J. Richard Wagner

Oxidation Reactions of Cytosine DNA Components by Hydroxyl Radical and One-Electron Oxidants in Aerated Aqueous Solutions

Indirect evidence strongly suggests that oxidation reactions of cytosine and its minor derivative 5-methylcytosine play a major role in mutagenesis and cancer. Therefore, there is an emerging necessity to identify the final oxidation products of these reactions, to search for their formation in cellular DNA, and to assess their mutagenic features. In this Account, we report and discuss the main *OH and one-electron-mediated oxidation reactions, two of the most potent sources of DNA damage, of cytosine and 5-methylcytosine nucleosides that have been recently characterized. The addition of *OH to the 5,6-unsaturated double bond of cytosine and 5-methylcytosine generates final degradation products that resemble those observed for uracil and thymine. The main product from the oxidation of cytosine, cytosine glycol, has been shown to undergo dehydration at a much faster rate as a free nucleoside than when inserted into double-stranded DNA. On the other hand, the predominant *OH addition at C5 of cytosine or 5-methylcytosine leads to the formation of 5-hydroxy-5,6-dihydro radicals that give rise to novel products with an imidazolidine structure. The mechanism of the formation of imidazolidine products is accounted for by rearrangement reactions that in the presence of molecular oxygen likely involve an intermediate pyrimidine endoperoxide. The reactions of the radical cations of cytosine and 5-methylcytosine are governed by competitive hydration, mainly at C6 of the pyrimidine ring, and deprotonation from the exocyclic amino and methyl group, leading in most cases to products similar to those generated by *OH. 5-Hydroxypyrimidines, the dehydration products of cytosine and uracil glycols, have a low oxidation potential, and their one-electron oxidation results in a cascade of decomposition reactions involving the formation of isodialuric acid, dialuric acid, 5-hydroxyhydantoin, and its hydroxyketone isomer. In biology, GC --> AT transitions are the most common mutations in the genome of aerobic organisms, including the lacI gene in bacteria, lacI transgenes in rodents, and the HPRT gene in rodents and humans, so a more complete understanding of cytosine oxidation is an essential research goal. The data and insights presented here shed new light on oxidation reactions of cytosine and 5-methylcytosine and should facilitate their validation in cellular DNA.

BIOLOGICAL ACTIVITIES OF PHTHALOCYANINES—VI. PHOTOOXIDATION OF L‐TRYPTOPHAN BY SELECTIVELY SULFONATED GALLIUM PHTHALOCYANINES: SINGLET OXYGEN YIELDS AND EFFECT OF AGGREGATION

Abstract— The photosensitized oxidation of L‐tryptophan by gallium phthalocyanines sulfonated to different degrees is studied as a function of both substrate and sensitizer concentrations in water and 50% MeOH‐H2O solutions. The maximum quantum yield of singlet oxygen was found to be nearly 0.5 for all sulfonated gallium complexes. The effect of adding sulfonate groups in the phthalocyanine backbone is to change the tendency of dye molecules to dimerize or aggregate in a particular solvent. A shift in the chemical equilibrium away from the monomeric state, which occurs at high dye concentrations and at lower degrees of dye sulfonation, results in a reduced photochemical yield. The variation of quantum yields in different solvent systems and at several wavelengths is similarly accounted for by the fraction of light absorbed by the productive monomer state.

BIOLOGICAL ACTIVITIES OF PHTHALOCYANINES—V. PHOTODYNAMIC THERAPY OF EMT‐6 MAMMARY TUMORS IN MICE WITH SULFONATED PHTHALOCYANINES

Abstract— We tested water‐soluble sulfonated phthalocyanine and three metal chelate derivatives for their tumoricidal effect on the EMT‐6 mammary tumor in mice exposed to red light. The metal‐free sulfophthalocyanine had little effect, whereas the aluminum complex and the lower sulfonated fraction of the gallium complex exhibited tumoricidal activity similar to hematoporphyrin‐based photosensitizer (Photofrin II). The higher sulfonated fractions of the gallium complex were less active as compared to the lower sulfonated fraction. The cerium complex was the most active sensitizer in terms of dye and light doses required to induce tumor necrosis and cure but also showed the highest phototoxicity towards healthy skin. These results suggest that sulfonated phthalocyanines will offer a new alternative in photodynamic therapy of light‐accessible neoplasms.

Biologically relevant oxidants and terminology, classification and nomenclature of oxidatively generated damage to nucleobases and 2-deoxyribose in nucleic acids.

Abstract A broad scientific community is involved in investigations aimed at delineating the mechanisms of formation and cellular processing of oxidatively generated damage to nucleic acids. Perhaps as a consequence of this breadth of research expertise, there are nomenclature problems for several of the oxidized bases including 8-oxo-7,8-dihydroguanine (8-oxoGua), a ubiquitous marker of almost every type of oxidative stress in cells. Efforts to standardize the nomenclature and abbreviations of the main DNA degradation products that arise from oxidative pathways are reported. Information is also provided on the main oxidative radicals, non-radical oxygen species, one-electron agents and enzymes involved in DNA degradation pathways as well in their targets and reactivity. A brief classification of oxidatively generated damage to DNA that may involve single modifications, tandem base modifications, intrastrand and interstrand cross-links together with DNA-protein cross-links and base adducts arising from the addition of lipid peroxides breakdown products is also included.

Low energy electron induced DNA damage: effects of terminal phosphate and base moieties on the distribution of damage.

Low energy electrons (LEE) induce DNA damage by dissociative electron attachment, which involves base release (N-glycosidic bond (N-C) cleavage) and the formation of strand breaks (phosphodiester-sugar bond (C-O) cleavage). The effect of terminal phosphate and base moieties was assessed by exposing DNA model compounds to LEE in the condensed phase followed by HPLC-UV analysis of products remaining on the surface. First, we report that the presence of terminal phosphate groups in monomers (pT, Tp, pTp) and dimers (pTpT, TpTp, pTpTp) increases overall damage by 2-3-fold while it decreases N-C and C-O bond cleavage by 2-10-fold. This suggests that the capture of LEE directly by the terminal phosphate does not contribute to N-C and C-O bond cleavage. Second, we report that terminal bases appear to shield the internal base from damage, resulting in a bias of damage toward the termini. In summary, the presence of terminal phosphate base moieties greatly affects the distribution of LEE induced damage in DNA model compounds.

Oxidatively generated base damage to cellular DNA by hydroxyl radical and one-electron oxidants: Similarities and differences

Hydroxyl radical (OH) and one-electron oxidants that may be endogenously formed through oxidative metabolism, phagocytosis, inflammation and pathological conditions constitute the main sources of oxidatively generated damage to cellular DNA. It is worth mentioning that exposure of cells to exogenous physical agents (UV light, high intensity UV laser, ionizing radiation) and chemicals may also induce oxidatively generated damage to DNA. Emphasis is placed in this short review article on the mechanistic aspects of OH and one-electron oxidant-mediated formation of single and more complex damage (tandem lesions, intra- and interstrand cross-links, DNA-protein cross-links) in cellular DNA arising from one radical hit. This concerns DNA modifications that have been accurately measured using suitable analytical methods such as high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Evidence is provided that OH and one-electron oxidants after generating neutral radicals and base radical cations respectively may partly induce common degradation pathways. In addition, selective oxidative reactions giving rise to specific degradation products of OH and one-electron oxidation reactions that can be used as representative biomarkers of these oxidants have been identified.

Increased susceptibility of low‐density lipoprotein (LDL) to oxidation by γ‐radiolysis with age

The susceptibility to oxidation of freshly isolated LDL from healthy normolipidemic individuals in three age groups was estimated by exposure of LDL to ionizing radiation followed by analyses of vitamin E, TBARS, conjugated dienes, and fluorescent products. The results clearly showed that LDL from elderly subjects was the most susceptible to oxidative damage in vitro. In particular, the greater susceptibility of LDL from elderly subjects in comparison to that from young subjects may be attributed to the much lower (4‐fold) concentration of LDL vitamin E in the elderly subjects. The present study reinforces the notion that the susceptibility of LDL to oxidation increases with age.

Phosphodiester and N-glycosidic bond cleavage in DNA induced by 4-15 eV electrons

Thin molecular films of the short single strand of DNA, GCAT, were bombarded under vacuum by electrons with energies between 4 and 15 eV. Ex vacuo analysis by high-pressure liquid chromatography of the samples exposed to the electron beam revealed the formation of a multitude of products. Among these, 12 fragments of GCAT were identified by comparison with reference compounds and their yields were measured as a function of electron energy. For all energies, scission of the backbone gave nonmodified fragments containing a terminal phosphate, with negligible amounts of fragments without the phosphate group. This indicates that phosphodiester bond cleavage by 4-15 eV electrons involves cleavage of the C-O bond rather than the P-O bond. The yield functions exhibit maxima at 6 and 10-12 eV, which are interpreted as due to the formation of transient anions leading to fragmentation. Below 15 eV, these resonances dominate bond dissociation processes. All four nonmodified bases are released from the tetramer, by cleavage of the N-glycosidic bond, which occurs principally via the formation of core-excited resonances located around 6 and 10 eV. The formation of the other nonmodified products leading to cleavage of the phosphodiester bond is suggested to occur principally via two different mechanisms: (1) the formation of a core-excited resonance on the phosphate unit followed by dissociation of the transient anion and (2) dissociation of the CO bond of the phosphate group formed by resonance electron transfer from the bases. In each case, phosphodiester bond cleavage leads chiefly to the formation of stable phosphate anions and sugar radicals with minimal amounts of alkoxyl anions and phosphoryl radicals.

One-electron oxidation reactions of purine and pyrimidine bases in cellular DNA

Abstract Purpose: The aim of this survey is to critically review the available information on one-electron oxidation reactions of nucleobases in cellular DNA with emphasis on damage induced through the transient generation of purine and pyrimidine radical cations. Since the indirect effect of ionizing radiation mediated by hydroxyl radical is predominant in cells, efforts have been made to selectively ionize bases using suitable one-electron oxidants that consist among others of high intensity UVC laser pulses. Thus, the main oxidation product in cellular DNA was found to be 8-oxo-7,8-dihydroguanine as a result of direct bi-photonic ionization of guanine bases and indirect formation of guanine radical cations through hole transfer reactions from other base radical cations. The formation of 8-oxo-7,8-dihydroguanine and other purine and pyrimidine degradation products was rationalized in terms of the initial generation of related radical cations followed by either hydration or deprotonation reactions in agreement with mechanistic pathways inferred from detailed mechanistic studies. The guanine radical cation has been shown to be implicated in three other nucleophilic additions that give rise to DNA-protein and DNA-DNA cross-links in model systems. Evidence was recently provided for the occurrence of these three reactions in cellular DNA. Conclusion: There is growing evidence that one-electron oxidation reactions of nucleobases whose mechanisms have been characterized in model studies involving aqueous solutions take place in a similar way in cells. It may also be pointed out that the above cross-linked lesions are only produced from the guanine radical cation and may be considered as diagnostic products of the direct effect of ionizing radiation.

2'-Deoxycytidine glycols, a missing link in the free radical-mediated oxidation of DNA.

2′-Deoxycytidine glycols (5,6-dihydroxy-5,6-dihydro-2′-deoxycytidine) are major products of the hydroxyl radical-induced oxidation of 2′-deoxycytidine resulting from either a Fenton reaction or exposure to ionizing radiation. Because of their instability, however, the glycols have not previously been characterized. Instead, the impetus has been placed on the primary decomposition products of 2′-deoxycytidine glycols, which includes 5-hydroxy-2′-deoxycytidine, 5-hydroxy-2′-deoxyuridine, and 2′-deoxyuridine glycols. Here, we have identified one of the four possible diastereomers of 2′-deoxycytidine glycols by product analyses of decomposition products, 1H NMR, and mass spectrometry. This glycol was observed to decompose with a half-life of 50 min at 37 °C in buffered neutral solutions and preferentially undergo dehydration to 5-hydroxy-2′-deoxycytidine. The rate of decomposition was strongly dependent on pH (2-10) and the concentration of phosphate ion (10–300 mm). Next, we report on the deamination of cytosine glycols to uracil glycols in oxidized DNA using acid hydrolysis and high performance liquid chromatography analysis with electrochemical detection to monitor 5-hydroxycytosine and 5-hydroxyuracil. The results showed that the lifetime of cytosine glycols is greatly enhanced in DNA (34-fold; half-life, 28 h), and that deamination accounts for at least one-third of the total decomposition. The relatively long lifetime of cytosine glycols in DNA suggests that this important class of DNA oxidation products will be significantly involved in repair and mutagenesis processes.