Deoxyribose Oxidation Research Articles

Dual mechanism of mangiferin protection against iron-induced damage to 2-deoxyribose and ascorbate oxidation

We studied mangiferin effects on the degradation of 2-deoxyribose induced by Fe(III)–EDTA/citrate plus ascorbate, in relation to ascorbate oxidation (measured at 265 nm). Results revealed that mangiferin was equally effective in preventing degradation of both 15 and 1.5 mM 2-deoxyribose. At a fixed Fe(III) concentration, increasing the concentration of ligands (either EDTA or citrate) caused a significant reduction in the protective effects of mangiferin. Interestingly, mangiferin strongly stimulated Fe(III)–EDTA ascorbate oxidation, but inhibited it when citrate was used as iron co-chelator. Mangiferin stimulated O 2 consumption due to Fe(II) (formed by Fe(III) ascorbate reduction) autoxidation when the metal ligand was EDTA, but inhibited it when citrate was used. These results suggest that mangiferin removes iron from citrate, but not from EDTA, forming an iron–mangiferin complex that cannot induce ascorbate oxidation effectively, thus inhibiting iron-mediated oxyradical formation. Taken together, these results indicate that mangiferin works mainly by a mechanism different from the classical hydroxyl radical scavengers, keeping iron in its ferric form, by complexing Fe(III), or stimulating Fe(II) autoxidation.

Long-patch Base Excision DNA Repair of 2-Deoxyribonolactone Prevents the Formation of DNA-Protein Cross-links with DNA Polymerase β

Oxidized abasic sites are a major form of DNA damage induced by free radical attack and deoxyribose oxidation. 2-Deoxyribonolactone (dL) is a C1'-oxidized abasic site implicated in DNA strand breakage, mutagenesis, and formation of covalent DNA-protein cross-links (DPCs) with repair enzymes such as DNA polymerase beta (polbeta). We show here that mammalian cell-free extracts incubated with Ape1-incised dL substrates under non-repair conditions give rise to DPCs, with a major species dependent on the presence of polbeta. DPC formation was much less under repair than non-repair conditions, with extracts of either polbeta-proficient or -deficient cells. Partial base excision DNA repair (BER) reconstituted with purified enzymes demonstrated that Flap endonuclease 1 (FEN1) efficiently excises a displaced oligonucleotide containing a 5'-terminal dL residue, as would be produced during long-patch (multinucleotide) BER. Simultaneous monitoring of dL repair and dL-mediated DPC formation demonstrated that removal of the dL residue through the combined action of strand-displacement DNA synthesis by polbeta and excision by FEN1 markedly diminished DPC formation with the polymerase. Analysis of the patch size distribution associated with DNA repair synthesis in cell-free extracts showed that the processing of dL residues is associated with the synthesis of >or=2 nucleotides, compared with predominantly single nucleotide replacement for regular abasic sites. Our observations reveal a cellular repair process for dL lesions that avoids formation of DPCs that would threaten the integrity of DNA and perhaps cell viability.

Antioxidant properties of casein-phosphopeptides

Caseinphosphopeptides (CPP) have been associated with binding of bivalent ions and enhanced solubility of many important minerals, such as calcium and iron. Less is known of the affinity of these bioactive peptides to prevent oxidation reactions through possible primary or secondary antioxidant mechanisms. A CPP preparation derived from spray-dried whole tryptic digests of bovine casein contained unidentified peptides with molecular weights less than 6 KDa and an affinity to sequester Fe 2+ . Associated with this activity, the CPP also effectively suppressed Fenton reaction-induced site-specific and non site-specific deoxyribose oxidation. In addition, CPP was effective at reducing 2,2′-azobis(2amidinopropane) dihydrochloride; (AAPH-) and Fe 2+ -induced liposomal peroxidation and showed direct scavenging affinity for the hydrophilic 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid; (ABTS) radical. It can be concluded that CPP derived from bovine casein has both primary and secondary antioxidant properties that specifically involve direct free radical scavenging and sequestering of potential metal prooxidants.

Protecting or promoting effects of spermine on DNA strand breakage induced by iron or copper ions as a function of metal concentration

Polyamines are ubiquitous polycations that participate in cellular processes such as growth, differentiation and cell death. Among the different functions ascribed to these organic cations, the polyamine spermine is known to protect DNA from the damage produced by reactive oxygen species (ROS) generated by different agents including copper ions. We have found that spermine exerts opposite effects on DNA strand breakage induced by Fenton reaction depending on metal concentration. Whereas at low concentration of the transition metals, 10 μM copper or 50 μM Fe(II), 1 mM spermine exerted a protective role, at metal concentrations higher than 25 μM copper or 100 μM Fe(II), spermine stimulated DNA strand breakage. The promotion of the damage induced by spermine was independent of DNA sequence but decreased by increasing the ionic concentration of the media or by the presence of metal-chelating agents. Moreover, spermine did not increase the oxidation of 2-deoxyribose by metal/H 2O 2 when DNA was substituted by 2-deoxyribose as a target for damage. Our results corroborate that spermine may protect DNA and 2-deoxyribose from the damage induced by ROS but also demonstrate that under certain conditions spermine may promote DNA strand breakage. The fact that this promoting effect of spermine on ROS-induced damage was observed only in the presence of DNA suggests that this polyamine under certain conditions may facilitate the interaction of copper and iron ions with DNA leading to the formation of ROS in close proximity to DNA.

The radioprotection and antioxidant properties of dehydrogingerdione

The reactions of hydroxyl and glutathiyl radicals with dehydrogingerdione (1) have been studied using biochemical assays and the pulse radiolysis technique in order to understand its radioprotective and antioxidant action. It was found to inhibit the Fenton-mediated 2-deoxyribose oxidation, as well as γ-ray-induced plasmid DNA strand breaks in a concentration-dependent fashion. The pulse radiolysis studies revealed its high reactivity with the hydroxyl and glutathiyl radicals. With the hydroxyl radicals, it initially formed three species, an adduct, a phenoxyl and a methylenic radical. The bimolecular rate constants for the formation of these species were the same (approx. 6.8 × 109 dm3/mol per s). The initially formed adduct also produced the phenoxyl radical at a later stage. In the case of glutathiyl-radical-induced oxidation, an adduct at the α–β double bond was formed as the sole species. Its identity as a carbon-centered radical was inferred from the result of the oxygen effect on the decay of the adduct radical. At a later stage, the adduct radical also produced the phenoxyl radical through an intramolecular radical transformation. A suitable mechanism for the oxidation reactions of 1 has been proposed.

Anti-oxidant activity of spermine and spermidine re-evaluated with oxidizing systems involving iron and copper ions

This study was designed to investigate the direction of redox reactions of spermine and spermidine in the presence of iron and copper. The redox activity of spermine and spermidine was assessed using a variety of methods, including their ability to: (1) reduce Fe 3+ to Fe 2+ ions; (2) protect deoxyribose from oxidation by Fe 2+–ethylene diaminetetraacetic acid, Fe 3+–ethylene diaminetetraacetic acid systems with and without H 2O 2; (3) protect DNA from damage caused by Cu 2+–H 2O 2, and Fe 2+–H 2O 2 with and without ascorbic acid; (4) inhibit H 2O 2-peroxidase-induced luminol dependent chemiluminescence; (5) scavenge diphenyl-picryl-hydrazyl radical. Spermine and spermidine at concentration 1 mM reduced 1.8 ± 0.3 and 2.5 ± 0.1 nmol of Fe 3+ ions during 20 min incubation. Both polyamines enhanced deoxyribose oxidation. The highest enhancement of 7.6-fold in deoxyribose degradation was found for combination of spermine with Fe 3+–ethylene diaminetetraacetic acid. An 10 mM spermine and spermidine decreased CuSO 4–H 2O 2–ascorbic acid- and FeSO 4–H 2O 2–ascorbic-induced DNA damage by 73 ± 6, 69 ± 4% and 90 ± 5, 53 ± 4%, respectively. They did not protect DNA from CuSO 4–H 2O 2 and FeSO 4–H 2O 2. Spermine apparently increased the CuSO 4–H 2O 2-dependent injury to DNA. Polyamines attenuated H 2O 2-peroxidase-induced luminol dependent chemiluminescence. Total light emission from specimens containing 10 mM spermine or spermidine was attenuated by 85.3 ± 1.5 and 87 ± 3.6%. During 20 min incubation 1 mM spermine or spermidine decomposed 8.1 ± 1.4 and 9.2 ± 1.8% of diphenyl-picryl-hydrazyl radical. These results demonstrate that polyamines of well known anti-oxidant properties may act as pro-oxidants and enhance oxidative damage to DNA components in the presence of free iron ions and H 2O 2.

Chemical and Biological Evidence for Base Propenals as the Major Sourceof the Endogenous M1dG Adduct in CellularDNA

The endogenous DNA adduct, M(1)dG, has been shown to arise in vitro in reactions of dG with malondialdehyde (MDA), a product of both lipid peroxidation and 4'-oxidation of deoxyribose in DNA, and with base propenals also derived from deoxyribose 4'-oxidation. We now report the results of cellular studies consistent with base propenals, and not MDA, as the major source of M1dG under biological conditions. As a foundation for cellular studies, M1dG, base propenals, and MDA were quantified in purified DNA treated with oxidizing agents known to produce deoxyribose 4'-oxidation. The results revealed a consistent pattern; Fe2+-EDTA and gamma-radiation generated MDA but not base propenals or M1dG, whereas bleomycin and peroxynitrite (ONOO-) both produced M1dG as well as base propenals with no detectable MDA. These observations were then assessed in Escherichia coli with controlled membrane levels of polyunsaturated fatty acids (PUFA). ONOO- treatment (2 mm) of cells containing no PUFA (defined medium with 18:0/stearic acid) produced 6.5 M1dG/10(7) deoxynucleotides and no detectable lipid peroxidation products, including MDA, as compared with 3.8 M1dG/10(7) deoxynucleotides and 0.07 microg/ml lipid peroxidation products with control cells grown in a mixture of fatty acids (0.5% PUFA) mimicking Luria-Bertani medium. In cells grown with linoleic acid (18:2), the level of PUFA rose to 54% and the level of MDA rose to 0.14 microg/ml, whereas M1dG fell to 1.4/10(7) deoxynucleotides. Parallel studies with gamma-radiation revealed levels of MDA similar to those produced by ONOO- but no detectable M1dG. These results are consistent with base propenals as the major source of M1dG in this model cell system.

Differential Oxidation of Deoxyribose in DNA by γ and α-Particle Radiation

Emerging evidence points to the importance of deoxyribose oxidation in the toxicity of oxidative DNA damage, including the formation of protein-DNA crosslinks and base adducts. With the goal of understanding the differences in deoxyribose oxidation chemistry known to occur with different oxidants, we have compared the formation of one product of 3'-oxidation of deoxyribose in DNA, 3'-phosphoglycolaldehyde (PGA) residues, in isolated DNA and cells exposed to ionizing radiations. A recently developed gas chromatography/negative chemical ionization mass spectrometry method was used to quantify PGA residues in purified DNA and in human TK6 lymphoblastoid cells exposed to gamma radiation (60Co) and alpha particles (241Am). The level of PGA residues was then correlated with the total quantity of deoxyribose oxidation determined by plasmid topoisomer analysis. Alpha-particle irradiation (0-100 Gy) of purified DNA in 50 mM potassium phosphate (pH 7.4) produced a linear dose response of 0.13 PGA residues per 10(6) nucleotides per gray. When normalized to an estimate of the total number of deoxyribose oxidation events (2.0 per 10(6) nucleotides per gray), PGA formation occurred in 7% (+/-0.5) of deoxyribose oxidation events produced by alpha-particle radiation. In contrast, the efficiency of PGA formation in gamma-irradiated DNA was found to be 1% (+/-0.02), which indicates a shift in the chemistry of deoxyribose oxidation, possibly as a result of the different track structures of the two types of ionizing radiation. Studies with gamma radiation were extended to TK6 cells, in which it was observed that gamma radiation produced a linear dose response of 0.0019 PGA residues per 10(6) nucleotides per gray. This is consistent with an approximately 1000-fold quenching effect in cells, similar to the results of other published studies of oxidative DNA damage in vivo.

Quantification of DNA strand breaks and abasic sites by oxime derivatization and accelerator mass spectrometry: Application to γ-radiation and peroxynitrite

We report a highly sensitive method to quantify abasic sites and deoxyribose oxidation products arising in damaged DNA. The method exploits the reaction of aldehyde- and ketone-containing deoxyribose oxidation products and abasic sites with [ 14C]methoxyamine to form stable oxime derivatives, as originally described by Talpaert-Borle and Liuzzi [Reaction of apurinic/apyrimidinic sites with [ 14C]methoxyamine. A method for the quantitative assay of AP sites in DNA, Biochim. Biophys. Acta 740 (1983) 410–416]. The sensitivity of the method was dramatically improved by the application of accelerator mass spectrometry to quantify the 14C, with a limit of detection of 1 lesion in 10 6 nucleotides in 1 μg of DNA. The method was validated using DNA containing a defined quantity of abasic sites, with a >0.95 correlation between the quantities of abasic sites and those of methoxyamine labels. The original applications of this and similar oxyamine derivatization methods have assumed that abasic sites are the only aldehyde-containing DNA damage products. However, deoxyribose oxidation produces strand breaks and abasic sites containing a variety of degradation products with aldehyde and ketone moieties. To assess the utility of methoxyamine labeling for quantifying strand breaks and abasic sites, the method was applied to plasmid DNA treated with γ-radiation and peroxynitrite. For γ-radiation, there was a 0.99 correlation between the quantity of methoxyamine labels and the quantity of strand breaks and abasic sites determined by a plasmid nicking assay; the abasic sites comprised less than 10% of the radiation-induced DNA damage. Studies with peroxynitrite demonstrate that the method, in conjunction with DNA repair enzymes that remove damaged bases to produce aldehydic sugar residues or abasic sites, is also applicable to quantifying nucleobase lesions in addition to strand break products. Compared to other abasic site quantification techniques, the modified method offers the advantage of providing a straightforward and direct measurement of aldehyde- and ketone-containing strand breaks and abasic sites, with the potential for direct labeling in cells prior to DNA isolation.

Antioxidant Properties of Carnosine Re-Evaluated with Oxidizing Systems Involving Iron and Copper Ions

Carnosine has antioxidant properties and is efficient in the treatment of chemically-induced inflammatory lesions in animals. However, some studies question its biological significance as antioxidant and show lack of protection and even pro-oxidant effect of carnosine in systems containing nickel and iron ions. The ability of carnosine to: (1) reduce Fe(3+) into Fe(2+) ions; (2) protect deoxyribose from oxidation by Fe(2+)-, Fe(3+)-, and Cu(2+)-H(2)O(2)-EDTA systems; (3) protect DNA from damage caused by Cu(2+)-, and Fe(2+)-H(2)O(2)-ascorbate systems; (4) inhibit HClO- and H(2)O(2)-peroxidase-induced luminol dependent chemiluminescence was tested in vitro. At concentration 10 mM carnosine reduced 16.6+/-0.5 nmoles of Fe(3+) into Fe(2+) ions during 20 min. incubation and added to plasma significantly increased its ferric reducing ability. Inhibition of deoxyribose oxidation by 10 mM carnosine reached 56+/-5, 40+/-11 and 30+/-11% for systems containing Fe(2+), Fe(3+) and Cu(2+) ions, respectively. The damage to DNA was decreased by 84+/-9 and 61+/-14% when Cu(2+)-, and Fe(2+)-H(2)O(2)-ascorbate systems were applied. Combination of 10 mM histidine with alanine or histidine alone (but not alanine) enhanced 1.3 and 2.3 times (P<0.05) the DNA damage induced by Fe(2+)-H(2)O(2)-ascorbate. These amino acids added to 10 mM carnosine decreased 3.1-fold (P<0.05) its protective effect on DNA. Carnosine at 10 and 20 mM decreased by more than 90% light emission from both chemiluminescent systems. It is concluded that carnosine has significant antioxidant activity especially in the presence of transition metal ions. However, hydrolysis of carnosine with subsequent histidine release may be responsible for some pro-oxidant effects.

Changing Selectivity of DNA Oxidation from Deoxyribose to Guanine by Ligand Design and a New Binuclear Copper Complex

A dinuclear copper complex [CuII2(PD'O)(H2O)2]3+ (1) (where PD'OH is a pyridylalkylamine containing binucleating ligand) promotes guanine oxidation in single-stranded DNA in the presence of 3-mercaptopropionic acid and dioxygen. This reaction is detected after subsequent piperidine treatment. Little spontaneous strand scission indicative of deoxyribose oxidation is observed in contrast to the results known for other copper complexes. Chemical characterization and nanospray ionization mass spectrometry analysis of oligodeoxynucleotides treated with 1 suggest conversion of guanine residues to their 2,6-diamino-5-formamidino-4-hydroxypyrimidine (+18 amu) and possibly 5,8-dihydroxy-7,8-dihydroguanine (+34 amu) derivatives. The selectivity toward nucleobase rather than deoxyribose oxidation is discussed in terms of the specific nature of the dicopper (hydro)peroxo species formed with the PD'OH ligand versus the intermediates formed in the presence of other binucleating ligands.

Effects of Peroxynitrite Dose and Dose Rate on DNA Damage and Mutation in thesupFShuttle Vector

Peroxynitrite (ONOO-) induces oxidative and nitrosative DNA damage, and previous studies by our group have shown that it is strongly mutagenic in the supF shuttle vector pSP189 replicated in Escherichia coli MBL50 cells. In those experiments, however, the pSP189 plasmid was exposed under unphysiological conditions to large single bolus doses of ONOO-, which limits extrapolation of the data to in vivo pathological states in which ONOO- may play a role. We have thus sought to define the effects of ONOO- dose and dose rate on the DNA damage and mutations induced in the supF gene by three different dosage mechanisms: (i) by infusion of ONOO- solution into suspensions of pSP189 at rates approximating those estimated to occur in inflamed tissues; (ii) by exposure to 3-morpholinosydnonimine (SIN-1), which generates ONOO- spontaneously during decomposition; and (iii) by bolus doses of ONOO- solution. In all cases, plasmid DNA was exposed in the presence of 25 mM bicarbonate, since the reaction of CO2 with ONOO- (to form nitrosoperoxycarbonate) has a major impact on mutagenic potency of ONOO- in this system. Nucleobase and deoxyribose damage were evaluated by a plasmid nicking assay immediately after ONOO- and SIN-1 exposures. Mutation frequency (MF) and mutational spectra in the supF gene were determined after plasmid pSP189 replicated in host E. coli cells. Bolus ONOO- addition caused the highest amount of DNA damage, including base and deoxyribose lesions, while infusion caused the least. SIN-1 was found to induce almost exclusively deoxyribose oxidation, while bolus addition generated a high percentage of base damage. MF increased in a dose-dependent manner following all treatments, but infused ONOO- and SIN-1 exposures were less mutagenic than bolus ONOO- exposure. MFs induced by infusion and by SIN-1 incubated for 100 min at the highest level (4 mM) were 63 and 43% less, respectively, than that induced by bolus. All mutational hot spots were located at G:C sites except for A121 and A177 induced by SIN-1 exposure. Hot spots at C108 and C168 were common to all exposures; G113, G115, and G116 were common to bolus and infused ONOO- exposures; and G129 was common to infused ONOO- and SIN-1 exposures. Almost all mutations were single base pair substitutions under all exposure conditions. Whereas those induced by infused or bolus ONOO- and SIN-1 consisted predominantly of G:C to T:A transversions (66, 65, and 51%, respectively), G:C to C:G mutations were much less frequent following infusion and SIN-1 (8 and 19%, respectively) than those induced by bolus exposure (29%). A:T to T:A mutations induced were detected only after ONOO- infusion and SIN-1 exposure (9 and 11%, respectively). In conclusion, both dose and dose rate at which a genetic target is exposed to ONOO- substantially influence the damage and mutational response, indicating that these parameters will need to be taken into account in assessing the potential effects of ONOO- in vivo. Furthermore, the results indicate that the chemistry of SIN-1-induced DNA damage differs substantially from native ONOO-, which suggests the need for caution in interpreting the biological relevance of SIN-1 as a surrogate for ONOO-.

Antioxidant activity of differently regioselective chitosan sulfates in vitro

Differently regioselective chitosan sulfates were prepared according to Hanno Baumann’s methods. Their antioxidant potencies were investigated employing various established in vitro systems, such as 1,1-diphenyl-2-picrylhydrazyl (DPPH)/superoxide/hydroxyl radicals scavenging, reducing power, iron ion chelating and total antioxidant activity. All kinds of sulfated chitosans (HCTS, TSCTS, SCTS, TCTS) showed strong inhibitory activity toward superoxide radical by the PMS-NADH system compared to Vc. According to the above-mentioned order their IC 50 were 0.012, 0.040, 0.015, 0.022 mg/mL, respectively, however, scavenging activity of Vc on superoxide radical was 68.19% at 2.0 mg/mL. Scavenging activity of superoxide radical was found to be in the order of HCTS > SCTS > TCTS > TSCTS > Vc. Furthermore, all kinds of sulfated chitosans exhibited strong concentration-dependent inhibition of deoxyribose oxidation. Except for HCTS, others had stronger scavenging activity on hydroxyl radical than Vc. Scavenging effect of TSCTS on 1,1-diphenyl-2-picrylhydrazyl radical was little lower than that of BHA, but better than that of others. All kinds of sulfated chitosans were efficient in the reducing power, especially TSCTS. TSCTS and TCTS showed considerable ferrous ion chelating potency. The data obtained in vitro models clearly establish the antioxidant potency of all kinds of sulfated chitosans. These in vitro results suggested the possibility that sulfated chitosans could be effectively employed as ingredient in health or functional food, to alleviate oxidative stress. However, comprehensive studies need to be conducted to ascertain the in vivo safety of sulfated chitosans in experimental animal models.

5‘-(2-Phosphoryl-1,4-dioxobutane) as a Product of 5‘-Oxidation of Deoxyribose in DNA: Elimination as trans-1,4-Dioxo-2-butene and Approaches to Analysis

Oxidation of deoxyribose in DNA leads to the formation of a spectrum of electrophilic products unique to each position in the sugar. For example, chemical reactions following abstraction of the C5'-hydrogen atom partition to form either a nucleoside 5'-aldehyde residue attached to the 5'-end of the DNA strand or a 5'-formyl phosphate residue attached to the 3'-end of the DNA strand that is accompanied by a four-carbon fragment on the 5'-end. We now present two approaches that both identify the latter fragment as 5'-(2-phosphoryl-1,4-dioxobutane) and provide a means to quantify the formation of this residue by different oxidizing agents. The first approach involves oxidation of DNA followed by reaction with O-benzylhydroxylamine to form stable dioxime derivatives of the putative 5'-(2-phosphoryl-1,4-dioxobutane) residues. The beta-elimination product of this dioxime proved to be the expected trans-1,4-dioxo-2-butene, as judged by gas chromatographic and mass spectrometric (GC/MS) comparison to authentic dioximes of cis- and trans-1,4-dioxo-2-butene, which revealed a unique pattern of three signals for each isomer, and by X-ray crystallography. Using a benzylhydroxylamine dioxime derivative of [2H4]-labeled cis-1,4-dioxo-2-butene as an internal standard, the dose-response for the formation of 5'-(2-phosphoryl-1,4-dioxobutane) was determined to be linear for gamma-radiation, with approximately 6 lesions per 10(6) nt per Gy, and nonlinear for Fe2+-EDTA. A comparison of 5'-(2-phosphoryl-1,4-dioxobutane) formation to total deoxyribose oxidation suggests that gamma-radiation produces approximately 0.04 lesions per deoxyribose oxidation event. As a positive control for 5'-oxidation of deoxyribose, the enediyne calicheamicin was observed to produce 5'-(2-phosphoryl-1,4-dioxobutane) at the rate of approximately 9 lesions per 10(6) nt per microM. A second approach to identifying and quantifying the sugar residue involved derivatization with hydrazine and beta-elimination to form pyridazine followed by quantification of the pyridazine by GC/MS. Using this approach, it was observed that the enediyne, neocarzinostatin, produced a linear dose-response for pyridazine formation, as expected given the ability of this oxidant to cause 1'-, 4'-, and 5'-oxidation of deoxyribose in DNA. The antitumor antibiotic, bleomycin, on the other hand, produced pyridazine at a 10-fold lower rate, which is consistent with 4'-chemistry as the predominant mode of deoxyribose oxidation by this agent. These results provide novel insights into the chemistry of deoxyribose oxidation in DNA and two approaches to quantifying the 5'-(2-phosphoryl-1,4-dioxobutane) precursor of trans-1,4-dioxo-2-butene, an electrophile known to react with nucleobases to form novel DNA adducts.

Reaction of 2′-deoxyribonucleosides with cis- and trans-1,4-dioxo-2-butene

cis-1,4-Dioxo-2-butene is a toxic metabolite of furan, while the trans-isomer is a product of deoxyribose oxidation in DNA. It has recently been reported that both cis- and trans-1,4-dioxo-2-butene react with the 2′-deoxynucleosides dC, dG, and dA to form novel diastereomeric oxadiazabicyclo(3.3.0)octaimine adducts. We have now extended these studies with kinetic and spectroscopic analyses of the reactions of cis- and trans-1,4-dioxo-2-butene, as well as the identification of novel adducts of dA. The reaction of dC with trans-1,4-dioxo-2-butene was observed to be nearly quantitative and produced two interchanging diastereomers with a second-order rate constant of 3.66×10−2M−1s−1, which is nearly 10-fold faster than the reaction with the cis-isomer (3.74×10−3M−1s−1). HPLC analyses of reactions of 1,4-dioxo-2-butene with both dA and 9-methyladenine (pH 7.4, 37°C) revealed multiple products including a novel pair of closely eluting fluorescent species of identical mass ([M+H] m/z 420), each of which contains two molecules of 1,4-dioxo-2-butene, and a more abundant but unstable and non-fluorescent species ([M+H] m/z 414). Partial structural characterization of the fluorescent adducts of dA revealed the presence of the oxadiazabicyclo(3.3.0)octaimine ring system common to the dC adducts. These results support the genotoxic potential of furan metabolites and products of deoxyribose oxidation.

Oxidation of 2-deoxyribose by benzotriazinyl radicals of antitumor 3-amino-1,2,4-benzotriazine 1,4-dioxides.

Tirapazamine (3-amino-1,2,4-benzotriazine 1,4-dioxide) is the lead bioreductive drug in clinical trials as an anticancer agent to kill refractory hypoxic cells of solid tumors. It has long been known that, upon metabolic one-electron reduction, tirapazamine induces lethal DNA double strand breaks in hypoxic cells. These strand breaks arise from radical damage to the ribose moiety of DNA, and in this pulse radiolysis and product analysis study we examine mechanistic aspects of the dual function of tirapazamine and analogues in producing radicals of sufficient power to oxidize 2-deoxyribose to form radicals, as well as the ability of the compounds to oxidize the resulting deoxyribose radicals to generate the strand breaks. Both the rate of oxidation of 2-deoxyribose and the radical yield increase with the one-electron reduction potentials of the putative benzotriazinyl radicals formed from the benzotriazine 1,4-dioxides. Subsequent oxidation of the 2-deoxyribose radicals by the benzotriazine 1,4-dioxides and 1-oxides proceeds through adduct formation followed by breakdown to form the radical anions of both species. The yield of the radical anions increases with increasing one-electron reduction potentials of the compounds. We have previously presented evidence that oxidizing benzotriazinyl radicals are formed following one-electron reduction of the benzotriazine 1,4-dioxides. The reactions reported in this work represent the kinetic basis of a short chain reaction leading to increased oxidation of 2-deoxyribose, a process which is dependent on the one-electron reduction potential of the benzotriazinyl radicals that are above a threshold value of ca. 1.24 V.

Analysis of 3‘-Phosphoglycolaldehyde Residues in Oxidized DNA by Gas Chromatography/Negative Chemical Ionization/Mass Spectrometry

Deoxyribose oxidation in DNA represents a biologically important facet of oxidative DNA damage that gives rise to protein-DNA cross-links and base adducts. Toward the goal of quantifying deoxyribose oxidation chemistry in cells, we report a method for the quantification of 3'-phosphoglycolaldehyde (PGA) residues, which likely arise from 3'-oxidation of deoxyribose in DNA. The method exploits the aldehyde moiety in PGA by derivatization as a stable oxime with pentafluorobenzylhydroxylamine, followed by solvent extraction and gas chromatography/negative chemical ionization/mass spectrometry. A stable isotopically labeled [(13)C(2)]PGA was synthesized and used as an internal standard. The assay showed a linear response over the range of 30 fmol to 300 pmol, and its precision was verified by analysis of a synthetic, PGA-containing oligodeoxynucleotide. The limit of detection in the presence of DNA was 30 fmol per sample, corresponding to two molecules of PGA in 10(6) nucleotides for 170 microg of DNA. Samples were exposed to 0-100 Gy of (60)Co gamma-radiation, which resulted in a linear dose-response of 1.5 PGA residues per 10(6) nucleotides per Gy and a radiation chemical yield (G-value) of 0.0016 micromol/J. When compared to the total quantity of deoxyribose oxidation occurring under the same conditions (141 oxidation events per 10(6) nucleotides per Gy; determined by plasmid topoisomer analysis), PGA formation occurs in 1% of deoxyribose oxidation events. This small fraction is consistent with current models of limited solvent accessibility of the 3'-position of deoxyribose, although partitioning of 3'-chemistry could lead to other damage products that would increase the fraction of oxidation at this site in deoxyribose.

Free radical scavenging activity of an aqueous extract of potato peel

The antioxidant potency of freeze-dried aqueous extract of potato peel was investigated employing various established in vitro systems, such as lipid peroxidation in rat liver homogenate, 1, 1-diphenyl-2-picrylhydrazyl (DPPH)/superoxide/hydroxyl radical scavenging, reducing power, and iron ion chelation. Freeze-dried aqueous extract of potato peel powder (PPE) showed strong inhibitory activity toward lipid peroxidation of rat liver homogenate induced by the FeCl 2–H 2O 2 system. Furthermore, PPE exhibited a strong concentration-dependent inhibition of deoxyribose oxidation. PPE also showed a considerable antioxidant activity in the DPPH radical assay system. The multiple antioxidant activity of PPE was evident as it showed strong reducing power, superoxide scavenging ability and also ferrous ion chelating potency. The data obtained in the in vitro models clearly establish the antioxidant potency of freeze-dried extract of potato peel. Considering that potato peels are discarded as waste and not effectively utilized, these in vitro results suggest the possibility that potato peel waste could be effectively employed as an ingredient in health or functional food, to alleviate oxidative stress. However, comprehensive studies need to be conducted to ascertain the in vivo safety of such extracts in experimental animal models.

Pro-oxidative properties of flavonoids in human lymphocytes.

The pro-oxidative properties of the four flavonoids, quercetin, morin, naringenin and hesperetin, in human lymphocyte system were investigated. Naringenin and hesperetin accelerated the oxidation of deoxyribose induced by Fe(3+)/H(2)O(2) in a concentration range of 0-200 microM, but quercetin and morin decreased it when the concentration was greater than 100 microM. The generation of hydrogen peroxide and the superoxide anion and the production of TBARS in lymphocytes were increased with increasing concentration of a flavonoid. Cell membrane protein thiols of the lymphocytes decreased when treated with the four flavonoids. Quercetin and hesperetin had no significant effect (p>0.05) on the activity of glutathione reductase, but morin and naringenin could inhibit the activity of the enzyme at a concentration of 200 microM, when compared to the control group. The glutathione S-transferase activity was slightly decreased by treatment with each of the four flavonoids only at a concentration of 200 microM. Therefore, the DNA damage in lymphocytes induced by the flavonoids in the model system might have been due to their stimulation of oxidative stress in the lymphocytes, which resulted in the decrease of cell membrane protein thiols, increase of lipid peroxidation in cell membrane and in the influence of the antioxidative enzyme activities.

Kinetic deoxyribose degradation assay and its application in assessing the antioxidant activities of phenolic compounds in a Fenton-type reaction system

Pro-oxidant properties of phenolic antioxidants, which are derived from their iron recycling reactivity, render the traditional deoxyribose degradation assay invalid to assess the hydroxyl radical-scavenging activity in Fenton-type reaction systems. In the present paper, we studied in detail the interactions between iron and phenolic compounds, and established a kinetic deoxyribose method by taking advantage of the distinct difference between the completion time of Fenton reaction and that of the iron-reducing process. With the newly established kinetic method, we investigated the effects of phenolics on hydroxyl radical formation in a Fenton-type system and determined successfully the second rate constants of hydroxyl radical-scavenging reactions. The site-specific and non-site-specific hydroxyl radical-scavenging ability suggested that both direct hydroxyl radical-scavenging potency and iron-chelating capacity accounted for their inhibitory effects on deoxyribose oxidation degradation. This method, more simple, time saving, and applicative than the traditional deoxyribose assay, produces as accurate results (RSD<0.05, with dynamic range from 7.5 to 575 μM) as typical methods, such as radiolysis technology, and may be of significance in evaluating and screen the hydroxyl radical-scavenging antioxidants.