. Illumination of cell-free DNA in the presence of 2-HPT and 4-HPT gave rise to damage profiles characteristic for hydroxyl ra dicals, i.e. single-strand breaks and the various endonuclease-sensiti ve modifications were formed in the same ratios as after exposure to e stablished hydroxyl radical sources. In contrast, HAT plus light gave rise to a completely different DNA damage profile, namely that charact eristic for singlet oxygen. Experiments with various scavengers (t-but anol, catalase, superoxide dismutase) and in D2O as solvent confirmed that hydroxyl radicals are directly responsible for the DNA damage cau sed by photoexcited 2-HPT and 4-HPT, while the damage by HAT plus ligh t is mediated by singlet oxygen and type I reactions. The type of DNA damage characteristic of hydroxyl radicals was also observed in L1210 mouse leukemia cells when treated with 2-HPT plus light or with H2O2 a t 0 degrees C. t-Butanol (2%) inhibited the cellular DNA damage by sim ilar to 50%. A dose of 2-HPT plus light that generated single-strand b reaks at a frequency of 5 x 10(-7)/bp was associated with 50% cell sur vival. No DNA damage and cytotoxicity was observed after treatment wit h 2-HPT in the dark. We propose that 2-HTP and 4-HTP may serve as new agents to study the consequences of DNA damage induced by hydroxyl rad icals in cells. In addition, the data provide direct evidence that hyd roxyl radicals are ultimately responsible for the genotoxic effects ca used by H2O2 in the dark.