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

Abstract— Isolated calf thymus DNA was treated with the 1,2-dioxetanes 3-acetoxymethyl-3,4,4-tri-methyl-1,2-dioxetane, 2,3-dimethylbenzofuran dioxetane, 3-hydroxymethyl-3,4,4-trimethyl-1,2-dioxeta-ne (HTMD), 3,3,4,4-tetramethyl-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 product 7,8-dihydro-8-oxoguanine (8-oxoGua), a sensitive and selective HPLC electrochemical assay was used after acidic hydrolysis (HF/pyridine) of the dioxetane-treated DNA. High yields of 8-oxoGua (up to ca 4% of the available guanine) were obtained for HTMD and TrMD. Both were investigated in detail with respect to effects of concentration, time and temperature. The oxidative reactivity of 1,2-dioxetanes was compared with several type I (benzophenone and riboflavin) and type II (methylene blue and rose bengal) photooxidants and disodium 1,4-etheno-2,3-ben-zodioxin-1,4-dipropionate as a chemical source of singlet oxygen. The persistence of 8-oxoGua towards oxidation by HTMD was examined in the reaction with 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodGuo) and with oxidized DNA. It was shown that, indeed, 8-oxoGua is consumed in the oxidized DNA on prolonged exposure to an excess of HTMD. The reaction of 8-oxodGuo with HTMD afforded the two 4R* and 4S* diastereomers of 9-(2-deoxy-ß-D-erythropentofuranosyl)-4,8-dihydro-4-hydroxy-8-oxoguanine as main oxidation products. Trapping experiments with teft-butanol confirmed that hydroxyl radicals are not involved, whereas the use of the triplet quenchers sodium 9,10-dibromo-anthra-cene-2-sulfonate and 2,3-diazabicyclo2.2.1]hept-2-ene established that triplet-excited states are mainly responsible for the observed DNA oxidation through type I action (electron transfer chemistry). The role of singlet oxygen was tested by means of deuterium isotope effects in D₂O versus H₂O, but no definitive conclusion could be reached in regard to the involvement of ¹0₂ in these oxidations. The present results reveal that 1,2-dioxetanes are efficient DNA oxidants and excellent tools to study photooxidation reactions of DNA in the dark.