Long-range charge transport through double-stranded DNA mediated by manganese or iron porphyrins

Guanine oxidation by electron transfer results in the formation of a guanine radical cation, which is at the origin of long-range charge transport through double-stranded DNA. It is possible to observe guanine lesions at a long distance from the oxidative reagent covalently bound to DNA owing to the migration of the positive hole in the DNA pi-stacks. This phenomenon of long-range hole transport is classically studied in the literature with photosensitizers used as one-electron oxidants. It is shown in the present work that the process of long-range charge transport and the concomitant formation of guanine lesions at a long distance can be observed also in the case of two-electron oxidants. This is the signature of the formation of a transient guanine radical cation in the course of the two-electron abstraction process and consequently evidence of the separated one plus one electron abstraction steps. Long-range charge transport is likely to be a universal mechanism for any two-electron oxidant acting by electron abstraction provided that the second electron abstraction is slower than hole transfer.     

Comparison of transition metal-mediated oxidation reactions of guanine in nucleoside and single-stranded oligodeoxynucleotide contexts

As the most readily oxidized of DNA’s four natural bases, guanine is a prime target for attack by reactive oxygen species (ROS) and transition metal-mediated oxidants. The oxidation products of a modified guanosine nucleoside and of a single-stranded oligodeoxynucleotide, 5′-d(TTTTTTTGTTTTTTT)-3′ have been studied using oxidants that include Co^II, Ni^II, and Ir^IV compounds as well as photochemically generated oxidants such as sulfate radical, electron-transfer agents and singlet oxygen. The oxidized lesions formed include spiroiminodihydantoin (Sp), guanidinohydantoin (Gh), imidazolone (Iz), oxazolone (Z) and 5-carboxamido-5-formamido-2-iminohydantion (2-Ih) nucleosides with a high degree of dependence on the exact oxidation system employed. Interestingly, a nickel(II) macrocyclic complex in conjunction with KHSO₅ leads to the recently reported 2-Ih heterocycle as the major product in both the nucleoside and oligonucleotide contexts.     

Physiological roles of animal succinate thiokinases Specific association of the guanine nucleotide-linked enzyme with haem biosynthesis

The discovery of two distinct succinate thiokinases in mammalian tissues, one (G-STK) specific for GDP/GTP and the other (A-STK) for ADP/ATP, poses the question of their differential metabolic roles. Evidence has suggested that the A-STK functions in the citric acid cycle in the direction of succinyl-CoA breakdown (and ATP formation) whereas one role of the G-STK appears to be the re-cycling of succinate to succinyl-CoA (at the expense of GTP) for the purpose of ketone body activation. A third metabolic participation of succinyl-CoA is in haem biosynthesis. This communication shows that in chemically induced hepatic porphyria, when the demand for succinyl-CoA is increased, it is the level of G-STK only which is elevated, that of A-STK being unaffected. The results implicate G-STK in the provision of succinyl-CoA for haem biosynthesis, a conclusion which is further supported by the observation of a high G-STK/A-STK ratio in bone marrow.     

Measuring oxidative damage to DNA and its repair with the comet assay

Background

Single cell gel electrophoresis, or the comet assay, was devised as a sensitive method for detecting DNA strand breaks, at the level of individual cells. A simple modification, incorporating a digestion of DNA with a lesion-specific endonuclease, makes it possible to measure oxidised bases.

Scope of review

With the inclusion of formamidopyrimidine DNA glycosylase to recognise oxidised purines, or Nth (endonuclease III) to detect oxidised pyrimidines, the comet assay has been used extensively in human biomonitoring to monitor oxidative stress, usually in peripheral blood mononuclear cells.

Major conclusions

There is evidence to suggest that the enzymic approach is more accurate than chromatographic methods, when applied to low background levels of base oxidation. However, there are potential problems of over-estimation (because the enzymes are not completely specific) or under-estimation (failure to detect lesions that are close together). Attempts have been made to improve the inter-laboratory reproducibility of the comet assay.

General significance

In addition to measuring DNA damage, the assay can be used to monitor the cellular or in vitro repair of strand breaks or oxidised bases. It also has applications in assessing the antioxidant status of cells. In its various forms, the comet assay is now an invaluable tool in human biomonitoring and genotoxicity testing. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Temperature-dependence of UV laser one-electron oxidative guanine modifications as a probe of local stacking fluctuations and conformational transitions

By monitoring R(pip)/R(Fpg), i.e. the relative sensitivity to hot piperidine and to formamidopyrimidine DNA glycosylase (Fpg protein) of the guanine lesions induced in DNA exposed to UV laser irradiation, we have previously observed that the formation of the two major types of one-electron oxidative guanine modifications, oxazolone and 7,8-dihydro-8-oxoguanine (8-oxodG), depends on DNA conformational features. While oxazolone is largely predominant at each site of single-stranded DNA (R(pip)>R(Fpg)), 8-oxodG is the major lesion at most of the sites of double-stranded DNA (R(pip)R(Fpg) at 20 degrees C and the ratio R(pip)/R(Fpg) does not vary significantly during the melting process. Interestingly, these guanine residues display a high sensitivity to dimethyl sulfoxide methylation while the opposite cytosine residues are unsensitive, suggesting that the prevalence of R(pip) over R(Fpg) is related not to base-pairing disruption but rather to the local helical alteration of the B-DNA stacking geometry. This leads us to propose that the slight variations in the ratios R(pip)/R(Fpg) observed, at individual sites, at temperatures below the helix-coil transition reflect local small-scale breathing motions, unstacking single dinucleotide steps prior to opening. Our results thus support the view that the temperature dependence of the ratio of R(pip)/R(Fpg) at sites of B-DNA provides a sensitive probe of the DNA internal local thermal stability and are discussed in relation with the mechanisms proposed for the intramolecular rearrangement of the guanyl radical.     

An electrochemical sensor based on single-stranded DNA-poly(sulfosalicylic acid) composite film for simultaneous determination of adenine, guanine, and thymine

Poly(sulfosalicylic acid) and single-stranded DNA composite (PSSA–ssDNA)-modified glassy carbon electrode (GCE) was prepared by electropolymerization and then successfully used to simultaneously determine adenine (A), guanine (G), and thymine (T). The characterization of electrochemically synthesized PSSA–ssDNA film was investigated by scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS). The modified electrode exhibited enhanced electrocatalytic behavior and good stability for the simultaneous determination of A, G, and T in 0.1 M phosphate buffer solution (PBS, pH 7.0). Well-separated voltammetric peaks were obtained among A, G, and T presented in the analyte mixture. Under the optimal conditions, the peak currents for A, G, and T increased linearly with the increase of analyte mixture concentration in the ranges of 6.5 × 10⁻⁸ to 1.1 × 10⁻⁶, 6.5 × 10⁻⁸ to 1.1 × 10⁻⁶, and 4.1 × 10⁻⁶ to 2.7 × 10⁻⁵ M, respectively. The detection limits (signal/noise = 3) for A, G, and T were 2.2 × 10^–8, 2.2 × 10^–8, and 1.4 × 10^–6 M, respectively.     

Preparation of guanine and diaminopurine from biuret. Part III

Because of their potential prebiotic origin and relative chemical stability, urea, biuret, formic acid, and glycine amide might have played a role in the assembly process of purine bases. In this paper, we describe a short reaction path to purine nucleobases from these acyclic precursors. The formation of different purines was verified by UV and NMR spectroscopy, as well as by mass spectrometry.     

Efficiency,specificity and DNA polymerase-dependence of translesion replication across the oxidative DNA lesion 8-oxoguanine in human cells

The oxidation product of guanine, 8-oxoguanine, is a major lesion formed in DNA by intracellular metabolism, ionizing radiation, and tobacco smoke. Using a recently developed method for the quantitative analysis of translesion replication, we have studied the bypass of 8-oxoguanine in vivo by transfecting human cells with a gapped plasmid carrying a site-specific 8-oxoguanine in the ssDNA region. The efficiency of bypass in the human large-cell lung carcinoma cell line H1299 was 80%, and it was similar when assayed in the presence of aphidicolin, an inhibitor of DNA polymerases alpha, delta and epsilon. A similar extent of bypass was observed also in XP-V cells, defective in pol eta, both in the absence and presence of aphidicolin. DNA sequence analysis indicated that the major nucleotide inserted opposite the 8-oxoguanine was the correct nucleotide C, both in H1299 cells (81%) and in XP-V cells (77%). The major mutagenic event was the insertion of an A, both in H1299 and XP-V cells, and it occurred at a frequency of 16-17%, significantly higher than previously reported. Interestingly, the misinsertion frequency of A opposite 8-oxoguanine was decreased in XP-V cells in the presence of aphidicolin, and misinsertion of G was observed. This modulation of the mutagenic specificity at 8-oxoguanine is consistent with the notion that while not essential for the bypass reaction, pol eta and pol delta, when present, are involved in bypass of 8-oxoguanine in vivo.     

The oxidative damage of plasmid DNA by ascorbic acid derivatives in vitro: the first research on the relationship between the structure of ascorbic acid and the oxidative damage of plasmid DNA

To study the structure-function relationship of the oxidative-damage effect of ascorbic acid, we have focused on the interaction between plasmid DNA pUC19 and a series of ascorbic acid derivatives modified on different OH groups in the presence of transition metal ions. Some ascorbic acid derivatives can selectively cleave plasmid DNA from Form I to Form II in the presence of low concentration of Cu2+ just like ascorbic acid itself, while other derivatives oxidatively damage plasmid DNA slightly. We found that those derivatives with unattached 2-OH and 3-OH groups retain the ability to cleave the plasmid DNA. The derivatives that have been methylated on 2-OH or 3-OH can only cleave plasmid DNA softly, and those derivatives that have been protected on both 2-OH and 3-OH can hardly exert an oxidative damage on plasmid DNA under the same condition. Form these results, we can draw the conclusion that 2-OH and 3-OH groups of the ascorbic acid molecule contribute most to this biological activity.     

Role of antioxidants in protecting cellular DNA from damage by oxidative stress

We have previously derived 2 V79 clones resistant to menadione (Md1 cells) and cadmium (Cd1 cells), respectively. They both were shown to be cross-resistant to hydrogen peroxide. There was a modification in the antioxidant repertoire in these cells as compared to the parental cells. Md1 presented an increase in catalase and glutathione peroxidase activities whereas Cd1 cells exhibited an increase in metallothionein and glutathione contents. The susceptibility of the DNA of these cells to the damaging effect of H₂O₂ was tested using the DNA precipitation assay. Both Md1 and Cd1 DNAs were more resistant to the peroxide action. In the case of Md1 cells it seems clear that the extra resistance is provided by the increase in the two H₂O₂ scavenger enzymes, catalase and glutathione peroxidase. In the case of Cd1 cells the activities of these enzymes as well as of superoxide dismutases (Cu/Zn and Mn) are unaltered as compared to the parental cells. The facts that parental cells exposed to 100 μM Zn²⁺ in the medium exhibit an increase in metallothionein but not in glutathione and that these cells become more resistant to the DNA-damaging effect of H₂O₂ suggest that this protein might play a protective role in vivo against the OH radical attack on DNA.     

Binding of transition metal complexes to guanine and guanine–cytosine: hydrogen bonding and covalent effects

Density functional calculations and Atoms in Molecules analysis are used to investigate the role of covalent and hydrogen bondings in determining the binding of transition metal complexes to guanine, and the subsequent effect on pairing with cytosine. Hydrogen bonding is ubiquitous, and typically contributes ca. 10% to overall binding, a value that varies with the coordination site on guanine, as well as metal and ligands. Early transition metals show a clear preference for the O6 position, while later ones prefer N7, the crossover point coming at the vanadium group. Metallation at N7 causes a redistribution of hydrogen bonding strength between guanine and cytosine, but does not greatly affect the overall pairing energy. In contrast, metallation at O6 strongly reduces the pairing energy, as may be expected given the role of O6 in pairing guanine with cytosine. This effect can be quantified using electron density properties, and seems to be due to both electrostatic repulsion from the positive metal centre and a redistribution of electron density within guanine itself. Qualitative agreement with experimental mass spectroscopic results is obtained.     

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.     

Comparative direct-acting mutagenicity of 1- and 2-nitropyrene: Evidence for 2-nitropyrene mutagenesis by both guanine and adenine adducts

The mutations and DNA adducts produced by the environmental pollutant 2-nitropyrene were examined in Salmonella typhimurium tester strains. 2-Nitropyrene was a stronger mutagen than its extensively studied structural isomer 1-nitropyrene in strains TA96, TA97, TA98, TA100, TA102, TA104 and TA1538. Both 1- and 2-nitropyrene were essentially inactive in TA1535. The mutagenicity of 1- and 2-nitropyrene in TA98 was much higher than in TA98NR and the activity of these compounds in TA100 was much higher than in TA100NR. While 1-nitropyrene exhibited similar mutagenicity in strains TA98 and TA98/1,8-DNP₆, the mutagenicity of 2-nitropyrene in TA98/1,8-DNP₆ was much lower than in TA98. Analysis of DNA from TA96 and TA104 incubated with 2-nitropyrene indicated the presence of two adducts, N-(deoxyguanosin-8-yl)-2-aminopyrene and N-deoxyadenosin-8-yl)-2-aminopyrene. The results suggest that 2-nitropyrene is metabolized by bacterial nitroreductase(s) to N-hydroxy-2-aminopyrene, and possibly by activation to a highly mutagenic O-acetoxy ester. DNA adduct formation with deoxyguanosine and deoxyadenosine correlates with the mutagenicity of 2-nitropyrene in tester strains possessing both G:C and A:T mutational targets.     

Optimization and oxidative stability of the microencapsulated conjugated linoleic acid

We used response surface methodology to optimize the preparation conditions of conjugated linoleic acid (CLA) microcapsules for maximum entrapment efficiency. Three independent variables were used: the ratio of CLA core material to agar and waxy corn starch wall material (X₁), the temperature of dispersion fluid (X₂), and the concentration of emulsifier (X₃). The optimized values of X₁, X₂, and X₃ were found to be 3.82:6.18, 19.97 °C, and 0.34%, respectively. The CLA oxidation stability was significantly protected by microencapsulation. These results suggest that CLA-loaded microcapsules can be used as a means to enhance not only the entrapment efficiency but also the oxidative stability of CLA.     

Molecular toxicity of nanoplastics involving in oxidative stress and desoxyribonucleic acid damage

Microplastic pollution attracted extensive attention because of its global presence and adverse effects on ecosystem. However, it is insufficient to clear the effects of nanoplastics on organisms at the molecular level. Herein, a nanopolystyrene (50 nm) was used to examine molecular responses of superoxide dismutase (SOD) and desoxyribonucleic acid (DNA) using spectroscopy (UV‐vis, circular dichroism spectra, and fluorescence measurements) and single cell gel electrophoresis methods. Results showed that nanopolystyrene induced oxidative stress, involving in the increase of SOD activity and malondialdehide (MDA) content, and DNA damage because of the significant increase of olive tail moment, head optical density, and tail DNA percentage in the groups at exposure concentrations above 5 × 10^?6 mol/L. The second structural and microenvironment of aromatic amino acids of SOD were changed with nanopolystyrene exposure. The fluorescence of SOD was quenched by nanopolystyrene at exposure concentration above 1 × 10^?5 mol/L, and the quenching mode could be ascribed to the static type. The results and the combined methods are favorable to explore the molecular toxicity of other nanoplastics and the interaction mechanism.     

Chronic low-dose lesion equilibrium along genes: measurement, molecular epidemiology, and theory of the minimal relevant dose

Spontaneous mutations seem to be caused almost entirely by endogenous lesions. The pattern of these lesions along a gene represents an equilibrium between damage and repair. A pattern can be measured using ligation-mediated PCR (polymerase chain reaction) and a large chronic dose of a suspected endogenous mutagen. A study using dimethylsulfate-induced 7^meGuanine lesions indicates that the exogenously induced pattern depends on how methyl purine glycosylase recognizes sequence context and, for this lesion, the pattern may be independent of the mutagen's dose.     

Mutagenicity and repair of oxidative DNA damage: insights from studies using defined lesions

Oxidative DNA damage has been implicated in mutagenesis, carcinogenesis and aging. Endogenous cellular processes such as aerobic metabolism generate reactive oxygen species (ROS) that interact with DNA to form dozens of DNA lesions. If unrepaired, these lesions can exert a number of deleterious effects including the induction of mutations. In an effort to understand the genetic consequences of cellular oxidative damage, many laboratories have determined the patterns of mutations generated by the interaction of ROS with DNA. Compilation of these mutational spectra has revealed that GC → AT transitions and GC → TA transversions are the most commonly observed mutations resulting from oxidative damage to DNA. Since mutational spectra convey only the end result of a complex cascade of events, which includes formation of multiple adducts, repair processing, and polymerase errors, it is difficult if not impossible to asses the mutational specificity of individual DNA lesions directly from these spectra. This problem is especially complicated in the case of oxidative DNA damage owing to the multiplicity of lesions formed by a single damaging agent. The task of assigning specific features of mutational spectra to individual DNA lesions has been made possible with the advent of a technology to analyze the mutational properties of single defined adducts, in vitro and in vivo. At the same time, parallel progress in the discovery and cloning of repair enzymes has advanced understanding of the biochemical mechanisms by which cells excise DNA damage. This combination of tools has brought our understanding of DNA lesions to a new level of sophistication. In this review, we summarize the known properties of individual oxidative lesions in terms of their structure, mutagenicity and repairability.     

Effects of boric acid feeding on the oxidative stress parameters in testes,sperm parameters and DNA damage in mice

This study was aimed to determine the effects of boric acid on oxidative stress, testicular tissue and spermatozoon DNA. Experiments were performed with Swiss Albino mice divided equally into two groups based on the tratment period: one for 4 and the other for 6-week duration. These groups were further divided into subgroups as Control and those administered daily at oral doses of 115 mg/kg, 250 mg/kg and 450 mg/kg of boric acid. Then, testicular tissue were examined postmortem and analyzed using ex-vivo biochemical tools for oxidative stress, spermatozoon membrane integrity, sperm motility and live cell rate (%). In both 4 and 6-week groups, v. seminalis weight, membrane integrity, motility, live cells and GSH levels exhibited a decreasing trent compared to the controls. In addition, 6-week group had a decrease in SOD level. MDA level was higher in controls in both 4 and 6-week groups. Spermatozoon DNA was intact in the 4-week group, but damaged in the 6-week group, and the degree of the damage dependent on the administered dose. Boric acid induces oxidative stress in testicular tissue, and its long-term application (only 6 weeks) caused damage in spermatozoon DNA.