Yield of DNA strand breaks after base oxidation of plasmid DNA

We have irradiated aerobic aqueous solutions of plasmid DNA with 137Cs gamma rays in the presence of inorganic radical scavengers including nitrite, iodide, azide, thiocyanate and bromide. These scavengers react with the strongly oxidizing hydroxyl radical (*OH) to produce less powerful oxidants. Of these scavengers, only thiocyanate and bromide result in the formation of oxidizing species [(SCN)2*- and Br2*-, respectively] which are capable of reacting with the bases in DNA. The oxidized bases were detected after incubation of the irradiated plasmid with the two E. coli DNA base excision repair endonucleases, formamidopyrimidine-DNA N-glycosylase and endonuclease III. Depending on the experimental conditions, the intermediate base radicals may ultimately form stable oxidized bases in very high yields (within an order of magnitude of the *OH yield), and possibly also single-strand breaks (SSBs) in much lower yield (between 0.1 and 1% of the total yield of base damage). By competing for (SCN)2*- with an additional species (nitrite), it was possible to estimate the second-order rate constant for the reaction of (SCN)2*- with DNA as 1.6 x 10(4) dm3 mol(-1) s(-1), and also to demonstrate a correlation between the large yield of damaged bases and the much smaller increase in the yield of SSBs over background levels due to *OH. The efficiency of transfer of damage from oxidized base to sugar is estimated as about 0.5% or 5%, depending on whether purine or pyrimidine base radicals are responsible for the base to sugar damage transfer.     

Glycoconjugated hypocrellin: photosensitized generation of free radicals (O2*-, *OH, and GHB*-) and singlet oxygen (1O2).

To improve water solubility and specific affinity for malignant tumors, glycoconjugated hypocrellin B (GHB) has been synthesized. Illumination of deoxygenated DMSO solution containing GHB generates a strong electron paramagnetic resonance (EPR) signal. The EPR signal is assigned to the semiquinone anion radical of GHB (GHB*-) based on a series of experimental results. Spectrophotometric measurements show that the absorption bands at 645 nm and 502 nm (pH 8.0) or 505 nm (pH 11.0) arise from the semiquinone anion radical (GHB*-) and hydroquinone (GHBH2) of GHB, respectively. GHBH2 is readily formed via the decay of GHB*- in water-contained solution. The increase of pH value of the reaction media promotes this process. When oxygen is present, superoxide anion radical (O2*-) is formed, via the electron transfer from GHB*-, the precursor, to ground state molecular oxygen. Hydroxyl radical can be readily detected by DMPO spin trapping when aerobic aqueous solution containing GHB is irradiated. As compared with the parent compound, hypocrellin B (HB), the efficiency of O2* and *OH generation by GHB photosensitization is enhanced significantly. Singlet oxygen (1O2) can be produced via the energy transfer from triplet GHB to ground state oxygen molecules, with a decreased quantum yield, i.e., 0.19. These findings suggest that the new GHB possesses an enhanced type I process and a decreased type II process as compared with hypocrellin B.     

Reduction in free-radical-induced DNA strand breaks and base damage through fast chemical repair by flavonoids

This paper provides evidence that dietary flavonoids can repair a range of oxidative radical damages on DNA, and thus give protection against radical-induced strand breaks and base alterations. We have irradiated dilute aqueous solutions of plasmid DNA in the absence and presence of flavonoids (F) in a "constant *OH radical scavenging environment", k of 1.5 x 10(7) s(-1) by decreasing the concentration of TRIS buffer in relation to the concentration of added flavonoids. We have shown that the flavonoids can reduce the incidence of single-strand breaks in double-stranded DNA as well as residual base damage (assayed as additional single-strand breaks upon post-irradiation incubation with endonucleases) with dose modification factors of up to 2.0+/-0.2 at [F] < 100 microM by a mechanism other than through direct scavenging of *OH radicals. Pulse radiolysis measurements support the mechanism of electron transfer or H* atom transfer from the flavonoids to free radical sites on DNA which result in the fast chemical repair of some of the oxidative damage on DNA resulting from *OH radical attack. These in vitro assays point to a possible additional role for antioxidants in reducing DNA damage.     

ESR evidence of the photogeneration of free radicals (GDHB*-, O2*-) and singlet oxygen ((1)O2) by 15-deacetyl-13-glycine-substituted hypocrellin B

15-Deacetyl-13-glycine-substituted hypocrellin B (GDHB) is a new type of hypocrellin derivative with an enhanced red absorption longer than 600 nm and water solubility. Visible light (> 470 nm) irradiation of an anaerobic aqueous solution of GDHB, the formation of GDHB*- was detected by an ESR method in the absence or presence of electron donor. When exposed to oxygen, superoxide anion radical and singlet oxygen were formed. The superoxide anion radical was generated by GDHB*- via electron transfer to oxygen and this process was significantly enhanced by the presence of electron donors. Singlet oxygen ((1)O2) was also formed in the photosensitization of GDHB in aerobic solution and 1,4-diazabicyclo [2,2,2] octane (DABCO), sodium azide (NaN3) and histidine inhibited the generation of (1)O2. A 9,10-diphenyl antracene (DPA)-bleaching method was used to determine the quantum yield of (1)O2 generated from GDHB photosensitization. The (1)O2 quantum yield was estimated to be 0.65. With the depletion of oxygen, the accumulation of GDHB*- would replace that of (1)O2. Evidence accumulated that the photodynamic action of GDHB may proceed via both type I and type II mechanisms and that a type II mechanism will be transformed into a type I mechanism as oxygen gets depleted.     

Mechanism of electron transfer processes photoinduced by lumazine

UV-A (320-400 nm) and UV-B (280-320 nm) radiation causes damage to DNA and other biomolecules through reactions induced by different endogenous or exogenous photosensitizers. Lumazines are heterocyclic compounds present in biological systems as biosynthetic precursors and/or products of metabolic degradation. The parent and unsubstituted compound called lumazine (pteridine-2,4(1,3H)-dione; Lum) is able to act as photosensitizer through electron transfer-initiated oxidations. To get further insight into the mechanisms involved, we have studied in detail the oxidation of 2'-deoxyadenosine 5'-monophosphate (dAMP) photosensitized by Lum in aqueous solution. After UV-A or UV-B excitation of Lum and formation of its triplet excited state ((3)Lum*), three reaction pathways compete for the deactivation of the latter: intersystem crossing to singlet ground state, energy transfer to O(2), and electron transfer between dAMP and (3)Lum* yielding the corresponding pair of radical ions (Lum˙(-) and dAMP˙(+)). In the following step, the electron transfer from Lum˙(-) to O(2) regenerates Lum and forms the superoxide anion (O(2)˙(-)), which undergoes disproportionation into H(2)O(2) and O(2). Finally dAMP˙(+) participates in subsequent reactions to yield products.     

Activation of neocarzinostatin chromophore and formation of nascent DNA damage do not require molecular oxygen.

Thiol-activated neocarzinostatin chromophore abstracts tritium from the 5', but not from the 1' or 2' positions of deoxyribose in DNA and incorporates it into a stable, non-exchangeable form. The abstracted tritium remains covalently associated with the chromophore or its degradation product after treatment with acid or alkali, respectively. Drug activation and the consequent hydrogen abstraction reaction, presumably generating a carbon-centered radical at C-5', do not require molecular oxygen but have a dose-dependent relation with thiol. Under aerobic conditions, where base release and DNA strand breaks with nucleoside 5'-aldehyde at the 5'-ends are produced, hydrogen abstraction from C-5' parallels these parameters of DNA damage. It is possible to formulate a reaction scheme in which the carbon- centered radical at C-5' is an intermediate in the formation of the various DNA damage products found under both aerobic and anaerobic conditions.     

Participation of oxygen in the reduction of methylviologen, photosensitized by chlorophyll, in an aqueous solution of detergent]

Dependence of chlorophyll "a" photosensitized reduction of methylviologene with tiourea on the temperature of reaction mixture was studied in aerobic conditions in triton X-100 aqueous solution. It was found that the reaction consisted of two stages: the light and dark ones. Photosensitized oxidation of tiourea with air oxygen proceeds at the temperatures up to -70 degrees C. Reduction of methylviologen is a dark stage for which diffusion processes are necessary. The role of hydrogen peroxide in the reaction studied has been investigated. It has been shown that hydrogen peroxide is not the "initiator" of the reaction which results in the reduction of methylviologen. Reduced glutation and the mixture of reduced and oxidized glutations were used as electron donors in photosensitized reaction in the presence of air oxygen. An increase of the depth and rate of the reduction of methylviologen under aerobic conditions as compared to anaerobic ones points to the formation of more active reducers than the initial electron donor.     

An analysis of the role of coenzyme Q in free radical generation and as an antioxidant.

The vital role of coenzyme Q in mitochondrial electron transfer and its regulation, and in energy conservation, is well established. However, the role of coenzyme Q in free oxyradical formation and as an antioxidant remains controversial. Demonstration of the existence of the semiquinone form of coenzyme Q during electron transport, coupled with recent evidence that hydrogen peroxide (but not molecular oxygen) may act as an oxidant of the semiquinone, suggests that the highly reactive OH. radical may be formed from the semiquinone. On the other hand, data exist implicating the Fe-S species as the source of electron transfer chain, free radical production. Additional data exist suggesting instead that the unpaired electron of the coenzyme Q semiquinone most likely dismutases superoxide radicals. These concepts and those arising from observations at several levels of organization including subcellular systems, intact animals, and human subjects in the clinical setting, supporting the concept of reduced coenzyme Q as an antioxidant, will be presented. The results of recent studies on the interaction between the two-electron quinone reductase--DT diaphorase and coenzyme Q10 will be presented. The possibility that superoxide dismutase may interact with reduced coenzyme Q, in conjunction with DT diaphorase inhibiting its autoxidation, will be described. The regulation of cellular coenzyme Q concentrations during oxidative stress accompanying aerobic exercise, resulting in increased protection from free radical damage, will also be presented.     

Studies on the photosensitized reduction of resorufin and implications for the detection of oxidative stress with Amplex Red

The photosensitized reduction of resorufin (RSF), the fluorescent product of Amplex Red, was investigated using electron spin resonance (ESR), optical absorption/fluorescence, and oxygen consumption measurements. Anaerobic reaction of RSF in the presence of the electron donor reduced nicotinamide adenine dinucleotide (NADH) demonstrated that during visible light irradiation (λ > 300 nm), RSF underwent one-electron reduction to produce a semiquinoneimine-type anion radical (RSF^• ‾) as demonstrated by direct ESR. Spin-trapping studies of incubations containing RSF, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and NADH demonstrated, under irradiation with visible light, the production of the superoxide dismutase (SOD)-sensitive DMPO/^•OOH adduct. Both absorption and fluorescence spectra of RSF in the presence of NADH demonstrated that the RSF^• ‾ was further reduced during irradiation with formation of its colorless dihydroquinoneimine form, dihydroresorufin (RSFH₂). Both RSF^• ‾ and RSFH₂, when formed in an aerobic system, were immediately oxidized by oxygen, which regenerated the dye and formed superoxide. Oxygen consumption measurements with a Clark-type oxygen electrode showed that molecular oxygen was consumed in a light-dependent process. The suppression of oxygen consumption by addition of SOD or catalase further confirmed the production of superoxide and hydrogen peroxide.     

Hydroxyl radical formation as a result of the interaction between primaquine and reduced pyridine nucleotides. Catalysis by hemoglobin and microsomes

Kinetic, circular dichroism, and NADH and NADPH fluorescence quenching studies indicate that these compounds interact with the antimalarial drug primaquine (PQ). The affinity of both pyridine nucleotides for PQ is similar. The data are in contrast with a previous report (Thornalley et al. (1983) Biochem. Pharmacol. 32, 3571-3575) suggesting specificity for the interaction with NADPH. The complex was seen to facilitate electron transfer from NAD(P)H to oxygen, generating oxygen-free radicals which were detected by the spin-trapping technique and to flavin nucleotides, giving rise to flavin semiquinone radicals which were demonstrated by direct ESR spectroscopy under anaerobic conditions. A twofold increase in oxygen uptake and hydroxyl radical generation by the NAD(P)H-PQ complex was observed in the presence of hemoglobin. This effect was independent of heme concentration (in the range 1 X 10(-5)-1 X 10(-4) M) and oxidation state of the iron. Under anaerobic conditions, the NAD(P)H-PQ complex reduces Fe-III to Fe-II hemoglobin, and under aerobic conditions about 65% of the heme chromophore is irreversibly destroyed. Superoxide dismutase inhibits hydroxyl radical generation by the NAD(P)H-PQ pair; this effect is not observed in the presence of hemoglobin. In the presence of microsomes there is a 10-fold increase in both oxygen consumption and hydroxyl radical generation by the NAD(P)H-PQ pair. The fact that both pyridine nucleotides are active, and the inability of SKF 525A in decreasing hydroxyl radical generation, suggests that microsomal reductases are involved in the catalysis.     

Visible-light promoted degradation of the commercial antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT): a kinetic study

Visible-light photo-irradiation of the commercial phenolic antioxidants (PhAs) butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), in the presence of vitamin B2 (riboflavin, Rf), in methanolic solutions and under aerobic conditions, results in the photo-oxidation of the PhAs. The synthetic dye photosensitiser Rose Bengal was also employed for auxiliary experiments. With concentrations of riboflavin and PhAs of ca. 0.02 mM and < 1 mM, respectively, the excited triplet state of the vitamin (3Rf*) is quenched by BHT in a competitive fashion with dissolved ground state triplet oxygen. From the quenching of 3Rf*, the semireduced form of the pigment is generated through an electron transfer process from BHT, with the subsequent production of superoxide anion radical (O2*-) by reaction with dissolved molecular oxygen. In parallel, the species singlet molecular oxygen, O2(1delta(g)), is also generated. Both reactive oxygen species produce the photodegradation of BHT. In the case of BHA, the lack of any effect exerted by superoxide dismutase drives out a significant participation of a O2(*-)-mediated mechanism. BHA mainly interacts with O2(1delta(g)) and exhibits a desirable property as an antioxidant--a relatively high capacity for O2(1delta(g)) de-activation and a low photodegradation efficiency by the oxidative species. Electrochemical determinations support the proposed photodegradative mechanism.     

The role of vitamin B6 as an antioxidant in the presence of vitamin B2-photogenerated reactive oxygen species. A kinetic and mechanistic study

We report on the photostability of a mixture of vitamins B6 and B2 (riboflavin, Rf) upon visible light irradiation and on the possible role of the vitamin B6 family (B6D) as deactivators of reactive oxygen species (ROS). The work is a systematic kinetic and mechanistic study under conditions in which only Rf absorbs photoirradiation. Pyridoxine, pyridoxal hydrochloride, pyridoxal phosphate and pyridoxamine dihydrochloride were studied as representative members of the vitamin B6 family. The visible light irradiation of dissolved Rf and B6D in pH 7.4 aqueous medium under aerobic conditions induces photoprocesses that mainly produce B6D degradation. The overall oxidative mechanism involves the participation of ROS. Photogenerated (3)Rf* is quenched either by oxygen, giving rise to O(2)((1)Δ(g)) by electronic energy transfer to dissolved ground state oxygen, or by B6D yielding, through an electron transfer process, the neutral radical RfH˙, and O(2)˙(-) in an subsequent step. B6D act as quenchers of O(2)((1)Δ(g)) and O(2)˙(-), the former in a totally reactive event that also inhibits Rf photoconsumption. The common chromophoric moiety of B6D represented by 3-hydroxypyridine, constitutes an excellent model that mimics the kinetic behavior of the vitamin as an antioxidant towards Rf-generated ROS. The protein lysozyme, taken as an O(2)((1)Δ(g))-mediated oxidizable biological target, is photoprotected by B6D from Rf-sensitized photodegradation through the quenching of electronically excited triplet state of the pigment, in a process that competes with O(2)((1)Δ(g)) generation.     

Mitochondrial damage induced by conditions of oxidative stress

Up to 2% of the oxygen consumed by the mitochondrial respiratory chain undergoes one electron reduction, typically by the semiquinone form of coenzyme Q, to generate the superoxide radical, and subsequently other reactive oxygen species such as hydrogen peroxide and the hydroxyl radical. Under conditions in which mitochondrial generation of reactive oxygen species is increased (such as in the presence of Ca²⁺ ions or when the mitochondrial antioxidant defense mechanisms are compromised), these reactive oxygen species may lead to irreversible damage of mitochondrial DNA, membrane lipids and proteins, resulting in mitochondrial dysfunction and ultimately cell death. The nature of this damage and the cellular conditions in which it occurs are discussed in this review article.     

Active site of Escherichia coli DNA photolyase: Asn378 is crucial both for stabilizing the neutral flavin radical cofactor and for DNA repair

Escherichia coli DNA photolyase repairs cyclobutane pyrimidine dimer (CPD) in UV-damaged DNA through a photoinduced electron transfer mechanism. The catalytic activity of the enzyme requires fully reduced FAD (FADH (-)). After purification in vitro, the cofactor FADH (-) in photolyase is oxidized into the neutral radical form FADH (*) under aerobic conditions and the enzyme loses its repair function. We have constructed a mutant photolyase in which asparagine 378 (N378) is replaced with serine (S). In comparison with wild-type photolyase, we found N378S mutant photolyase containing oxidized FAD (FAD ox) but not FADH (*) after routine purification procedures, but evidence shows that the mutant protein contains FADH (-) in vivo as the wild type. Although N378S mutant photolyase is photoreducable and capable of binding CPD in DNA, the activity assays indicate the mutant protein is catalytically inert. We conclude that the Asn378 residue of E. coli photolyase is crucial both for stabilizing the neutral flavin radical cofactor and for catalysis.     

Mitochondrial free radical generation, oxidative stress, and aging

Mitochondria have been described as "the powerhouses of the cell" because they link the energy-releasing activities of electron transport and proton pumping with the energy conserving process of oxidative phosphorylation, to harness the value of foods in the form of ATP. Such energetic processes are not without dangers, however, and the electron transport chain has proved to be somewhat "leaky." Such side reactions of the mitochondrial electron transport chain with molecular oxygen directly generate the superoxide anion radical (O2*-), which dismutates to form hydrogen peroxide (H2O2), which can further react to form the hydroxyl radical (HO*). In addition to these toxic electron transport chain reactions of the inner mitochondrial membrane, the mitochondrial outer membrane enzyme monoamine oxidase catalyzes the oxidative deamination of biogenic amines and is a quantitatively large source of H2O2 that contributes to an increase in the steady state concentrations of reactive species within both the mitochondrial matrix and cytosol. In this article we review the mitochondrial rates of production and steady state levels of these reactive oxygen species. Reactive oxygen species generated by mitochondria, or from other sites within or outside the cell, cause damage to mitochondrial components and initiate degradative processes. Such toxic reactions contribute significantly to the aging process and form the central dogma of "The Free Radical Theory of Aging." In this article we review current understandings of mitochondrial DNA, RNA, and protein modifications by oxidative stress and the enzymatic removal of oxidatively damaged products by nucleases and proteases. The possible contributions of mitochondrial oxidative polynucleotide and protein turnover to apoptosis and aging are explored.     

Spectroscopic properties and reactivity of free radical forms of A2E

A pyridinium bisretinoid (A2E) is the only identified blue-absorbing chromophore of retinal lipofuscin that has been linked to its aerobic photoreactivity and phototoxicity. Pulse radiolysis has been used to study both the one-electron oxidation and the one-electron reduction of A2E in aqueous micellar solutions. The reduction to the semireduced A2E (lambda(max) broad and between 500 and 540 nm) was achieved with formate radicals and the subsequent decay of A2E* was slow (over hundreds of milliseconds) via complex kinetics. The long lifetime of the A2E* should facilitate its reactions with other biomolecules. For example, with oxygen, the A2E* produced the superoxide radical anion with a rate constant of 3 x 10(8) M(-1) s(-1). The A2E was also reduced by the NAD radical, the corresponding rate constant being 2.3 x 10(8) M(-1) s(-1). Other experiments showed that the one-electron reduction potential of A2E lies in the range -640 to -940 mV. The semioxidized form of A2E (lambda(max) 590 nm) was formed via oxidation with the Br2*- radical and had a much shorter lifetime than the semireduced form. With strongly oxidizing peroxyl radicals (CCl3O2*) our kinetic data suggest the formation of a radical adduct followed by dissociation to the semioxidized A2E. With milder oxidizing peroxyl radicals such as that from methanol, our results were inconclusive. In benzene we observed an efficient oxidation of zeaxanthin to its radical cation by the A2E radical cation; this may be relevant to a detrimental effect of A2E in vision.     

Biochemical reactivity of melatonin with reactive oxygen and nitrogen species

Melatonin (N-acetyl-5-methoxytryptamine), an endogenously produced indole found throughout the animal kingdom, was recently reported, using a variety of techniques, to be a scavenger of a number of reactive oxygen and reactive nitrogen species both in vitro and in vivo. Initially, melatonin was discovered to directly scavenge the high toxic hydroxyl radical (*OH). The methods used to prove the interaction of melatonin with the *OH included the generation of the radical using Fenton reagents or the ultraviolet photolysis of hydrogen peroxide (H202) with the use of spin-trapping agents, followed by electron spin resonance (ESR) spectroscopy, pulse radiolysis followed by ESR, and several spectrofluorometric and chemical (salicylate trapping in vivo) methodologies. One product of the reaction of melatonin with the *OH was identified as cyclic 3-hydroxymelatonin (3-OHM) using high-performance liquid chromatography with electrochemical (HPLC-EC) detection, electron ionization mass spectrometry (EIMS), proton nuclear magnetic resonance (1H NMR) and COSY 1H NMR. Cyclic 3-OHM appears in the urine of humans and other mammals and in rat urine its concentration increases when melatonin is given exogenously or after an imposed oxidative stress (exposure to ionizing radiation). Urinary cyclic 3-OHM levels are believed to be a biomarker (footprint molecule) of in vivo *OH production and its scavenging by melatonin. Although the data are less complete, besides the *OH, melatonin in cell-free systems has been shown to directly scavenge H2O2, singlet oxygen (1O2) and nitric oxide (NO*), with little or no ability to scavenge the superoxide anion radical (O2*-) In vitro, melatonin also directly detoxifies the peroxynitrite anion (ONOO-) and/or peroxynitrous acid (ONOOH), or the activated form of this molecule, ONOOH*; the product of the latter interaction is proposed to be 6-OHM. How these in vitro findings relate to the in vivo antioxidant actions of melatonin remains to be established. The ability of melatonin to scavenge the lipid peroxyl radical (LOO*) is debated. The weight of the evidence is that melatonin is probably not a classic chain-breaking antioxidant, since its ability to scavenge the LOO* seems weak. Its ability to reduce lipid peroxidation may stem from its function as a preventive antioxidant (scavenging initiating radicals), or yet unidentified actions. In sum, in vitro melatonin acts as a direct free radical scavenger with the ability to detoxify both reactive oxygen and reactive nitrogen species; in vivo, it is an effective pharmacological agent in reducing oxidative damage under conditions in which excessive free radical generation is believed to be involved.     

Generation of superoxide by the mitochondrial Complex I

Superoxide production by inside-out coupled bovine heart submitochondrial particles, respiring with succinate or NADH, was measured. The succinate-supported production was inhibited by rotenone and uncouplers, showing that most part of superoxide produced during succinate oxidation is originated from univalent oxygen reduction by Complex I. The rate of the superoxide (O2*-)) production during respiration at a high concentration of NADH (1 mM) was significantly lower than that with succinate. Moreover, the succinate-supported O2*- production was significantly decreased in the presence of 1 mM NADH. The titration curves, i.e., initial rates of superoxide production versus NADH concentration, were bell-shaped with the maximal rate (at 50 microM NADH) approaching that seen with succinate. Both NAD+ and acetyl-NAD+ inhibited the succinate-supported reaction with apparent Ki's close to their Km's in the Complex I-catalyzed succinate-dependent energy-linked NAD+ reduction (reverse electron transfer) and NADH:acetyl-NAD+ transhydrogenase reaction, respectively. We conclude that: (i) under the artificial experimental conditions the major part of superoxide produced by the respiratory chain is formed by some redox component of Complex I (most likely FMN in its reduced or free radical form); (ii) two different binding sites for NADH (F-site) and NAD+ (R-site) in Complex I provide accessibility of the substrates-nucleotides to the enzyme red-ox component(s); F-site operates as an entry for NADH oxidation, whereas R-site operates in the reverse electron transfer and univalent oxygen reduction; (iii) it is unlikely that under the physiological conditions (high concentrations of NADH and NAD+) Complex I is responsible for the mitochondrial superoxide generation. We propose that the specific NAD(P)H:oxygen superoxide (hydrogen peroxide) producing oxidoreductase(s) poised in equilibrium with NAD(P)H/NAD(P)+ couple should exist in the mitochondrial matrix, if mitochondria are, indeed, participate in ROS-controlled processes under physiologically relevant conditions.     

The effect of reductant levels on the formation of damage lesions derived from a 2-deoxyribose radical in ssDNA

Thiols play a major role in the outcome of oxidative damage to DNA when it is initiated through cellular exposure to ionizing radiation. DNA radicals formed under aerobic conditions are converted to peroxyl radicals through trapping by oxygen at a diffusion-controlled rate. As a primary source of cellular reductant, thiols are responsible for the conversion of these DNA-derived peroxyl radicals to their corresponding hydrogen peroxides and subsequent strand breaks. Through the use of modified nucleotides, which act as precursors to nucleic acid radicals, we have investigated the effect of varying amounts of the cellular thiol glutathione (GSH) on the distribution of damage products produced from a 2-deoxyribose radical in DNA: the C3'-thymidinyl radical. The C3'-thymidinyl radical results from the abstraction of a hydrogen atom from the C3'-position of DNA oligomers at a thymidine residue, and is known to deliver several DNA damage lesions including the 3'-phosphoglycolaldehyde, 3'-phosphoglycolate and a 5'-aldehyde. Here we show that the level of GSH present has an impact on the level of production of these C3'-thymidinyl radical derived damage products.