Ozone-Induced DNA Damage: A Pandora's Box of Oxidatively Modified DNA Bases.

Ozone is a major component of air pollution and carries potentially mutagenic and harmful affects to health. The oxidation of isolated calf thymus DNA (CT-DNA) led to the nearly quantitative loss of normal DNA 2'-deoxyribonucleosides in the following order: T > G > C ≫ A. The major modification of pyrimidines (T, C, and 5-methylcytosine (5mC)) was the corresponding 5-hydroxyhydantoin derivative after complete digestion of DNA to its component 2'-deoxyribonucleosides. The oxidation of 5mC was 2.5-fold more susceptible than C considering the relative mole fraction of 5mC to C in CT-DNA. Other common oxidation products of pyrimidines (e.g., 5,6-dihydroxy-5,6-dihydropyrimidines, the so-called pyrimidine 5,6-glycols) were formed with a lower yield than 5-hydroxyhydantoin derivatives. In addition, several common oxidation products of G were observed (e.g., 8-oxo-7,8-dihydroguanine (8oxoG)) albeit with relatively minor yields. The sum of individual products was notably less than the loss of 2'-deoxyribonucleosides from which they were derived. In a search for additional products, we discovered the formation of pyrimidine ring fragments, predominantly N-formamide and N-urea, which were measured as a dinucleotide next to a nonmodified nucleotide upon partial digestion of oxidized DNA. Interestingly, the latter fragments were also observed in dinucleotides containing 8oxoG, indicating the formation of tandem lesions during ozonolysis of DNA. The oxidation of DNA upon exposure to ozone can be explained by reactions of an intermediate ozonide. These studies underline the complexity of ozone-induced DNA damage and provide valuable information to assess the formation of this damage in cellular DNA.

DNA Base Modifications Mediated by Femtosecond Laser-Induced Cold Low-Density Plasma in Aqueous Solutions.

Applications based on near-infrared femtosecond laser-induced plasma in biological materials involve numerous ionization events that inevitably mediate physicochemical effects. Here, the physical chemistry underlying the action of such plasma is characterized in a system of biological interest. We have implemented wavefront shaping techniques to control the generation of laser-induced low electron density plasma channels in DNA aqueous solutions, which minimize the unwanted thermo-mechanical effects associated with plasma of higher density. The number of DNA base modifications per unit of absolute energy deposited by such cold plasma is compared to those induced by either ultraviolet or standard ionizing radiation (γ-rays). Analyses of various photoinduced, oxidative, and reductive DNA base products show that the effects of laser-induced cold plasma are mainly mediated by reactive radical species produced upon the ionization of water, rather than by the direct interaction of the strong laser field with DNA. In the plasma environment, reactions among densely produced primary radicals result in a dramatic decrease in the yields of DNA damages relative to sparse ionizing radiation. This intense radical production also drives the local depletion of oxygen.

Thymidine decomposition induced by low-energy electrons and soft X rays under N2 and O2 atmospheres.

A novel technique has been employed to investigate the simultaneous damage to DNA components induced by soft X rays (1.5 keV) and low-energy electrons (0-30 eV) in thin films of thymidine deposited on glass and tantalum substrates and irradiated under atmospheric pressure and temperature. The films were surrounded by either an N2 or O2 environment. The formation of four radiation-induced products is reported in this article: base release, 5-hydroxymethyl-2'-deoxyuridine (5-HMdUrd), 5-formyl-2'-deoxyuridine (5-FordUrd) and 5,6-dihydrothymidine (5,6-DHThd). Analysis with LC-MS/MS shows larger damage yields in the samples deposited on tantalum than in those deposited on glass, which is attributed to the interaction of the additional low-energy electrons that are photoemitted from the metal surface. From a comparison of the results obtained from N2 and O2 environment, we report a dramatic effect from 6 O2: an approximately threefold increase in the yield of products, attributed to the reaction of O2 with initial carbon-centered thymidine radicals generated in the film during irradiation.

Hydroxyl-radical-induced oxidation of 5-methylcytosine in isolated and cellular DNA.

The methylation and oxidative demethylation of cytosine in CpG dinucleotides plays a critical role in the regulation of genes during cell differentiation, embryogenesis and carcinogenesis. Despite its low abundance, 5-methylcytosine (5mC) is a hotspot for mutations in mammalian cells. Here, we measured five oxidation products of 5mC together with the analogous products of cytosine and thymine in DNA exposed to ionizing radiation in oxygenated aqueous solution. The products can be divided into those that arise from hydroxyl radical (•OH) addition at the 5,6-double bond of 5mC (glycol, hydantoin and imidazolidine products) and those that arise from H-atom abstraction from the methyl group of 5mC including 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC). Based on the analysis of these products, we show that the total damage at 5mC is about 2-fold greater than that at C in identical sequences. The formation of hydantoin products of 5mC is favored, compared to analogous reactions of thymine and cytosine, which favor the formation of glycol products. The distribution of oxidation products is sequence dependent in specific ODN duplexes. In the case of 5mC, the formation of 5hmC and 5fC represents about half of the total of •OH-induced oxidation products of 5mC. Several products of thymine, cytosine, 5mC, as well as 8-oxo-7,8-dihydroguanine (8oxoG), were also estimated in irradiated cells.

Side-by-side comparison of DNA damage induced by low-energy electrons and high-energy photons with solid TpTpT trinucleotide.

The genotoxic effects of high-energy ionizing radiation have been largely attributed to the ionization of H2O leading to hydroxyl radicals and the ionization of DNA leading mostly to damage through base radical cations. However, the contribution of low-energy electrons (LEEs; ≤ 10 eV), which involves subionization events, has been considered to be less important than that of hydroxyl radicals and base radical cations. Here, we compare the ability of LEEs and high-energy X-ray photons to induce DNA damage using dried thin films of TpTpT trinucleotide as a simple and representative model for DNA damage. The main radiation-induced damage of TpTpT as measured by high-performance liquid chromatography (HPLC) with UV detection and HPLC coupled to tandem mass spectrometry analyses included thymine release (-Thy), strand breaks (pT, Tp, pTpT, TpTp, and TpT), and the formation of base modifications [5,6-dihydrothymine (5,6-dhT), 5-hydroxymethyluracil (5-hmU), and 5-formyluracil (5-fU)]. The global profile of products was very similar for both types of radiation indicating converging pathways of formation. The percent damage of thymine release, fragmentation, and base modification was 20, 19, and 61 for high-energy X-rays, respectively, compared to 35, 13, and 51 for LEEs (10 eV). Base release was significantly lower for X-rays. In both cases, phosphodiester bond cleavage gave mononucleotides (pT and Tp) and dinucleotides (pTpT and TpTp) containing a terminal phosphate as the major fragments. For base modifications, the ratio of reductive (5,6-dhT) to oxidative products (5-hmU plus 5-fU) was 0.9 for high-energy X-rays compared to 1.7 for LEEs. These results indicate that LEEs give a similar profile of products compared to ionizing radiation.

Generation of guanine-thymine cross-links in human cells by one-electron oxidation mechanisms.

The one-electron oxidation of cellular DNA in cultured human HeLa cells initiated by intense nanosecond 266 nm laser pulse irradiation produces cross-links between guanine and thymine bases (G*-T*), characterized by a covalent bond between C8 guanine (G*) and N3 thymine (T*) atoms. The DNA lesions were quantified by isotope dilution LC-MS/MS methods in the multiple reaction-monitoring mode using isotopically labeled [(15)N, (13)C]-nucleotides as internal standards. Among several known pyrimidine and 8-oxo-7,8-dihydroguanine lesions, the G*-T* cross-linked lesions were detected at levels of ~0.21 and 1.19 d(G*-T*) lesions per 10(6) DNA bases at laser intensities of 50 and 280 mJ/cm(2)/pulse, respectively.

Radiation-induced formation of 2',3'-dideoxyribonucleosides in DNA: a potential signature of low-energy electrons.

We have identified a series of modifications of the 2'-deoxyribose moiety of DNA arising from the exposure of isolated and cellular DNA to ionizing radiation. The modifications consist of 2',3'-dideoxyribonucleoside derivatives of T, C, A, and G, as identified by enzymatic digestion and LC-MS/MS. Under dry conditions, the yield of these products was 6- to 44-fold lower than the yield of 8-oxo-7,8-dihydroguanine. We propose that 2',3'-dideoxyribonucleosides are generated from the reaction of low-energy electrons with DNA, leading to cleavage of the C3'-O bond and formation of the corresponding C3'-deoxyribose radical.

Profiling cytosine oxidation in DNA by LC-MS/MS.

Spontaneous and oxidant-induced damage to cytosine is probably the main cause of CG to TA transition mutations in mammalian genomes. The reaction of hydroxyl radical (·OH) and one-electron oxidants with cytosine derivatives produces numerous oxidation products, which have been identified in large part by model studies with monomers and short oligonucleotides. Here, we developed an analytical method based on LC-MS/MS to detect 10 oxidized bases in DNA, including 5 oxidation products of cytosine. The utility of this method is demonstrated by the measurement of base damage in isolated calf thymus DNA exposed to ionizing radiation in aerated aqueous solutions (0-200 Gy) and to well-known Fenton-like reactions (Fe(2+) or Cu(+) with H(2)O(2) and ascorbate). The following cytosine modifications were quantified as modified 2'-deoxyribonucleosides upon exposure of DNA to ionizing radiation in aqueous aerated solution: 5-hydroxyhydantoin (Hyd-Ura) > 5-hydroxyuracil (5-OHUra) > 5-hydroxycytosine (5-OHCyt) > 5,6-dihydroxy-5,6-dihydrouracil (Ura-Gly) > 1-carbamoyl-4,5-dihydroxy-2-oxoimidazolidine (Imid-Cyt). The total yield of cytosine oxidation products was comparable to that of thymine oxidation products (5,6-dihydroxy-5,6-dihydrothymine (Thy-Gly), 5-hydroxy-5-methylhydantotin (Hyd-Thy), 5-(hydroxymethyl)uracil (5-HmUra), and 5-formyluracil (5-ForUra)) as well as the yield of 8-oxo-7,8-dihydroguanine (8-oxoGua). The major oxidation product of cytosine in DNA was Hyd-Ura. In contrast, the formation of Imid-Cyt was a minor pathway of DNA damage, although it is the major product arising from irradiation of the monomers, cytosine, and 2'-deoxycytidine. The reaction of Fenton-like reagents with DNA gave a different distribution of cytosine derived products compared to ionizing radiation, which likely reflects the reaction of metal ions with intermediate peroxyl radicals or hydroperoxides. The analysis of the main cytosine oxidation products will help elucidate the complex mechanism of oxidative degradation of cytosine in DNA and probe the consequences of these reactions in biology and medicine.