FORMATION OF 7,8-DIHYDRO-8-OXOGUANINE IN THE 1,2-DIOXETANE-INDUCED OXIDATION OF CALF THYMUS DNA - EVIDENCE FOR PHOTOSENSITIZED DNA-DAMAGE BY THERMALLY GENERATED TRIPLET KETONES IN THE DARK

Isolated calf thymus DNA was treated with the 1,2-dioxetanes 3-acetoxy methyl-3,4,4-trimethyl-1,2-dioxetane, 2,3-dimethylbenzofuran dioxetane , 3-hydroxymethyl-3,3,4-trimethyl-1,2-dioxetane (HTMD), 3,3,4,4-tetram ethyl-1,2-dioxetane and 3,4,4-trimethyl-1,2-dioxetane (TrMD), which on thermal decomposition generate triplet-excited carbonyl products. To monitor quantitatively the formation of the mutagenic oxidation produc t 7,8-dihydro-8-oxoguanine (8-oxoGua), a sensitive and selective HPLC electrochemical assay was used after acidic hydrolysis (HF/pyridine) o f the dioxetane-treated DNA. High yields of 8-oxoGua (up to ca 4% of t he available guanine) were obtained for HTMD and TrMD. Both were inves tigated in detail with respect to effects of concentration, time and t emperature. The oxidative reactivity of 1,2-dioxetanes was compared wi th several type I (benzophenone and riboflavin) and type II (methylene blue and rose bengal) photooxidants and disodium 1,4-etheno-2,3-benzo dioxin-1,4-dipropionate as a chemical source of singlet oxygen. The pe rsistence of 8-oxoGua towards oxidation by HTMD was examined in the re action with 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodGuo) and with o xidized DNA. It was shown that, indeed, 8-oxoGua is consumed in the ox idized DNA on prolonged exposure to an excess of HTMD. The reaction of 8-oxodGuo with HTMD afforded the two 4R and 4S* diastereomers of pen tofuranosyl)-4,8-dihydro-4-hydroxy-8-oxoguanine as main oxidation prod ucts. Trapping experiments with tert-butanol confirmed that hydroxyl r adicals are not involved, whereas the use of the triplet quenchers sod ium 9,10-dibromo-anthracene-2-sulfonate and 2,3-diazabicyclo[2.2.1]hep t-2-ene established that triplet-excited states are mainly responsible for the observed DNA oxidation through type I action (electron transf er chemistry). The role of singlet oxygen was tested by means of deute rium isotope effects in D2O versus H2O, but no definitive conclusion c ould be reached in regard to the involvement of O-1(2) in these oxidat ions. The present results reveal that 1,2-dioxetanes are efficient DNA oxidants and excellent tools to study photooxidation reactions of DNA in the dark.