Experimental and theoretical investigation effect of flavonols antioxidants on DNA damage

A new electrochemical biosensor was developed to demonstrate the effect of Acridine Orange (AO) on DNA damage. Then, the biosensor was used to check the inhibitors effect of three flavonols antioxidants (myricetin, fisetin and kaempferol) on DNA damage. Acridine Orange (AO) was used as a damaging agent because it shows a high affinity to nucleic acid and stretch of the double helical structure of DNA. Decreasing on the oxidation signals of adenine and guanine (in the DNA) in the presence of AO were used as probes to study the antioxidants power, using DNA-modified screen printed graphene electrode (DNA/SPGE). The results of our study showed that the DNA-biosensor could be suitable biosensor to investigate the inhibitors ability of the flavonols antioxidants on the DNA damage. The linear dependency was detected in the two regions in the ranges of 1.0–15.0 and 15.0–500.0 pmol L⁻¹. The detection limit was found 0.5 pmol L⁻¹ and 0.6 pmol L⁻¹ for guanine and adenine, respectively. To confirm the electrochemical results, Uv–Vis and fluorescence spectroscopic methods were used too. Finally molecular dynamic (MD) simulation was performed on the structure of DNA in a water box to study any interaction between the antioxidant, AO and DNA.     

DNA damage by fasicularin

Fasicularin is a structurally novel thiocyanate-containing alkaloid isolated from the ascidian Nephteis fasicularis. Early biological experiments suggested that this compound's cytotoxic properties may stem from its ability to damage cellular DNA. Sequence gel analysis reveals that treatment of a 5'-32P-labeled DNA duplex with fasicularin in pH 7.0 buffer causes strand cleavage selectively at guanine residues. Further experiments indicate that production of these base-labile lesions in DNA involves alkylation of guanine residues by a fasicularin-derived aziridinium ion. This work reveals fasicularin as the first natural product found to generate a DNA-alkylating aziridinium ion via a mechanism analogous to the clinically used anticancer drugs mechlorethamine, melphalan, and chlorambucil.     

Duocarmycins binding to DNA investigated by molecular simulation

Duocarmycins are a potent class of antitumor agents, whose activity arises through their covalent binding to adenine nucleobases of DNA.(1-3) Here, we perform molecular dynamics (MD) and hybrid Car-Parinello QM/MM simulations to investigate aspects of duocarmycin binding to the d(pGpApCpTpApApTpTpGpApC) oligonucleotide. We focus on the derivatives (+)-duocarmycin SA (DSA) and (+)-duocarmycin SI (DSI), for which structural information of the covalent complex with the oligonucleotide is available, as well as on the related, but less reactive, NBOC-duocarmycin SA (NBOC-DSA), interacting with the same oligonucleotide. Comparison is made with adenine alkylation reaction in water performed by the smallest of these compounds (NBOC-DSA). The MD calculations suggest that, in noncovalent complexes, (i) drug binding causes a partial dehydration of the minor groove, without inducing a significant conformational changes, and (ii) DSA and DSI occupy a more favorable position for nucleophilic attack than NBOC-DSA, consistently with the lower reactivity of the latter. The QM/MM calculations, which are used to investigate the first step of the alkylation reaction, turn out to provide strongly underestimated free energy barriers. Within these approximations, our calculations suggest that an important ingredient for the experimentally observed DNA catalytic power is the polarization of the drug by the biomolecular scaffold.     

Porphyrin binding to DNA investigated by cyclodextrin supramolecular system

The interactive mode of meso-tetrakis- (4- N-trimethylaminobenzyl) porphyrin (TAPP) with DNA has been investigated by the cyclodextrin (CD)-porphyrin supramolecular system. The binding of TAPP with DNA is inhibited by the anion CD derivative, sulfurbutyl-beta-cyclodextrin (SB-beta-CD); however, the neutral CD cannot influence the binding. As for the inclusion procedure of the CDs-TAPP system, the (1)H-NMR data suggests that the hydrophobic segment of TAPP enters into the cavity of CD, which means that the hydrophobic part of TAPP is not the binding site in the TAPP-DNA interaction. Therefore, the binding model cannot be the intercalation and is not related to the grooves of DNA. In addition, experimental data show that the charge attraction, which exists in the inclusion procedure of SB-beta-CD and TAPP hampers the binding of TAPP with DNA. The negative groups of SB-beta-CD compete with the phosphate groups of the DNA backbone, and have an attraction to the positive groups of TAPP. This competitive attraction supports the theory that the binding mode of TAPP with DNA is "electrostatic binding". Furthermore, there are two binding sites between one TAPP molecule and one DNA standard. We produce a possible binding structure (a suprahelical structure of DNA) for this TAPP-DNA complex. This structure is in good agreement with the literature.     

Protein binding has a large effect on radical mediated DNA damage

Oxidative DNA damage is important in aging and a variety of diseases. Significant advances have been made in our understanding of the chemistry of radical mediated DNA damage. These studies have been carried out on DNA in the absence of proteins. However, in cells DNA is typically bound by proteins such as in chromatin and transiently by proteins that regulate biochemical processes. How and whether protein binding affects DNA radical reactivity is not well understood. The effect of the DNA binding protein Hbb on the reactivity of the 5-(2'-deoxyuridinyl)methyl radical (1) and 5-(2'-deoxycytidinyl)methyl radical (2) was studied. Hbb bends DNA and disrupts base stacking at the sites of kinking. The reactivity of 1 and 2 are significantly affected when they are generated at the kinking site in the presence of Hbb. The increased conformational mobility of the radicals results in significantly higher yields of DNA interstrand cross-links. These studies provide the first specific data on how protein binding affects the reactivity of a DNA radical and bring us closer to understanding oxidative DNA damage in cells.     

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.     

Thermodynamic analysis of DNA binding by a Bacillus single stranded DNA binding protein

Background

Single-stranded DNA binding proteins (SSB) are essential for DNA replication, repair, and recombination in all organisms. SSB works in concert with a variety of DNA metabolizing enzymes such as DNA polymerase.

Results

We have cloned and purified SSB from Bacillus anthracis (SSB_BA). In the absence of DNA, at concentrations ??100 ??g/ml, SSB_BA did not form a stable tetramer and appeared to resemble bacteriophage T4 gene 32 protein. Fluorescence anisotropy studies demonstrated that SSB_BA bound ssDNA with high affinity comparable to other prokaryotic SSBs. Thermodynamic analysis indicated both hydrophobic and ionic contributions to ssDNA binding. FRET analysis of oligo(dT)₇₀ binding suggested that SSB_BA forms a tetrameric assembly upon ssDNA binding. This report provides evidence of a bacterial SSB that utilizes a novel mechanism for DNA binding through the formation of a transient tetrameric structure.

Conclusions

Unlike other prokaryotic SSB proteins, SSB_BA from Bacillus anthracis appeared to be monomeric at concentrations ??100 ??g/ml as determined by SE-HPLC. SSB_BA retained its ability to bind ssDNA with very high affinity, comparable to SSB proteins which are tetrameric. In the presence of a long ssDNA template, SSB_BA appears to form a transient tetrameric structure. Its unique structure appears to be due to the cumulative effect of multiple key amino acid changes in its sequence during evolution, leading to perturbation of stable dimer and tetramer formation. The structural features of SSB_BA could promote facile assembly and disassembly of the protein-DNA complex required in processes such as DNA replication.     

Colorimetric detection of peroxynitrite-induced DNA damage using gold nanoparticles, and on the scavenging effects of antioxidants

We describe a rapid and convenient colorimetric method for the detection of oxidative DNA damage caused by peroxynitrite (ONOO^?) using unmodified gold nanoparticles (AuNPs). AuNPs are stable in the presence of single-stranded DNA (ssDNA) against the aggregation induced by a high ionic strength. If adsorbed ssDNA are cleaved by ONOO^? to form smaller fragments, the AuNPs rapidly aggregate due to electrostatic attraction. As a result, the color of the solution changes from red to blue, and this can be seen with bare eyes. We also have evaluated the activity of the antioxidants gallic acid, ascorbic acid and caffeic acid to scavenge ONOO^?. This method therefore also can be applied to screen for anti-oxidation drugs and agents.

Ferric-ion-photosensitized damage to DNA by hydroxyl and non-hydroxyl radical mechanisms.

Iron(III) and UVA (320-400 nm) light strongly diminished the transforming activity of Haemophilus influenzae DNA in the presence of oxygen. Iron(III) alone in the absence of light had no measurable effect on the transforming activity. The chelating agent ethylenediaminetetraacetic acid (EDTA) conferred virtually complete protection, but hydroxyl radical scavengers (mannitol, methanol, ethanol, isopropanol and dimethyl sulfoxide) inhibited only a small fraction of the inactivation. Treatment of plasmid DNA (pBR322) with iron(III) results in the conversion of the covalently closed circular form of the plasmid to open circles and ultimately to the linear form. Concomitant with the alteration in the conformation of the plasmid, the ability to transform Escherichia coli was reduced. In model systems, iron(III) photoreacted with the DNA backbone causing nicking and double-strand breakage. The results are consistent with a mechanism involving a preliminary complexation of iron(III) by DNA followed by the generation of reactive free radicals other than .OH. We suggest that bound iron, or other UV-absorbing transition metal complexes, may be chromophores capable of causing DNA damage in the long-wave near-UV region.     

The ability of low level laser therapy to prevent muscle tissue damage induced by snake venom

Antivenom therapy has been ineffective in neutralizing the severe local fast developing tissue damage following snakebite envenoming. Herein, some effects of in situ helium neon (HeNe) laser irradiation on rat nerve-muscle preparation injected with Bothrops jararacussu venom are described. The tibialis anterior muscle was injected with venom diluted in 0.9% saline solution (60 microg/0.02 mL) or saline solution alone. Sixty minutes after venom injection, laser (HeNe) treatment was administered at three incident energy densities: dose 1, a single exposure of 3.5 J cm(-2); dose 2, three exposures of 3.5 J cm(-2); dose 3, a single exposure of 10.5 J cm(-2). Muscle function was assessed through twitch tension recordings whereas muscle damage was evaluated through histopathologic analysis, morphometry of area of tissue affected and creatine kinase (CK) serum levels, and compared to unirradiated muscles. Laser application at the dose of 3.5 J cm(-2) reduced the area of injury by 64% (15.9 +/- 1.5%vs 44.2 +/- 5.7%), decreased the neuromuscular blockade (NMB) by 62% (11.5 +/- 2.5%vs 30.4 +/- 5.2%) and reduced CK levels by 58% (from 455 +/- 4.5% to 190.3 +/- 23.4%) when compared with unirradiated controls. Dose 2 showed a poorer benefit than dose 1, and dose 3 was ineffective in preventing the venom effects. Measurements of the absorbance of unirradiated and irradiated venom solution showed no difference in absorption spectra. In addition, no difference in the intensity of partial NMB in nerve-muscle preparation was shown by unirradiated and irradiated venom. The results indicate that the laser light did not alter venom toxicity. We conclude that HeNe laser irradiation at a dosage of 3.5 J cm(-2) effectively reduces myonecrosis and the neuromuscular transmission blocking effect caused by B. jararacussu snake venom. Thus, low level laser therapy may be a promising tool to minimize the severity of some of the local effects of snake envenoming.     

Sodium-ion binding to DNA: detection by ultrafast time-resolved stokes-shift spectroscopy

The nature of the counterion is shown to have a pronounced effect on the apparent dynamic polarity in the interior of DNA. Time-resolved Stokes-shift measurements in the 80 ps to 40 ns time range were made on a polarity-sensitive fluorophore (coumarin 102) that replaces a base pair in an oligonucleotide. With sodium counterions, the emission spectrum narrows with time, whereas with tetrabutylammonium counterions, it does not. Our interpretation is that a subpopulation of helices have sodium cations bound in a fashion that slows the normal dynamics.     

Oxidative damage to DNA: counterion-assisted addition of water to ionized DNA

Oxidative damage to DNA, implicated in mutagenesis, aging, and cancer, follows electron loss that generates a radical cation that migrates to a guanine, where it may react with water to form 8-oxo-7,8-dihydroguanine (8-OxoG). Molecular dynamics and ab initio quantum simulations on a B-DNA tetradecamer reveal activated reaction pathways that depend on the local counterion arrangement. The lowest activation barrier, 0.73 eV, is found for a reaction that starts from a configuration where a Na(+) resides in the major groove near the N7 atoms of adjacent guanines, and evolves through a transition state where a bond between a water oxygen atom and a carbon atom forms concurrently with displacement of a proton toward a neighboring water molecule. Subsequently, a bonded complex of a hydronium ion and the nearest backbone phosphate group forms. This counterion-assisted proton shuttle mechanism is supported by experiments exploiting selective substitution of backbone phosphates by methylphosphonates.     

Predicting the activity of phenolic antioxidants: theoretical method, analysis of substituent effects, and application to major families of antioxidants.

A procedure based on density functional theory is used for the calculation of the gas-phase bond dissociation enthalpy (BDE) and ionization potential for molecules belonging to the class of phenolic antioxidants. We show that use of locally dense basis sets (LDBS) vs full basis sets gives very similar results for monosubstituted phenols, and that the LDBS procedure gives good agreement with the change in experimental BDE values for highly substituted phenols in benzene solvent. Procedures for estimating the O--H BDE based on group additivity rules are given and tested. Several interesting classes of phenolic antioxidants are studied with these methods, including commercial antioxidants used as food additives, compounds related to Vitamin E, flavonoids in tea, aminophenols, stilbenes related to resveratrol, and sterically hindered phenols. On the basis of these results we are able to interpret relative rates for the reaction of antioxidants with free radicals, including a comparison of both H-atom-transfer and single-electron-transfer mechanisms, and conclude that in most cases H-atom transfer will be dominant.     

DNA damage by sulfite autoxidation catalyzed by cobalt complexes

DNA damage was investigated in the presence of sulfite, dissolved oxygen and cobalt(II) complexes with glycylglycylhistidine, glycylhistidyllysine, glycylglycyltyrosylarginine and tetraglycine. These studies indicated that only Co(II) complexed with glycylglycylhistidine (GGH) induced DNA strand breaks at low sulfite concentrations (1-80 microM) via strong oxidants formed in the reaction. In the presence of the other complexes, some damage occurred only in the presence of high sulfite concentrations (0.1-2.0 mM) after incubation for 4 h. In the presence of GGH, Co(II) and dissolved O2, DNA damage must involve a reactive high-valent cobalt complex. The damaging effect was increased by adding S(IV), due to the oxysulfur radicals formed as intermediates in S(IV) autoxidation catalyzed by the complex. SO3 -, HO and H radicals were detected by EPR-spin trapping experiments with DMPO (5,5-dimethyl-1-pyrroline N-oxide). The results indicate that Co(II) binds O2 in the presence of GGH, and leads to the formation of a DMPO-HO adduct without first forming free superoxide or hydroxyl radical, supporting the participation of a reactive high-valent cobalt complex.     

Molecular forceps from combinatorial libraries prevent the farnesylation of Ras by binding to its carboxyl terminus

INTRODUCTION: Ras is one of the major oncogenes. In order to function properly it has to undergo post-translational processing at its carboxyl terminus. It has been shown that inhibitors of farnesyl transferase, the first enzyme in the processing chain, can suppress the transforming activity of oncogenic Ras. RESULTS: We have identified molecular forceps, branched peptidic molecules, from combinatorial libraries that bind to the carboxyl terminus of Ras and interfere with its farnesylation without inhibiting the farnesyl transferase. The active molecules were selected by a screening against the carboxy-terminal octapeptide of Ras. CONCLUSIONS: The implications of our findings are twofold. First, we demonstrate that it is possible to prevent enzymatic transformations by blocking the enzyme's access to its substrate using a synthetic small molecule to mask the substrate. Second, we show that it is feasible to derive molecules from combinatorial libraries that bind a specific epitope on a protein by selecting these molecules with the isolated peptide epitope.     

Spectral properties of topical retinoids prevent DNA damage and apoptosis after acute UV-B exposure in hairless mice

We showed in a recent study that topical retinyl palmitate prevented UV-B-induced DNA damage and erythema in humans. Given that retinyl palmitate is a precursor of retinoic acid, the biological form of vitamin A that acts through nuclear receptors, we wondered whether these protective effects toward UV-B exposure were either receptor dependent or linked to other properties of the retinoid molecule such as its spectral properties. We determined the epidermal retinoid profile induced by topical retinoic acid in hairless mice and analyzed its effect on markers of DNA photodamage (thymine dimers) and apoptosis following acute UV-B exposure; we compared these effects to those induced by other natural topical retinoids (retinaldehyde, retinol and retinyl palmitate) which do not directly activate the retinoid receptors. We then analyzed the direct action of these retinoids on UV-B-induced DNA damage and apoptosis in cultured A431 keratinocytes. Topical retinoic acid significantly decreased (approximately 50%) the number of apoptotic cells, as well as the formation of thymine dimers in the epidermis of mice exposed to acute UV-B. Interestingly, the other topical retinoids decreased apoptosis and DNA damage in a similar way. On the other hand, neither retinoic acid nor the other retinoids interfered with the apoptotic process in A431 keratinocytes exposed to UV-B, whereas DNA photodamage was slightly decreased. We conclude that the decrease of apoptotic cells in hairless mouse epidermis following topical retinoids and UV-B irradiation reflects a protection of the primary targets of UV-B (DNA) by a mechanism independent of the activation of retinoid nuclear receptors, rather than a direct inhibition of apoptosis.     

Predicting methylation status of human DNA sequences by pseudo-trinucleotide composition

DNA methylation plays a key role in the regulation of gene expression. The most common type of DNA modification consists of the methylation of cytosine in the CpG dinucleotide. The detections of DNA methylation have been determined mostly by experimental methods; however, these methods were time-consuming, expensive, and difficult to meet the requirements of modern large-scale sequencing technology. Accordingly, it is necessary to develop automatic and reliable prediction methods for DNA methylation.In this study, the pseudo-trinucleotide composition was proposed, and a novel method was developed by support vector machine (SVM) with the pseudo-trinucleotide composition as input parameter to represent DNA sequence for DNA methylation prediction. The model was evaluated on two datasets, including a dataset of Rollins (dataset_1) and a dataset collected healthy human records from the MethDB database (dataset_2). For dataset_1, the Matthews correlation coefficient (MCC) and accuracy (ACC) by jackknife validation were 0.8051 and 0.6098, respectively. For dataset_2, the MCC and ACC were 0.8500 and 0.7203, respectively. The good prediction results reveal that the pseudo-trinucleotide composition is an effective representation method for DNA sequence and plays a very important role in the prediction of DNA function.