Monitoring of DNA oxidative damage with piezoelectric quartz crystal method

The piezoelectric quartz crystal (PQC) sensor was used to detect the whole process of DNA damage oxidized by H(2)O(2) system containing Cu(2+) or Zn(2+) based on the density-viscosity change of the detected solution. The PQC impedance method has been applied to further investigate the damage process of DNA oxidized by H(2)O(2) system containing Cu(2+). It was found that the ratio of DeltaR(1) to Deltaf(0) or DeltaL(1) coincided well with that calculated from Martin's equations reflecting the solution density-viscosity effect, suggesting that the continuing change in liquid loading on to the PQC surface caused the significant variation of Deltaf(0), DeltaR(1) and DeltaL(1). It has also been found that the H(2)O(2) system containing Cu(2+) could oxidize DNA more completely than that containing Zn(2+), and a trace Cu(2+) in the system could cause DNA damage. However, no significant breakage in the DNA backbone was observed if the system contained only H(2)O(2). The DNA concentration was linearly related to Deltaf(0s), which is the different between the initial frequency and the frequency after a reaction time of 45 min, in the range of 50-1000 mug ml(-1). The effect of H(2)O(2) or Cu(2+) concentration of was also investigated. The above-mentioned results that the H(2)O(2) system containing Cu(2+) or Zn(2+) could break the DNA backbone were also supported by the agarose gel electrophoresis technique.     

DNA oxidation in anionic reverse micelles: ruthenium-mediated damage at Guanine in single- and double-stranded DNA

One-electron guanine oxidation in DNA has been investigated in anionic reverse micelles (RMs). A photochemical method for generating Ru3+ from the ruthenium polypyridyl complex tris(2-2'-bipyridine)ruthenium(II) chloride ([Ru(bpy)3]Cl2) is combined with high-resolution polyacrylamide gel electrophoresis (PAGE) to quantify piperidine-labile guanine oxidation products. As characterized by emission spectroscopy of Ru(bpy)3(2+), the addition of DNA to RMs containing Ru(bpy)3(2+) does not perturb the environment of Ru(bpy)3(2+). The steady-state quenching efficiency of Ru(bpy)3(2+) with K3[Fe(CN)6] in buffer solution is approximately 2-fold higher than that observed in RMs. Consistent with the difference in quenching efficiency in the two media, a 1.5-fold higher yield of piperidine-labile damage products as monitored by PAGE is observed for duplex oligonucleotide in buffer vs RMs. In contrast, a 13-fold difference in the yield of PAGE-detected G oxidation products is observed when single-stranded DNA is the substrate. Circular dichroism spectra showed that single-stranded DNA undergoes a structural change in anionic RMs. This structural change is potentially due to cation-mediated adsorption of the DNA phosphates on the anionic headgroups of the RMs, leading to protection of the guanine from oxidatively generated damage.     

Dynamical signature of abasic damage in DNA

Time-dependent Stokes shift (TDSS) responses in proteins and DNA exhibit a broad range of long time scales (>10 ps) that are not present in bulk aqueous solution. The physical interpretation of the long TDSS time scales in biomolecular systems is a matter of considerable debate because of the many different components present in the sample (water, biomolecule, counterions), which have highly correlated motions and intrinsically different abilities to adapt to local perturbations. Here we use molecular dynamics (MD) simulations to show that the surprisingly slow (～10 ns) TDSS response of coumarin 102 (C102), a base pair replacement, reflects a distinct dynamical signature for DNA damage. When the C102 molecule is covalently incorporated into DNA, an abasic site is created on the strand opposite the C102 probe. The abasic sugar exhibits a reversible interchange between intra- and extrahelical conformations that are kinetically stable on a nanosecond time scale. This conformational change, only possible in damaged DNA, was found to be responsible for the long time scales in the measured TDSS response. For the first time, a TDSS measurement has been attributed to a specific biomolecular motion. This finding directly contradicts the prevailing notion that the TDSS response in biomolecular contexts is dominated by hydration dynamics. It also suggests that TDSS experiments can be used to study ultrafast biomolecular dynamics that are inaccessible to other techniques.     

Modulation of photo-oxidative DNA damage by cationic surfactant complexation

The natural packaging of DNA in the cell by histones provides a particular environment affecting its sensitivity to oxidative damage. In this work, we used the complexation of DNA by cationic surfactants to modulate the conformation, the dynamics, and the environment of the double helix. Photo-oxidative damage initiated by benzophenone as the photosensitizer on a plasmid DNA complexed by dodecyltrimethylammonium chloride (DTAC), tetradecyltrimethylammonium chloride (TTAC), cetyltrimethyammonium chloride (CTAC) and bromide (CTAB) was detected by agarose gel electrophoresis. By fluorescent titration in the presence of ethidium bromide (EB) and agarose gel electrophoresis, we experimentally confirmed the complexation diagrams with a critical aggregation concentration on DNA matrix (CAC DNA) delimiting two regions of complexation, according to the DNA-phosphate concentration. The study of the photo-oxidative damage shows, for the first time, a direct correlation between the DNA complexation by these surfactants and the efficiency of DNA cleavage, with a maximum corresponding to the CAC DNA for DTAC and CTAC, and to DNA neutralization for CTAC and CTAB. The localization of a photosensitizer having low water solubility, such as benzophenone, inside the hydrophobic domains formed by the surfactant aggregated on DNA, locally increases the photoinduced cleavage by the free radical oxygen species generated. The inefficiency of a water-soluble quencher of hydroxyl radicals, such as mannitol, confirmed this phenomenon. The detection of photo-oxidative damage constitutes a new tool for investigating DNA complexation by cationic surfactants. Moreover, highlighting the drastically increased sensitivity of a complexed DNA to photo-oxidative damage is of crucial importance for the biological use of surfactants as nonviral gene delivery systems.     

DNA damage in nucleosomes

Eukaryotic genomic DNA is packed into chromatin, whose fundamental structural unit is the nucleosome. As DNA-histone protein complexes, nucleosomes show different properties toward exogenous and endogenous DNA-damaging agents. This review summarizes nucleosome DNA damage due to different sources, including alkylating agents, radicals, UV radiation and reactive DNA damage intermediates. In most cases, the histone core protects the associated DNA against damage via its structure and/or scavenging of damaging agents. In contrast, histones react with damaged DNA and, in some instances, catalyze DNA damage in the nucleosome. The biological consequence of nucleosome DNA damage and future prospects in this field are briefly discussed.     

Hole transfer in DNA and photosensitized DNA damage: importance of adenine oxidation

Photosensitized DNA damage reactions were investigated for two well-known DNA-damaging photosensitizers (Sens), naphthalimide (NI) and napthaldiimide (NDI), which have similar photophysical properties but differ in their redox properties. NI and NDI derivatives (NIN, NDIN), which have cationic side chains and electrostatically binding to DNA due to favorable electrostatic interactions between the negatively charged phosphate groups of DNA and cationic groups, and NIP and NDIP, which possess phosphate groups and do not bind to DNA, were synthesized. NIN and NDIN can oxidize A and G via their singlet excited state, and NDIP oxidizes A and G via its triplet excited state, whereas NIP oxidizes only G. A combination of laser flash photolysis kinetic studies and quantitative HPLC analyses of photosensitized DNA damage was performed for several DNA sequences in the presence of Sens. NIN, NDIN, and NDIP, which oxidizes A, caused significant DNA damage upon photoirradiation, and DNA damage yield increased with the length of the consecutive A stretch. In contrast, NIP, which oxidizes only G, caused only moderate damage to DNA and showed no preference for the consecutive A sequences. These results clearly demonstrate the importance of A-oxidation, especially in consecutive A sequences, which triggers the rapid hole transfer between A's.     

Characterization of UVC-induced DNA damage in bloodstains: forensic implications

The ability to detect DNA polymorphisms using molecular genetic techniques has revolutionized the forensic analysis of biological evidence. DNA typing now plays a critical role within the criminal justice system, but one of the limiting factors with the technology is that DNA isolated from biological stains recovered from the crime scene is sometimes so damaged as to be intractable to analysis. Potential remedies for damaged DNA are likely to be dependent upon the precise nature of the DNA damage present in any particular sample but, unfortunately, current knowledge of the biochemical nature, and the extent, of such DNA damage in dried biological stains is rudimentary. As a model for DNA damage assessment in biological stains recovered from crime scenes, we have subjected human bloodstains and naked DNA in the hydrated and dehydrated states to varying doses of UVC radiation. It was possible to damage the DNA sufficiently in a bloodstain to cause a standard autosomal short tandem repeat (STR) profile to be lost. However, a detailed analysis of the process, based upon assays developed to detect bipyrimidine photoproducts (BPPPs), single- and double-strand breaks, and DNA–DNA crosslinks, produced some unexpected findings. Contrary to the situation with living tissues or cells in culture, the predominant UVC-induced damage to DNA in bloodstains appears not to be pyrimidine dimers. Although some evidence for the presence of BPPPs and DNA crosslinks was obtained, the major form of UVC damage causing genetic profile loss appeared to be single-strand breaks. It was not possible, however, to preclude the possibility that a combination of damage types was responsible for the profile loss observed. We demonstrate here that a significant measure of protection against UVC-mediated genetic profile loss in dried biological stain material is afforded by the dehydrated state of the DNA and, to a lesser extent, the DNA cellular milieu.     

DNA photo-oxidative damage hazard in transfection complexes

Complexes of DNA with various cationic vectors have been largely used for nonviral transfection, and yet the photochemical stability of DNA in such complexes has never been considered. We studied, for the first time, the influence of DNA complexation by a cationic lipid and polymers on the amount of damage induced by benzophenone photosensitization. The localization of benzophenone inside the hydrophobic domains formed by a cationic lipid, DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride), and close to DNA, locally increases the photoinduced cleavage by the reactive oxygen species generated. The same effect was found in the case of DNA complexation with an amphiphilic polymer (polynorbornenemethyleneammonium chloride). However, a decrease in DNA damage was observed in the case of complexation with a hydrophilic polymer (polyethylenimine). The DNA protection in this case was because of the absence of benzophenone hydrophobic incorporation into the complex, and to DNA compaction which decreased the probability of radical attack. These results underline the importance of the chemical structure of the nonviral transfection vector in limiting the risks of photo-oxidative damage of the complexed DNA.     

The breakage and damage of plasmid DNA photocatalized by TiO2/carbon nanotube composites

In this paper, the plasmid DNA was used as a target to evaluate the bioeffect of TiO₂/Carbon Nanotube (CNT) composites. The conformational change and breakage of DNA induced by the composites were characterized by the integrated tools of electrochemistry, circular dichroism (CD), atomic force microscopy (AFM), and DNA electrophoresis. At the early stage of incubation, the DNA double helix conformation was substantially changed by TiO₂/CNT composites. Both electrophoresis and electrochemistry results suggested the breakage and damage appeared on the native DNA molecules. When DNA was treated longer by TiO₂/CNT, DNA molecules were broken into fragment. AFM images confirmed the process. The DNA damage was deemed to be a gradual process: supercoiled plasmid DNA was first damaged to nicked‐circle structure, then further to linear form, and then DNA fragment. Copyright © 2011 John Wiley & Sons, Ltd.     

2-Aminopurine hairpin probes for the detection of ultraviolet-induced DNA damage

Nucleic acid exposure to radiation and chemical insults leads to damage and disease. Thus, detection and understanding DNA damage is important for elucidating molecular mechanisms of disease. However, current methods of DNA damage detection are either time-consuming, destroy the sample, or are too specific to be used for generic detection of damage. In this paper, we develop a fluorescence sensor of 2-aminopurine (2AP), a fluorescent analogue of adenine, incorporated in the loop of a hairpin probe for the quantification of ultraviolet (UV) C-induced nucleic acid damage. Our results show that the selectivity of the 2AP hairpin probe to UV-induced nucleic acid damage is comparable to molecular beacon (MB) probes of DNA damage. The calibration curve for the 2AP hairpin probe shows good linearity (R² = 0.98) with a limit of detection of 17.2 nM. This probe is a simple, fast and economic fluorescence sensor for the quantification of UV-induced damage in DNA.     

Label-free and rapid colorimetric detection of DNA damage based on self-assembly of a hemin-graphene nanocomposite

Hemin-graphene nanosheets (H-GNs) can be controllably assembled by target DNA via a hybridization process. This results in a color change from dark blue-green to light blue-green. The degree of aggregation is dependent on DNA concentration and very sensitive to base mismatch. The formation of the blue-green color can be detected with bare eyes or a spectrometer. The method is simple, rapid, and works over the concentration range from 1.0 to 100 nM. The detection limit for target DNA is 0.2 nM. Excellent selectivity is also found in that a DNA with a single base mismatch can be discriminated. This was exploited to detect DNA damage as induced by styrene oxide, sodium arsenite, Fenton’s reagent, or UV radiation. We presume that this method represents a promising tool for evaluating genotoxicity. Figure

Detection of DNA damage based on DNA-directed self-assembly of H-GNs     

Comparison between X-ray photon and secondary electron damage to DNA in vacuum

Both monolayer and thick (20 microm) films of dry pGEM-3Zf(-) plasmid DNA deposited on tantalum foil were exposed to Al Kalpha X-rays (1.5 keV) for various times in an ultrahigh vacuum chamber. For monolayer DNA, the damage was induced mainly by low energy secondary electrons (SEs) emitted from the tantalum. For the thick films, DNA damage was induced chiefly by X-ray photons. Different forms of plasmid DNA were separated and quantified by agarose gel electrophoresis. The exposure curves for the formation of nicked circular (single strand break, SSB), linear (double strand break, DSB), and interduplex cross-link forms 1 and 2 were obtained for both monolayer and thick films of DNA, respectively. The lower limits of G values for SSB and DSB induced by SEs were derived to be 86 +/- 2 and 8 +/- 2 nmol J(-1), respectively. These values are 1.5 and 1.6 times larger than those obtained with 1.5 keV photons. The projected X-ray energy dependence of the low energy electron (LEE) enhancement factor for the SSB and DSB in monolayer DNA is also discussed. This new method of investigation of the SE-induced damage to large biomolecules allows direct comparison of the yield of products induced by high energy photons and LEEs under identical experimental conditions.     

Ferrocenecarboxaldehyde labeled DNA probe for the study on DNA damage and protection

A ferrocenecarboxaldehyde (FCA) labeled DNA probe is used for the first time in the study of DNA damage and protection. The electrochemically active reagent FCA was labeled successfully on to a denatured calf-thymus DNA by 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide (EDC). The FCA labeled DNA probe was used to hybridize with the sample DNA sequence accumulated on the surface of a graphite electrode. The anodic peaks of the FCA bound to the double-stranded DNA (dsDNA) by differential pulse voltammetry (DPV) were used for the detection of DNA damage and protection. Thiourea, sodium benzoic acid and isopropanol can decrease DNA damage by hydroxyl radicals, and their protection efficiencies are discussed.     

Efficient photoinduced DNA damage by coralyne

The photoinduced DNA damage by the berberine derivative coralyne is presented. The irradiation of coralyne in the presence of plasmid DNA namely, pBR322, leads to remarkably fast DNA damage by single-strand cleavage, as determined by agarose-gel electrophoresis. Even upon exposure to sunlight, almost all of the supercoiled plasmid is converted to the open circular form in less than a minute [c(pBR322) = 3.5 x 10(-9) M; c(coralyne) = 4.3 x 10(-5) M]. The efficiency of the DNA strand cleavage is not decreased in the presence of radical-trapping reagents such as tert-butanol or DMSO. Moreover, the extent of the DNA damage is the same under aerobic conditions and at reduced oxygen concentration. Thus, the formation of reactive intermediates such as hydroxyl radicals or singlet oxygen is excluded. These results show that the exposure of coralyne and derivatives thereof to light, even with moderate light intensity, needs to be avoided during experiments in which their biological activity is assessed by plasmid unwinding assays.     

Capillary electrophoresis-laser induced fluorescence analysis of endogenous damage in mitochondrial and genomic DNA

Reactive oxygen molecules are formed in vivo as by-products of normal aerobic metabolism. All organisms dependent on oxygen are inevitably exposed to these species so that DNA damage can occur in both genomic and mitochondrial DNA (mtDNA). In order to determine endogenous DNA damage we have developed an analytical method that involves the isolation and hydrolysis of genomic DNA or mtDNA, the labeling of modified and unmodified nucleotides and micellar electrokinetic chromatography with laser-induced fluorescence detection. With this method we have found etheno-adenine, thymine glycol, uracil, hypoxanthine, and 5-methylcytosine. These were identified by the addition of internal standards to the genomic or mtDNA. There are a large number of other signals in the electropherograms of mtDNA that we have never found in genomic DNA analysis because they are at lower concentration in the genome. In the DNA of untreated patients with chronic lymphocytic leukemia (CLL), uracil and high levels of etheno-adenine were found, which can be explained by antioxidant enzyme alterations and oxidative stress in the CLL lymphocytes.     

Ferrocenecarboxaldehyde labeled DNA probe for the study on DNA damage and protection

A ferrocenecarboxaldehyde (FCA) labeled DNA probe is used for the first time in the study of DNA damage and protection. The electrochemically active reagent FCA was labeled successfully on to a denatured calf-thymus DNA by ¶1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide (EDC). The FCA labeled DNA probe was used to hybridize with the sample DNA sequence accumulated on the surface of a graphite electrode. The anodic peaks of the FCA bound to the double-stranded DNA (dsDNA) by differential pulse voltammetry (DPV) were used for the detection of DNA damage and protection. Thiourea, sodium benzoic acid and isopropanol can decrease DNA damage by hydroxyl radicals, and their protection efficiencies are discussed.     

Chemopreventive action of xanthone derivatives on photosensitized DNA damage

Photosensitized DNA damage participates in solar-UV carcinogenesis, photogenotoxicity and phototoxicity. A chemoprevention of photosensitized DNA damage is one of the most important methods for the above phototoxic effects. In this study, the chemopreventive action of xanthone (XAN) derivatives (bellidifolin [BEL], gentiacaulein [GEN], norswertianin [NOR] and swerchirin [SWE]) on DNA damage photosensitized by riboflavin was demonstrated using [32P]-5'-end-labeled DNA fragments obtained from genes relevant to human cancer. GEN and NOR effectively inhibited the formation of piperidine-labile products at consecutive G residues by photoexcited riboflavin, whereas BEL and SWE did not show significant inhibition of DNA damage. The four XAN derivatives decrease the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), an oxidative product of G, by photoexcited riboflavin. The preventive action for the 8-oxodGuo formation of these XAN derivatives increased in the following order: GEN>NOR>BEL>SWE. A fluorescence spectroscopic study and ab initio molecular orbital calculations suggested that the prevention of DNA photodamage is because of the quenching of the triplet excited state of riboflavin by XAN derivatives through electron transfer. This chemoprevention is based on neither antioxidation nor a physical sunscreen effect; rather, it is based on the quenching of a photosensitizer. In conclusion, XAN derivatives, especially GEN, may act as novel chemopreventive agents by the quenching mechanism of an excited photosensitizer.     

Photosensitized DNA damage and its protection via a novel mechanism

UVA, which accounts for approximately 95% of solar UV radiation, can cause mutations and skin cancer. Based mainly on the results of our study, this paper summarizes the mechanisms of UVA-induced DNA damage in the presence of various photosensitizers, and also proposes a new mechanism for its chemoprevention. UVA radiation induces DNA damage at the 5'-G of 5'-GG-3' sequence in double-stranded DNA through Type I mechanism, which involves electron transfer from guanine to activated photosensitizers. Endogenous sensitizers such as riboflavin and pterin derivatives and an exogenous sensitizer nalidixic acid mediate DNA photodamage via this mechanism. The major Type II mechanism involves the generation of singlet oxygen from photoactivated sensitizers, including hematoporphyrin and a fluoroquinolone antibacterial lomefloxacin, resulting in damage to guanines without preference for consecutive guanines. UVA also produces superoxide anion radical by an electron transfer from photoexcited sensitizers to oxygen (minor Type II mechanism), and DNA damage is induced by reactive species generated through the interaction of hydrogen peroxide with metal ions. The involvement of these mechanisms in UVA carcinogenesis is discussed. In addition, we found that xanthone derivatives inhibited DNA damage caused by photoexcited riboflavin via the quenching of its excited triplet state. It is thus considered that naturally occurring quenchers including xanthone derivatives may act as novel chemopreventive agents against photocarcinogenesis.     

Photohydrolysis of methotrexate produces pteridine,which induces poly-G-specific DNA damage through photoinduced electron transfer

Methotrexate (MTX), an antineoplastic agent, demonstrates phototoxicity. The mechanism of damage to biomacromolecules induced by photoirradiated MTX was examined using 32P-labeled DNA fragments obtained from a human gene. Photoirradiated MTX caused DNA cleavage specifically at the underlined G in 5'-GG and 5'-GGG sequences in double-stranded DNA only when the DNA fragments were treated with piperidine, which suggests that DNA cleavage was caused by base modification with little or no strand breakage. With denatured single-stranded DNA the damage occurred at most guanine residues. The amount of formation of 8-hydroxy-2'-deoxyguanosine (8-oxodGuo), an oxidative product of 2'-deoxyguanosine, in double-stranded DNA exceeded that in single-stranded DNA. These results suggest that photoirradiated MTX participates in 8-oxodGuo formation at the underlined G in 5'-GG and 5'-GGG sequences in double-stranded DNA through electron transfer, and then 8-oxodGuo undergoes further oxidation into piperidine-labile products. Fluorescence measurement, high-pressure liquid chromatography and mass spectrometry have demonstrated that photoexcited MTX is hydrolyzed into 2,4-diamino-6-(hydroxymethyl)pteridine (DHP). DNA damage induced by DHP was observed in a similar manner as was the damage induced by MTX. The extent of DNA damage and the formation of 8-oxodGuo by DHP were much larger than those induced by MTX. The kinetic analysis, based on the time course of DNA oxidation by photoirradiated MTX, suggests that DNA damage is caused by photoexcited DHP rather than by photoexcited MTX. In conclusion, photoexcited MTX undergoes hydrolysis through intramolecular electron transfer, resulting in the formation of DHP, which exhibits a phototoxic effect caused by oxidation of biomacromolecules through photoinduced electron transfer.     

Mechanisms and measurements of nanomaterial-induced oxidative damage to DNA

Many of the current investigations on the environmental and human health risks of engineered nanomaterials focus on their short-term acute toxicity. However, the long-term chronic effects of nanomaterials on living systems, and in particular, on the genetic components of living systems, also warrant attention. An increasing number of nanomaterial safety studies include an assessment of genotoxicity as part of the overall risk evaluation. The potential of nanomaterials to directly or indirectly promote the formation of reactive oxygen species is one of the primary steps in their genotoxic repertoire. The subsequent modification of genomic DNA by reactive oxygen species could lead to the development of mutagenesis, carcinogenesis, or other age-related diseases if the DNA damage is not repaired. This review focuses on the interactions of nanomaterials with DNA and specifically on the capacity of some nanomaterials to induce oxidative damage to DNA. A critical assessment of the analytical methodology and the potential biochemical mechanisms involved in nanomaterial induction of oxidative damage to DNA is presented, results obtained for the various studies with each nanomaterial are compared, and recommendations for future research are discussed. Researchers should consider, among other experimental recommendations, (1) the application of more chromatography-based and mass-spectrometry-based analytical techniques to the assessment of oxidative damage to DNA to facilitate an enhanced understanding of DNA damage mechanisms and (2) the verification of cellular viability before conducting genotoxicity assays to reduce the impact of fragmented DNA, formed as a consequence of cell death, on DNA damage measurements.