Daniel Ballmaier

Photochemical and Photobiotogical Studies with Acridine and Phenanthridine Hydroperoxides in Cell‐free DNA

Abstract— The acridine and phenanthridine hydroperoxides 3 and 7 were synthesized as photochemical hydroxyl radical sources for oxidative DNA damage studies. The generation of hydroxyl radicals upon UVA irradiation (Λ. = 350 nm) was verified by trapping experiments with 5,5‐di‐methyl‐1‐pyrroline N‐oxide and benzene. The enzymatic assays of the damage in cell‐free DNA from bacteriophage PM2 caused by the acridine and phenanthridine hydroperoxides 3 and 7 under near‐UVA irradiation revealed a wide range of DNA modifications. Particularly, extensive single‐strand break formation and DNA base modifications sensitive to formamidopyrimidine DNA glycosylase (Fpg protein) were observed. In the photooxida‐tion of calf thymus DNA, up to 0.69±0.03% 8‐oxo‐7,8‐dihydroguanine was formed by the hydroperoxides 3 and 7 on irradiation, whose yield was reduced up to 40% in the presence of the hydroxyl radical scavengers mannitol and fert‐butanol. The acridine and phenanthridine hydroperoxides 3 and 7 also induce DNA damage through the type I photooxidation process, for which photoinduced electron transfer from 2′‐deoxyguanosine to the singlet states of 3 and 7 was estimated by the Rehm‐Weller equation to possess a negative Gibbs free energy of cα ‐5 kcal/ mol. Control experiments with the sensitizers acridine 1 and the acridine alcohol 4 in calf thymus and PM2 DNA confirmed the photosensitizing propensity of the UVA‐ab‐sorbing chromophores. The present study emphasizes that for the development of selective and efficient photochemical hydroxyl radical sources, chromophores with low photosensitizing ability must be chosen to avoid type I and type II photooxidation processes.

Photochemical and Photobiological Studies of a Furonaphthopyranone as a Benzo-spaced Psoralen Analog in Cell-free and Cellular DNA

Abstract— Photobiological activities of the benzo‐spaced psoralen analog furonaphthopyranone 3 have been investigated in cell‐free and cellular DNA. The molecular geometry parameters of 3 suggest that it should not form interstrand crosslinks with DNA. With cell‐free DNA no evidence for crosslinking but also not for monoadduct formation was obtained; rather, the unnatural furocoumarin 3 induces oxidative DNA modifications under near‐UVA irradiation. The enzymatic assay of the photosensitized damage in cell‐free PM2 DNA revealed the significant formation of lesions sensitive to formamidopyrimidine DNA glyco‐sylase (Fpg protein). In the photooxidation of calf thymus DNA by the furonaphthopyranone 3, 0.29±0.02% 8‐oxo‐7,8‐dihydroguanine (8‐oxoGua) was observed. With 2′‐deoxyguanosine (dGuo), the guanidine‐releasing photooxidation products oxazolone and oxoimidazolidine were formed predominately, while 8‐oxodGuo and 4‐HO‐8‐oxodGuo were obtained in minor amounts. The lack of a significant D2O effect in the photooxidation of DNA and dGuo reveals that singlet oxygen (type II process) plays a minor role; control experiments with tert‐butanol and mannitol confirm the absence of hydroxyl radicals as oxidizing species. The furonaphthopyranone 3 (Ered= ‐1.93±0.03V) should act in its singlet‐excited state as electron acceptor for the photooxidation of dGuo (δGETca– kcal/mol), which corroborates photoinduced electron transfer (type I) as a major DNA‐oxidizing mechanism. A comet assay in Chinese hamster ovary (CHO) AS52 cells demonstrated that the psoralen analog 3 damages cellular DNA upon near‐UVA irradiation; however, no photosensitized mutagenicity was observed in CHO AS52 cell cultures

Oxidative DNA Damage Profiles in Mammalian Cells

Reactive oxygen species (ROS) are formed inside cells not only under the influence of exogenous agents (visible light, ionizing radiation, and many oxidants such as peroxides or quinones), but also under normal (physiological) conditions as byproducts of oxygen metabolism and other cellular redox reactions (Pryor 1986; Halliwell and Gutteridge 1986; Sies 1986; Clayson et al. 1994). ROS such as hydroxyl radicals and singlet oxygen are a serious threat to the integrity of the cellular genome, since they efficiently react with DNA to generate many types of DNA modifications, at least some of which are pre- mutagenic (Breimer 1990; Halliwell and Aruoma 1991; Epe 1991; Feig et al. 1994). Steady-state levels of 8-hydroxyguanine (8-oxoG) and other oxidative DNA base modifications observed in untreated cells indicate that the various cellular defense and DNA repair systems (Demple and Harrison 1994) do not completely eliminate the mutagenic risk associated with ROS formation even under normal growth conditions. This led to the assumption that oxidative DNA damage is a causal or ancillary risk factor for the development of cancer and several age-correlated degenerative diseases (Ames 1983; Wallace 1992; Gutteridge 1993). A strategy to verify this hypothesis and to quantify the mutagenic risk associated with oxidative DNA damage could be to determine (a) what type of oxidative DNA damage profile (pattern of DNA modifications) is generated in the cells under the conditions of interest and (b) the mutagenicity associated with this damage profile. Then, the quantification of any suitable marker modification of this damage profile should allow an estimation of the mutagenicity to be expected.

Oxidative DNA Damage Induced by Dioxetanes, Photosensitizing Ketones, and Photo-Fenton Reagents

In the last decade, the importance of oxidative DNA damage in mutagenesis, carcinogenesis, aging, and various diseases has prompted intensive investigations of chemical, biochemical, and biological aspects of DNA oxidation caused by reactive species, which are involved in oxidative stress (Sies 1991). Oxidative degradation of DNA causes mutations predominantly at GC base pairs to yield single base substitutions and G to T transversions (Piette 1991). Consequently, the DNA transformation efficiency is diminished and replication is inhibited.