Photolysis of N-hydroxpyridinethiones: 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 leukemia cells and in isolated DNA from bacteriophage PM2. DNA single-strand breaks and modifications sensitive to various repair endonucleases (Fpg protein, endonuclease III, exonuclease III, T4 endonuclease V) were quantified. Illumination of cell-free DNA in the presence of 2-HPT and 4-HPT gave rise to damage profiles characteristic for hydroxyl radicals, i.e. single-strand breaks and the various endonuclease-sensit…

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

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 glycosylase (Fpg protein). In the photooxidation of calf thymus DNA by the furonaphthopyranone 3, 0.2…

DNA damage by peroxynitrite characterized with DNA repair enzymes.

The DNA damage induced by peroxynitrite in isolated bacteriophage PM2 DNA was characterized by means of several repair enzymes with defined substrate specificities. Similar results were obtained with peroxynitrite itself and with 3-morpholinosydnonimine (SIN-1), a compound generating the precursors of peroxynitrite, nitric oxide and superoxide. A high number of base modifications sensitive to Fpg protein which, according to HPLC analysis, were mostly 8-hydroxyguanine residues, and half as many single-strand breaks were observed, while the numbers of oxidized pyrimidines (sensitive to endonuclease III) and of sites of base loss (sensitive to exonuclease III or T4 endonuclease V) were relativ…

[33] Use of repair endonucleases to assess DNA damage by peroxynitrite

Publisher Summary This chapter discusses the use of repair endonucleases to assess DNA damage by peroxynitrite. Repair endonucleases allow a convenient quantification of various types of oxidative modifications induced by peroxynitrite, both in cultured cells and in cell-free DNA. The high sensitivity of the assays allows highly ectotoxic exposure conditions to be avoided—as well as the generation of secondary DNA modifications—that often become a problem at high levels of damage because primary DNA oxidation products can be orders of magnitude more reactive than the original bases, as demonstrated for the reaction of 8-hydroxyguanine with singlet oxygen. The ratio of the various types of m…

Photochemical and photobiological studies with acridine and phenanthridine hydroperoxides in cell-free DNA.

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 (lambda = 350 nm) was verified by trapping experiments with 5,5-dimethyl-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 photooxidat…

Contributions - C: Carcinogenic Factors: Endogenous

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-…

DNA damage by bromate: Mechanism and consequences

Abstract Exposure of mammalian cells to bromate (BrO3−) generates oxidative DNA modifications, in particular 7,8-dihydro-8-oxo-guanine (8-oxoG). The damaging mechanism is quite unique, since glutathione, which is protective against most oxidants and alkylating agents, mediates a metabolic activation, while bromate itself does not react directly with DNA. Neither enzymes nor transition metals are required as catalysts in the activation. The ultimate DNA damaging species has not yet been established, but experiments under cell-free conditions suggest that neither molecular bromine nor reactive oxygen species such as superoxide, hydrogen peroxide or singlet oxygen are involved. Rather bromine …