Photo-induced hole transfer from base to sugar in DNA: relationship to primary radiation damage

This work presents the hypothesis that photo-excitation of G.+ in DNA and model systems results in the same electronic states expected from direct ionization of the sugar phosphate backbone and that these states lead to specific sugar radicals on the DNA sugar phosphate backbone. As evidence we show that visible photo-excitation of guanine cation radicals (G.+) in the dinucleoside phosphate TpdG results in high yields (about 85%) of deoxyribose sugar radicals at the C1' and C3' sites. Further, we have calculated transition energies of hole transfer from G.+ in TpdG using time-dependent density functional theory (TD-DFT) at the B3LYP/6-31G(d) level in gas phase as well as in a solvated environment. These calculations clearly predict that visible excitation of G.+ in TpdG causes transitions from only inner-shell filled molecular orbitals (MOs) to the singly occupied molecular orbital (SOMO) that effectively result in hole transfer from guanine either to the sugar phosphate backbone or to the adjacent base, thymine. The hole transfer is followed by rapid deprotonation from the sugar to form C1' and C3' radicals. These experimental and theoretical results are in agreement with our previously published experimental and theoretical results that photo-excitation of G.+ results in high yields of deoxyribose sugar radicals in DNA, guanine deoxyribonucleosides and deoxyribonucleotides. Photo-excitation of G.+ therefore provides a convenient method to produce and study sugar radicals that are expected to be formed in gamma-irradiated DNA systems unencumbered by the many other pathways involved in direct ionization.     

Oxygen inhibition of nitroreductase: electron transfer from nitro radical-anions to oxygen.

The inhibition of many nitroreductases by oxygen has been explained by Mason and Holtzman in terms of electron transfer to oxygen from the nitro radical-anions, which have been identified as the first intermediate in some reductase systems. We have used the pulse radiolysis technique to measure the bimolecular rate constants of this electron-transfer reaction for over 20 nitro compounds, including substituted 2- and 5-nitroimidazoles of interest as antiprotozoal drugs and radiosensitizers, nitrofurans in use as antibacterial agents, and substituted nitrobenzenes previously used as model substrates for nitroreductases. The logarithm of the rate constant for the reaction of the nitro radical-anion with oxygen is linearly related to the one-electron reduction potential of the nitro compound.     

Oxygen effect in heat-mediated damage to DNA

The effects of gassing conditions in DNA solution on the major types of heat-mediated DNA damage (depurination of DNA, generation of 8-oxoguanine, cytosine deamination with the formation of uracil) have been studied by ELISA, column liquid chromatography, and spectrophotometry. It was found that the number of DNA lesions depends on oxygen concentration in solution; i.e., the oxygen effect takes place. The heat-induced generation of hydrogen peroxide in water increased after the addition of D20 and decreased by the action of various 1O2 quenchers, suggesting that singlet oxygen is involved in the heat-induced production of reactive oxygen species (ROS) in water. The data obtained favor the hypothesis that all the types of heat-induced damage to DNA are due to a common mechanism associated with the heat-mediated generation of reactive oxygen species in solution.     

8-Chloroadenine: a novel product formed from hypochlorous acid-induced damage to calf thymus DNA

Hypochlorous acid (HOCl) is formed by the action of the enzyme myeloperoxidase on hydrogen peroxide and chloride ions. It has been shown to be highly bactericidal and cytotoxic by a variety of mechanisms, one of which, may be the modification of DNA. Previously we have demonstrated by GC-MS analysis that exposure of calf thymus DNA to HOCl causes extensive pyrimidine modification, including 5-chlorocytosine formation. Using GC-MS analysis, we now demonstrate the formation of an additional chlorinated base product, 8-Cl adenine. The addition of 50 μM HOCl was sufficient to produce a significant increase in this product. The reaction of HOCl with adenine in calf thymus DNA was shown to be rapid with the reaction complete after 1 min. pH-dependence studies suggest HOCl rather than its conjugate base (OCl-) to be responsible for 8-Cl adenine formation. Other commercially available chlorinated base products, 6-Cl guanine or 2-Cl adenine were not detected. Therefore, 8-Cl adenine might prove a useful biomarker for studying the role of reactive chlorine species (RCS) during inflammatory processes.     

8-Chloroadenine: a novel product formed from hypochlorous acid-induced damage to calf thymus DNA

Hypochlorous acid (HOCl) is formed by the action of the enzyme myeloperoxidase on hydrogen peroxide and chloride ions. It has been shown to be highly bactericidal and cytotoxic by a variety of mechanisms, one of which, may be the modification of DNA. Previously we have demonstrated by GC-MS analysis that exposure of calf thymus DNA to HOCl causes extensive pyrimidine modification, including 5-chlorocytosine formation. Using GC-MS analysis, we now demonstrate the formation of an additional chlorinated base product, 8-Cl adenine. The addition of 50 μM HOCl was sufficient to produce a significant increase in this product. The reaction of HOCl with adenine in calf thymus DNA was shown to be rapid with the reaction complete after 1 min. pH-dependence studies suggest HOCl rather than its conjugate base (OCl-) to be responsible for 8-Cl adenine formation. Other commercially available chlorinated base products, 6-Cl guanine or 2-Cl adenine were not detected. Therefore, 8-Cl adenine might prove a useful biomarker for studying the role of reactive chlorine species (RCS) during inflammatory processes.     

Stereoselective synthesis of the thermodynamically less stable manno isomers from a nitro sugar

Reaction of methyl 4,6-O-benzylidene-2,3-dideoxy-3-nitro-o--D-erythro-hex-2-enopyranoside (2) with hydrazoic acid, hydrogen cyanide, theophylline, and 2,6-dichloropurine under weakly acidic or neutral conditions resulted in the stereo-selective formation of addition products having the thermodynamically less-stable manno configuration, whereas reaction of 2 with more-basic reagents yielded mainly products having the more stable gluco configuration.     

The radiation modification of the sugar fragment in DNA: the formation of breaks, a change in polymer conformation and the transfer of the damage to the base]

Comparative analysis of the author's own data and data reported in the literature on the nature of the intermediate and end molecular products of radiolysis of DNA, its precursors and substances stimulating certain DNA fragments, allows to attribute the formation of breaks and alkaline-labile sites in DNA strands and changes in the polymer conformation, to the formation and transformations of some types of primary radicals of the sugar fragment. In order to explain certain effects induced by irradiation of DNA and its precursors (a balance of basic products of radiation destruction of DNA and ESR data concerning low temperature radiolysis of bases, nucleotides and nucleosides) the author proposes a model of the transfer of a damage (free valence) from 2-deoxyribosyl to a base within one nucleotide.     

Oxygen transfer from silicone hollow fiber membrane to water

Silicone rubber hollow fiber was able to enrich the oxygen concentration in air by about 30%, and oxygen was transferred sufficiently from the membrane to water. When an aeration tank was filled with hollow fiber to up to 10% of its volume, the oxygen utilization rate, Rr, was about seven times as much as the value in the standard activated sludge method. It is suggested that there is some possibility of improving the efficiency of aerobic wastewater treatment by using this system.     

DNA damage by the sulfate radical anion: hydrogen abstraction from the sugar moiety versus one-electron oxidation of guanine

The products of oxidative damage to double-stranded (ds) DNA initiated by photolytically generated sulfate radical anions SO₄^?? were analyzed using reverse-phase (RP) high-performance liquid chromatography (HPLC). Relative efficiencies of two major pathways were compared: production of 8-oxoguanine (8oxoG) and hydrogen abstraction from the DNA 2-deoxyribose moiety (dR) at C1,′ C4,′ and C5′ positions. The formation of 8oxoG was found to account for 87% of all quantified lesions at low illumination doses. The concentration of 8oxoG quickly reaches a steady state at about one 8oxoG per 100 base pairs due to further oxidation of its products. It was found that another guanine oxidation product identified as 2-amino-5-(2′-alkylamino)-4H-imidazol-4-one (X) was released in significant quantities from its tentative precursor 2-amino-5-[(2′-deoxy-β-d-erythro-pentofuranosyl)amino]-4H-imidazol-4-one (dIz) upon treatment with primary amines in neutral solutions. The linear dose dependence of X release points to the formation of dIz directly from guanine and not through oxidation of 8oxoG. The damage to dR was found to account for about 13% of the total damage, with majority of lesions (33%) originating from the C4′ oxidation. The contribution of C1′ oxidation also turned out to be significant (17% of all dR damages) despite of the steric problems associated with the abstraction of the C1′-hydrogen. However, no evidence of base-to-sugar free valence transfer as a possible alternative to direct hydrogen abstraction at C1′ was found.     

Nonenzymatic reduction of nitro derivative of a heterocyclic amine IQ by NADH and Cu(II) leads to oxidative DNA damage.

Nitro derivative (nitro-IQ) of a carcinogenic heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) is known to be a potent mutagen as well as IQ, and nitro-IQ is believed to be activated enzymatically by nitroreductase. We investigated nonenzymatic reduction of nitro-IQ by an endogenous reductant NADH and the ability of inducing DNA damage by nitro-IQ. Nitro-IQ caused DNA damage including 8-oxo-7,8-dihydro-2'-deoxyguanosine in the presence of NADH and Cu(II). Catalase and bathocuproine, a Cu(I)-specific chelator, inhibited the DNA damage, suggesting the involvement of H2O2 and Cu(I). Nitro-IQ induced DNA cleavage frequently at thymine and cytosine residues in the presence of NADH and Cu(II). UV-vis spectroscopic study showed that no spectral change of Nitro-IQ and NADH was observed in the absence of Cu(II), while rapid spectral change was observed in the presence of Cu(II), suggesting that Cu(II) mediated redox reaction of nitro-IQ and NADH. These results suggest that nitro-IQ can be reduced nonenzymatically by NADH in the presence of Cu(II), and the redox reaction resulted in oxidative DNA damage due to the copper-oxygen complex, derived from the reaction of Cu(I) with H2O2. We conclude that nonenzymatic reduction of nitro-IQ and resulting in oxidative DNA damage can play a role in carcinogenesis of IQ.     

Formation of a transfer product from stevioside by the cultures of Actinomycete.

An Actinomycete, strain K-128, which was isolated from soil, formed a transfer product when the strain was cultured in a medium containing both stevioside and curdlan. The transfer product in culture broth was separated by DIAION HP-20 column and TOYOPEARL HW-40F column chromatographies. From structural studies, the transfer product was identified as C13-O-beta-6(2)-beta-glucosylsophorosyl-C19-O-beta-glucopyranosyl steviol. This product was the first to be obtained by a culture of an Actinomycete.     

Cytogenetic monitoring of a group of Italian floriculturists: no evidence of DNA damage related to pesticide exposure

The induction of sister chromatid exchanges (SCE), structural chromosome aberrations (CA) or micronuclei (MN) was investigated in peripheral lymphocytes of a group of Italian floriculturists exposed to a mixture of pesticides. No statistically significant difference in the frequencies of cytogenetic damage was detected between exposed and control subjects. Assessment of the effect of confounding factors indicated that smoking affected both SCE and CA frequencies. Multiple regression analysis showed that in heavy smokers (≥ 20 cigarettes/day), SCE and CA levels increased significantly by 17% and 54%, respectively, as compared to non-smokers.     

o-Diphenol: Oxygen oxidoreductase from leaves of sugar cane

A non-particulate o-diphenol: O₂ oxidoreductase (phenolase) has been isolated from leaves of sugar cane. Gel filtration produced two fractions MW 32000 and 130000. The preferred substrate was chlorogenic acid. Other o-diphenols (caffeic acid, catechol, pyrogallol, dihydroxyphenylalanine) all of which were slowly oxidized when tested alone, increased the rates of O₂ consumption obtained with catalytic amounts of chlorogenic acid. Both enzyme fractions were inhibited by thiols; thioglycollate, which acted in a non-competitive manner, was most effective.     

Nuclear dynamics of RAD52 group homologous recombination proteins in response to DNA damage

Recombination between homologous DNA molecules is essential for the proper maintenance and duplication of the genome, and for the repair of exogenously induced DNA damage such as double-strand breaks. Homologous recombination requires the RAD52 group proteins, including Rad51, Rad52 and Rad54. Upon treatment of mammalian cells with ionizing radiation, these proteins accumulate into foci at sites of DNA damage induction. We show that these foci are dynamic structures of which Rad51 is a stably associated core component, whereas Rad52 and Rad54 rapidly and reversibly interact with the structure. Furthermore, we show that the majority of the proteins are not part of the same multi-protein complex in the absence of DNA damage. Executing DNA transactions through dynamic multi-protein complexes, rather than stable holo-complexes, allows flexibility. In the case of DNA repair, for example, it will facilitate cross-talk between different DNA repair pathways and coupling to other DNA transactions, such as replication.     

A role of oxidative DNA sugar damage in mutagenesis by neocarzinostatin and bleomycin

The anti-tumor antibiotics neocarzinostatin and bleomycin specifically oxidize deoxyribose in DNA at the C-5' and C-4' positions, respectively. The resulting DNA lesions include strand breaks and apyrimidinic sites. Both agents are broad specificity mutagens, inducing, in various systems, base substitutions, frameshifts and deletions. Sequencing studies in bacterial systems have suggested that the base substitutions may result primarily from replicative bypass of the oxidized apyrimidinic sites.