the effects of nitroxide radicals on oxidative dna damage

Original Contribution

THE EFFECTS OF NITROXIDE RADICALS ON OXIDATIVE DNA DAMAGE

ELISABETTA DAMIANI ,* BEATA KALINSKA ,† ADRIANA CANAPA,‡ STEFANIA CANESTRARI,* M ICHAL WOZNIAK,†

ETTORE OLMO,‡ and LUCEDIO GRECI**Dipartimento di Scienze dei Materiali e della Terra, Via Brecce Bianche, Universita`, I-60131 Ancona, Italy;†Department of

General Chemistry, Medical University of Gdansk, 80-211, Debinki 1, Poland; and‡Istituto di Biologia e Genetica, Via BrecceBianche, Universita`, I-60131 Ancona, Italy

(Received13 December1999;Revised7 March 2000;Accepted9 March 2000)

Abstract—The indolinonic and quinolinic aromatic nitroxides synthesized by us are a novel class of biologicalantioxidants, which afford a good degree of protection against free radical-induced oxidation in different lipid andprotein systems. To further our understanding of their antioxidant behavior, we thought it essential to have moreinformation on their effects on DNA exposed to free radicals. Here, we report on the results obtained after exposure ofplasmid DNA and calf thymus DNA to peroxyl radicals generated by the water-soluble radical initiator, 2,29-azobis(2-amidinopropane)dihydrochloride (AAPH), and the protective effects of the aromatic nitroxides and their hydroxy-lamines, using a simple in vitro assay for DNA damage. In addition, we also tested for the potential of these nitroxidesto inhibit hydroxyl radical-mediated DNA damage inflicted by Fenton-type reactions using copper and iron ions. Thecommercial aliphatic nitroxides 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), 4-hydroxy-2,2,6,6-tetramethylpiperi-dine-1-oxyl (TEMPOL), and bis(2,2,6,6-tetramethyl-1-oxyl-piperidin-4-yl)sebacate (TINUVIN 770) were included forcomparison. The results show that the majority of compounds tested protect: (i) both plasmid DNA and calf thymusDNA against AAPH-mediated oxidative damage in a concentration-dependent fashion (1–0.1 mM), (ii) both Fe(II) andCu(I) induced DNA oxidative damage. However, all compounds failed to protect DNA against damage inflicted by thepresence of the transition metals in combination with H2O2. The differences in protection between the compounds arediscussed in relation to their molecular structure and chemical reactivity. © 2000 Elsevier Science Inc.

Keywords—Aromatic nitroxides, Aliphatic nitroxides, Peroxyl radicals, DNA, Antioxidants, Transition metals, Hy-droxyl radical, Free radicals

INTRODUCTION

In this past decade, chemists, biologists, and food scien-tists have increasingly focused their attention on re-searching and testing for new and efficient antioxidants(natural and synthetic) aimed at reducing and/or inhibit-ing free-radical reactions that mediate damage to biomol-ecules [1,2]. In this context, our group has been studyingthe chemical and biochemical characteristics of a newclass of emerging antioxidants: nitroxide radicals [3,4].These stable radicals, which generally serve as spinprobes and labels for studies on membranes and wholecells [5] and as contrast agents for magnetic resonanceimaging [6], are endowed with versatile antioxidant

properties—a characteristic recognized almost three de-cades ago, where it was observed that nitroxides inhib-ited lipid peroxidation [7]. The most frequently em-ployed nitroxides are the piperidines, oxazolidines,pyrrolines, and pyrrolidines (Fig. 1). However, our stud-ies have concentrated on indolinonic and quinolinic ni-troxides synthesized by us (see Fig. 1). The chemistry ofthese two latter types of nitroxides has revealed a distinctreactivity toward a wide range of free radicals generallyinvolved in the oxidation of biomolecules such as alkyl[8,9], peroxyl [10], alkoxyl [11], hydroxyl [12], super-oxide [12], and thiyl [13]. Based on this property, severalapplications in biological systems exposed to free radicalinsult have been undertaken. These have included studieson peroxidation of linolenic acid [14], methyl linoleate[15], rat liver microsomes [16,17], low density lipopro-teins [18], albumin [19,20], and trout erythrocytes [21],and the results obtained have all been promising. How-

Address correspondence to: Dr. Elisabetta Damiani, Dipartimento diScienze dei Materiali e della Terra, Via Brecce Bianche, Universita`,I-60131 Ancona, Italy; Tel:139-0712204416; Fax:139-0712204714;E-Mail: [email protected].

Free Radical Biology & Medicine, Vol. 28, No. 8, pp. 1257–1265, 2000Copyright © 2000 Elsevier Science Inc.Printed in the USA. All rights reserved

0891-5849/00/$–see front matter

PII S0891-5849(00)00242-2

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ever, little information is available on their ability toprotect DNA exposed to free radical damage. Recently,we demonstrated that they were capable of protectingDNA damage in stressed trout nucleated erythrocytesusing the comet assay [22] and to protect DNA fromdamage when illuminated in vitro in the presence of acommon ultraviolet-A-absorbing sunscreen [23]. To fur-ther our understanding of the antioxidant behavior of thisclass of nitroxide compounds, and before extending theiruse into more complex biological systems such as viablecells and whole animals, we thought it essential to havemore information on their effects on DNA exposed tofree radicals.

Among the different kinds of free radicals that takepart in the degradation of cellular and nuclear mem-branes and proteins implicated in the pathogenesis of anumber of human diseases, the peroxyl radical plays aprominent role. It is the prime species involved in chainpropagation reactions in the autoxidation of lipids [24]and is considered to be responsible for the inactivation ofcertain enzymes [25]. However, it has recently beendemonstrated that it is able to cleave the phosphodiesterbackbone of DNA resulting in single and double strandbreaks [26], to oxidize thymidine yielding highly muta-genic 5-methyl oxidation products [26], and to causetransversion at deoxyguanosine [27]. The peroxyl radi-cal-induced DNA damage, therefore, provides an oppor-tunity to study a variety of agents, specifically antioxi-dants, as protectors against oxidative DNA damage.

Here we report on the results obtained after exposureof plasmid DNA and calf thymus DNA to peroxyl rad-icals generated by the water-soluble radical initiator,2,29-azobis(2-amidinopropane)dihydrochloride (AAPH),

and the protective effects of the aromatic nitroxides andtheir corresponding reduced forms (hydroxylamines,.N-OH) synthesized by us, using a simple in vitro assayfor DNA damage. In addition, we also tested for thepotential of these nitroxides to inhibit hydroxy radical-mediated DNA damage inflicted by Fenton-type reac-tions using copper and iron ions. The commercial ali-phatic nitroxides TEMPO, TEMPOL, and TINUVIN 770were also included for comparison.

MATERIALS AND METHODS

All reagents were of pure analytical grade. Calf Thy-mus DNA (D-3664, sodium salt) was purchased fromSigma Chemical Co. (St. Louis, MO, USA), hydrogenperoxide was an Acros Organics product (Geel, Bel-gium), copper(II) sulfate and iron(II) sulfate were pur-chased from Fluka Chemie (Zurich, Switzerland), cop-per(I) chloride, iron(III) chloride and solvents werepurchased from Carlo Erba (Milan, Italy), whereas allother compounds were from Aldrich Chemical Co. (Mil-waukee, WI, USA). Plasmid DNA was the plasmidpMOSBlue (2887 base pairs (bp)) produced by growingpreviously transformedEscherichia coliMOSBlue cells(Amersham, Braunschweig, Germany):endA1,hsdR17(rk12

2mk121), supE44, thi-1, recA1, gyrA96,

relA1, lac[F9proA1B1lac9ZDM15:Tn10(TcR)]. Theplasmid was harvested using the Wizard Plus Maxiprepkit (Promega, Madison, WI, USA) and analyzed on aga-rose gels according to Maniatis et al. [28]. TINUVIN 770was a gift from Ciba Specialty Chemicals (Basel, Swit-zerland), whereas the nitroxides1, 2, 3, 4, and7 and thehydroxylamines corresponding to nitroxides2 and 4were synthesized in our laboratory as described previ-ously by Berti et al. [29,30]. The nitroxides 1,2-dihydro-2,2-diphenyl-3H-indol-3-ethoxyimino-1-oxyl (5) and1,2-dihydro-2-butyl-2-phenyl-3H-indol-3-ethoxyimino-1-oxyl (6) were also synthesized according to thismethod by reacting C6H5MgBr for nitroxide 5 andC4H9MgBr for nitroxide6 with 1,2-dihydro-2-phenyl-3-ethoxyimino-3H-indol-1-oxide. Compounds5 and 6were purified by silica gel column chromatography,eluted with benzene, and then crystallized from hotethanol from which red crystals precipitated. 1,2-Dihy-dro-2-phenyl-3-ethoxyimino-3H-indol-1-oxide was syn-thesized from 1,2-dihydro-2-phenyl-3-hydroxyimino-3H-indol-1-oxide (0.365 g) by reacting it with sodiumhydride and ethyl bromide in the ratio 1:3:3, respec-tively, in 30 ml of tetrahydrofuran (THF) for 24 h. Thereaction solution was poured into ice-cold water, ex-tracted with ethyl acetate, dried, and concentrated. Theorange solid obtained was crystallized from petroleumether. 1,2-Dihydro-2-phenyl-3-hydroxyimino-3H-indol-1-oxide, already described by Angeli et al. [31] was

Fig. 1. Scheme showing the chemical structures of the compoundsstudied.

1258 E. DAMIANI et al.

synthesized from 2-phenyl-1-hydroxyindole. The latter(3 g) was dissolved in 50 ml of ethanol to which sodiumethanoate was added (0.35 g of sodium in 10 ml ofethanol). The reaction was cooled in an ice bath and asolution of amyl nitrate (1.5 g in 10 ml of ethanol) wasadded. After 12 h the reaction was diluted with 60 ml ofwater and acidified with 2 ml of acetic acid from which1,2-dihydro-2-phenyl-3-hydroxyimino-3H-indol-1-oxideprecipitated out. The identity and purity of the com-pounds were checked by thin-layer chromatography, bymass spectroscopy on a Carlo Erba QMD 1000 spec-trometer (Milan, Italy) in EI1 mode, by electron spinresonance (ESR) spectroscopy on a spectrometer (E4,Varian, Sunnyvale, CA, USA), by infra-red (IR) spec-troscopy on an IR spectrometer (model 298, Perkin-Elmer, Norwalk, CT, USA), and by1H and 13C NMRspectra in CDCl3 solution on a Varian Gemini 200 spec-trometer (d in ppm referenced to Me4Si). The ESRspectra of nitroxides5 and6 were simulated on the basisof the following hyperfine coupling constants in Gauss:nitroxide5, aN (NO•) 5 8.86, aH (H4) 5 3.08, aH (H6) 52.94, aH (H3) 5 1.00, aH (H5) 5 0.99, aN (CN) 5 0.71;nitroxide6, aN (NO•) 5 8.85, aH (H4) 5 3.13, aH (H6) 52.97, aH (H3) 5 1.03, aH (H5) 5 0.98, aN (CN) 5 0.76,aH (-CH2-) 5 0.37. Their mass spectra showed the fol-lowing: nitroxide5, calculated for C22H19N2O2, 343.41,found:m/e(relative intensity), 343 (M1, 100), 297 (84),281 (65), 269 (86); nitroxide 6, calculated forC20H23N2O2, 323.41, found:m/e(relative intensity), 323(M1, 36), 267 (100), 239 (29).

To assay for DNA single strand breaks (ssb) inducedby peroxyl radicals, supercoiled plasmid DNA (0.3mg)in 10 mM sodium phosphate, pH 7.4, was incubated at37°C for 1 h with 50 mM AAPH solution in buffer withor without different concentrations of test compounds, ina total volume of 10ml. Test compounds were added toproduce the desired final concentrations as acetonitrilesolutions (1–2ml) The final concentration of acetonitrilein the 10ml reaction system was 1.9 M. The reaction wasstopped by addition of 4ml of loading buffer (0.25%bromphenol blue, 0.25% xylene cyanole, 30% glycerol),and the samples were directly analyzed using electro-phoresis in 1% agarose gels stained with ethidium bro-mide, in 5 3 TBE buffer (45 mM Tris-borate, 1 mMEDTA, pH 8) for 1 h at 8V/cm. Following electrophore-sis, the gels were illuminated with a UV transilluminator(300 nm) and photographed using 665 Polaroid positive-negative film. The extent of ssb was determined bycomparing the intensities of the migration bands of su-percoiled and relaxed DNA. Experiments with calf thy-mus DNA (1 mg) were carried out as described aboveand electrophoresed for 1 h at 6 V/cm. Appropriatecontrols were carried out throughout.

For the Fenton reaction, aqueous solutions of CuCl

and FeSO4.7H2O were always prepared immediately be-fore use and added alone or in combination with H2O2, atdifferent concentrations, to plasmid DNA. The metalsalts were always added after the addition of H2O2 to thesample mixture. Aqueous solutions of CuSO4.5H2O andFeCl3 were also tested alone or in combination withH2O2 at different concentrations. The experiments werecarried out and assayed as described above, after incu-bation for 1 h atroom temperature.

RESULTS

Among the different lesions that free radicals inducein DNA, such as single base modifications, tandem le-sions where two adjacent bases are modified, and intras-trand cross links, single- and double-strand breaks arealso included [32]. In the present study we assayed forstrand breaks using the plasmid nicking assay. These arerelatively simple, yet sensitive and quantitative assaysbased on the relaxation (due to single strand breaks) andlinearization (due to double strand breaks) of supercoiledDNA. These three forms can be separated by agarose gelelectrophoresis, because their electrophoretic mobilitiesare altered. Under our experimental conditions, super-coiled DNA migrated further than the linear form, whichin turn migrated more than the relaxed form.

A typical gel electrophoretogram is illustrated in Fig.2. In this figure it can be observed that when plasmidDNA is incubated for 1 h at37°C in the presence of 50mM AAPH (a well-known azo-initiator that producestertiary carbon radicals upon thermolysis, which in thepresence of oxygen generates a constant flux of peroxylradicals), it is converted into the relaxed form. Thisconversion is dependent on the concentration of AAPH(results not shown). The extent of DNA scission is evi-dent by comparing untreated DNA (control) with treatedDNA. The effects of several nitroxides, hydroxylamines,

Fig. 2. Agarose gel electrophoresis of supercoiled pMOSBlue DNAtreated with AAPH in the presence of free radical scavengers. Super-coiled pMOSBlue DNA (30mg/ml) (control) was incubated with 50mM AAPH in 10 mM phosphate buffer (pH 7.4) at 37°C for 1 h, in thepresence of the compounds reported in the figure at the final concen-trations of 1 mM (A) or 0.1 mM (B).

1259Nitroxides and DNA damage

and vitamin E on the DNA cleavage were investigated.Figure 2A shows that the majority of compounds testedinhibited DNA nicking to various extents at 1 mM finalconcentration, except in the case of compound7 whereonly a small amount of supercoiled DNA remains. More-over, it can be clearly observed that a small degree ofnicking is present in those lanes with compounds5, 2a,and4a in contrast to TEMPO, TEMPOL, TINUVIN, andcompounds1–4. Figure 2B shows the results of the sameexperiment as above, but using 100mM final concentra-tion of compounds. In this case, only the piperidinenitroxides, nitroxides1 and 2 and hydroxylamine2aexerted protection against AAPH-induced DNA damage.Hydroxylamine4a provided protection to some extent,whereas the remaining nitroxides3–7 did not. Acetoni-trile, in which the compounds were dissolved, has noeffect on the plasmid nicking induced by AAPH.

The outcome obtained with plasmid DNA subjectedto radical flux generation from AAPH in the presence orabsence of nitroxides was confirmed when calf thymusDNA (CT-DNA), a nonsupercoiled substrate, wastreated in a similar manner. In fact, Fig. 3 shows that,even when using this nonsupercoiled substrate, 50 mMAAPH is capable of inducing DNA damage [compareuntreated DNA (control) with treated DNA]. The piper-idine nitroxides TEMPO and TEMPOL, indolinonic ni-troxides 1 and 2, and hydroxylamine2a, completelyprotected CT-DNA from oxidative damage. It is impor-tant to observe that also quinolinic nitroxide7 exertedcomplete protection for CT-DNA exposed to radicalinsult as compared to the result obtained with plasmidDNA in which it conferred no protection (Fig. 2).

To test for the potential of nitroxides to inhibit hy-droxy radical-mediated DNA damage, plasmid DNA wasexposed to the oxidizing properties of the Fenton reac-

tion using both copper and iron ions. Thus, an initialpriority was to establish the conditions that would allowfor a significant and detectable level of damage in oursystem. Figure 4 shows that Cu(I) is stronger than Cu(II)(compare lane 2 with 3) at damaging DNA and that thisdamage is concentration-dependent (compare lane 2 with6). In addition, Fe(II) is a more effective mediator ofDNA damage than Fe(III) (compare lane 4 with 5) andthis damage is also dependent on the concentration (com-pare lane 4 with lane 8). Fe(III) at the final concentra-tions tested (1 and 10 mM) induced no damage. Further-more, Cu(I) appears to be more potent than Fe(II) atnicking DNA (compare lane 6 with 8), because the linearform of plasmid DNA is produced with the former.Lanes 10–15 show that exposure of plasmid DNA to 1mM Cu(I) or Cu(II) or 1 mM Fe(II) or Fe(III) withdifferent concentrations of H2O2 leads to more damagethan when each ingredient is used alone. Furthermore,the results indicate that the incubation mixture with cop-per ions leads to more DNA degradation than with ironions (compare lanes 10 and 11 with 12 and 13), whereasthe combination of Fe(III) plus H2O2 seems to mediatemore damage than Fe(II) plus H2O2 (compare lane 12with 13 and lane 14 with 15).

Before testing the outcome of the nitroxides on plas-mid nicking induced by Fenton-like reactions, we wantedto see whether the nitroxides could protect against dam-age induced by the metal alone. The reasoning behindthis stems from the fact that one of the antioxidantproperties attributed to nitroxides is that of maintainingmetal ions in their oxidized forms thus inhibiting theirparticipation in metal-catalyzed free radical reactions[33]. This was determined by exposure of plasmid DNAto Cu(I) and Fe(II), because these were the two transitionmetals that at the concentrations tested (1 and 10 mM)produced more damage; lower concentrations of the met-

Fig. 3. Agarose gel electrophoresis of CT-DNA treated with AAPH inthe presence of free radical scavengers. CT-DNA (100mg/ml) (control)was incubated with 50 mM AAPH in 10 mM phosphate buffer (pH 7.4)and 37°C for 1 h, in the presence of the compounds reported in thefigure at 1 mM final concentration.

Fig. 4. Agarose gel electrophoresis of supercoiled pMOSBlue DNAtreated with transition metals and in the presence or absence of hydro-gen peroxide. Supercoiled pMOSBlue DNA (30mg/ml) (control) wasincubated in 10 mM phosphate buffer (pH 7.4) at room temperature for1 h, in the presence of different concentrations of copper or iron ionsand in combination with different concentrations of hydrogen peroxide.


als were ineffective in producing substantial DNA dam-age in our system.

Figure 5 shows the results obtained when plasmidDNA was incubated with 1 mM Fe(II) (Fig. 5A) and 10mM Fe(II) (Fig. 5B) in the presence of 2 mM TEMPO,TEMPOL, nitroxides1, 2, and7 and hydroxylamine2a.The electrophoretogram shows that, at both concentra-tions of Fe(II) tested, the DNA damage is entirely inhib-ited by the above-mentioned compounds, except in thecase of hydroxylamine2a and nitroxide 7. Figure 6shows the results of a similar experiment as above, butusing Cu(I). In this case, protection was observed onlywhen the lowest concentration of Cu(I) (1 mM) wasused, whereas no protection was conferred by the nitrox-

ides using 10 mM Cu(I). Moreover, nitroxide7 seems tobe the least effective among the compounds tested inthese two systems (Figs. 5 and 6). Higher concentrationsof nitroxides could not be examined in this system forsolubility reasons. When the same compounds were in-cubated with plasmid DNA in the presence of the system1 mM Cu(I)/Fe(III) plus 1 mM H2O2, there was noprotection by any of the compounds tested (results notshown).

DISCUSSION

The main objective of this investigation was to exam-ine the damage produced by peroxyl radicals and metal-catalyzed generation of hydroxyl radicals on DNA, andthe effects of aromatic and aliphatic nitroxides on thisoxidative damage.

As Fig. 2 shows, all the piperidinic and indolinonicnitroxides tested, including the newly synthesized deriv-atives5 and6, protect against peroxyl radical attack onplasmid DNA, and this protection is concentration-de-pendent. Using a 10-fold less concentration (100mM),differences can be observed between the various com-pounds. Although the piperidinic nitroxides, includingTINUVIN 770 and the indolinonic nitroxides bearing acarbonyl group in position 3 (compounds1 and2) stillretain their ability to protect, the remaining compoundswhich bear either an arylimino or an ethoxyimino groupin position 3 (compounds3–6) do not. These differencesin protection at lower concentration may be explained bythe variabilities in affinity of the nitroxides for plasmidDNA, likely caused by structural effects. In fact, com-pounds3–5 possess an extra phenyl group and com-pound6 bears an hydrophobic group in position 2, whichmakes these compounds more lipid-soluble and thereforeprobably less compatible with the system under study.This hypothesis may be further confirmed by the factthat, at the lowest concentration used, hydroxylamine2a(bearing a carbonyl group in position 3) is a more effi-cient protector than hydroxylamine4a (bearing an ary-limino group in the same position). The quinolinic ni-troxide 7 failed to inhibit DNA damage at bothconcentrations tested. Again, we believe that this is dueto structural effects as explained above. Compound7possesses two 6-membered rings and hydrophobicgroups, which probably makes this compound less com-patible with the system under study.

Figure 3 shows that when a nonsupercoiled DNAsubstrate was used, in this case CT-DNA, besides theprotection conferred by the most efficient nitroxides andhydroxylamine observed in the previous case with plas-mid DNA (TEMPO, TEMPOL, nitroxides1 and2, andhydroxylamine2a), quinolinic nitroxide7 was also ca-pable of protecting DNA damage in this system at the

Fig. 5. Agarose gel electrophoresis of supercoiled pMOSBlue DNAtreated with Fe(II) in the presence of free radical scavengers. Super-coiled pMOSBlue DNA (30mg/ml) (control) was incubated with 1 mMFe(II) (A) or 10 mM Fe(II) (B) in 10 mM phosphate buffer (pH 7.4) andat room temperature for 1 h, in the presence of the compounds reportedin the figure at the final concentration of 2 mM.

Fig. 6. Agarose gel electrophoresis of supercoiled pMOSBlue DNAtreated with Cu(I) in the presence of free radical scavengers. Super-coiled pMOSBlue DNA (30mg/ml) (control) was incubated with 1 mMCu(I) (A) or 10 mM Cu(I) (B) in 10 mM phosphate buffer (pH 7.4) andat room temperature for 1 h, in the presence of the compounds reportedin the figure at the final concentration of 2 mM.


concentration tested. This suggests that nitroxide7 ismore compatible with this DNA system and that it has agreater binding affinity for this type of biomolecule;therefore it can exert its protective action.

The variabilities observed and discussed above musttherefore be related to the different structural featuresthat limit mobility and/or affinity to the target site, andthis seems to be the main factor determining the antiox-idant capacity of compounds in biological systems. Infact, we previously observed differences in the antioxi-dant activity of different indolinonic nitroxide radicalsand vitamin E and Trolox in both protein and lipidsystems that were related to their structural features [17].Hahn et al. [34] have also reported differences betweenthe variable protection of various piperidinic and pyrro-lidinic nitroxides against radiation-induced cytotoxicityin Chinese hamster V79 cells that were attributable to thepresence of amine groups in the structure of the nitrox-ides. Similarly, Niki et al. [35] have recently stated thatthe antioxidant efficacy of vitamin E becomes less sig-nificant in micelles and membranes than in solution,primarily because of its reduced mobility, with respect toits model compound, 2,2,5,7,8-pentamethyl-6-chromanolwithout phytyl side chain, which exerted a much higherantioxidant capacity. Therefore, our findings and those ofothers substantiate the principle that the antioxidant ac-tivity of nitroxide radicals and of all compounds ingeneral, may differ completely according to the type ofsubstrate and the microenvironment on which they arebeing tested, and this must always be borne in mindwhen designing and selecting new antioxidants targetedfor a specific use.

The probable mechanism of protection of the nitrox-ide radicals against AAPH-induced DNA damage is dueto scavenging of the initial carbon-centered radicals gen-erated upon thermal decomposition of the azo-initiator,which in the presence of oxygen provide peroxyl radicalsin a kinetically reproducible fashion [36]. In fact, thepiperidine nitroxides such as TEMPO, TEMPOL, andTINUVIN 770 are capable of scavenging only carbon-centered radicals at the nitroxide function to give alky-lated hydroxylamines [37], whereas indolinonic andquinolinic nitroxides have two possibilities for protec-tion: trapping of carbon-centered radicals at the nitroxidefunction [8,9] like the piperidines, and scavenging ofperoxyl radicals on the conjugated benzene ring by asubstitution reaction to give nonparamagnetic species(Fig. 7) [10]. Based on this different chemical reactivitytoward radical species, it was expected that the aromaticnitroxides should protect against AAPH-induced DNAdamage, whereas the piperidines should have no effect.However, because no differences in protection were ob-served between the piperidines and the aromatic nitrox-ides, and as they both react with carbon-centered radicals

at an almost diffusion-controlled rate, it appears thatprotection is due principally to trapping these alkyl rad-icals, thereby reducing the possibility of direct attack onDNA and of indirect attack via reaction with molecularoxygen to give peroxyl radicals. If only peroxyl radicalswere formed and were responsible for the DNA damage,then protection should have been observed only by thearomatic nitroxides. However, because the aromatic ni-troxides also trap oxygen-centered radicals [10–12], theyprovide an additional protective mechanism in thosesystems where they are likely to be generated. An im-portant observation worth bearing in mind is that the rateof the reaction of nitroxides with alkyl radicals must behigher than the one for the reaction between alkyl radi-cals and molecular oxygen, otherwise the results reportedabove would most probably not be observed. The mech-anism of protection by hydroxylamines2a and4a can beexplained by the classical hydrogen-donation reactionlike vitamin E, leading to the generation of the corre-sponding nitroxide. This latter, in turn, possesses its ownintrinsic radical-scavenging properties, which have beenmentioned previously. The fact that2a at 0.1 mM pro-tected better than compounds3–7 (Fig. 2B) could berelated to the structural effects discussed above for thenitroxides, because2a is first converted into its parentnitroxide during its antioxidant activity. In Fig. 2, it is

Fig. 7. Scheme showing the scavenging of alkyl (R•) and peroxylradicals (ROO•) by aliphatic and aromatic nitroxide radicals.


worth noticing that this hydroxylamine at 1 mM pro-vided the plasmid DNA slightly less protection than thatconferred by 0.1 mM. A possible explanation for thisunexpected effect could be the result of the spontaneousoxidation by molecular oxygen in the reaction solution ofthe hydroxylamine to the parent nitroxide, with the for-mation of the hydroperoxyl radical (Eqn. 1).

{}NOOH 1 O23

{}NOO z 1 HOO z (1)

The hydroperoxyl radical can act as a hydrogen abstrac-tor on the DNA system triggering oxidation reactions.Consequently, a higher concentration of hydroxylaminecould likely translate into a higher production of hy-droperoxyl radicals and therefore more DNA damage.Hence, the degree of protection by the hydroxylaminecould most likely be influenced by the extent of itsspontaneous oxidation.

To add to our understanding of the role of nitroxideradicals in protecting DNA damage, we also investigatedthe ability of these compounds to protect against thedegradation inflicted by another radical of biologicalimportance: the highly reactive hydroxyl radical. Themost important pathway for hydroxyl radical formationin biological systems is definitely the reaction of hydro-gen peroxide with one-electron-donating transition metalions, of which the best known is the Fenton reactioninvolving Fe(II) [38]. For this study, we selected bothcopper and iron ions, because these are the two majortransition metals in the biological milieu that can cata-lyze extensive DNA damage in vitro and in vivo [39,40],which is most likely accomplished by the possible bind-ing of the free metal ions to DNA itself. In fact, it isworth bearing in mind that an ATP-dependent iron trans-port system has been recently described in isolated cellnuclei [41].

The results reported in Fig. 4 seem to indicate thatcopper ions are potentially more reactive in inducing ssband double strand breaks than iron ions when testedeither alone or in combination with hydrogen peroxide.These findings are in agreement with those of others[42–44], and the differences in characteristics of damageinduction by these two metals have been discussed else-where in detail [38] and will not be dealt with here.However, at present we have no explanation to accountfor the fact that Fe(III) plus hydrogen peroxide appearsto cause more oxidative damage in our experimentalsystem than does Fe(II) plus hydrogen peroxide.

The results given in Figs. 5 and 6, which show that allthe nitroxides except for nitroxide7 and hydroxylamine2a effectively protect against the DNA damage inducedby Fe(II) and Cu(I), are in line with the antioxidant

behavior of nitroxide compounds. In fact, several studieshave shown that nitroxides protect against cytotoxicitypromoted by hydrogen peroxide [45,46] and quinone-based xenobiotics [47,48], and this effect was suggestedto occur via nitroxide-induced oxidation of reduced met-als, thus pre-empting the production of the toxic hy-droxyl radical.

In this present study, the mechanism whereby DNA isimpaired by the metals alone, most probably proceedsthrough the production of superoxide via the autoxida-tion of these transition metals as shown in Eqn. 2 [49],although other oxidizing species such as the perferryl ionmay not be excluded [50].

Xn1 1 O2 º X~n11!1 1 O2•– (2)

Any system producing superoxide is expected to alsoproduce hydrogen peroxide, via either nonenzymatic orenzyme-catalyzed disproportionation, and this can thenreact in another one-electron reduction reaction by thetransition metal ions to give the hydroxyl radical. Theprobable mechanisms of nitroxide protection observed inFigs. 5 and 6 are most likely due to the oxidation of thereduced metals and/or to their reaction with superoxideradical, because one of the well-known antioxidant prop-erties of these compounds is their superoxide dismutase(SOD)-mimicking activity [33,51]. In fact, superoxidemay either reduce or oxidize the nitroxides according totheir redox potential, and it can act as an oxidizing agentfor hydroxylamines that are converted to the parent ni-troxides. Hydroxylamine2a, during its 1 h incubationwith DNA and transition metals, may also undergo oxi-dation by molecular oxygen to the nitroxide, and there-fore, the apparent protective effect demonstrated by thiscompound could actually be due to its fraction whichundergoes oxidation.

However, if nitroxides protected solely by oxidizingthe reduced metal, one would not expect protection whenthe metal is in excess. The fact that protection by 2 mMnitroxide is observed with 10 mM Fe(II) (Fig. 5B) sug-gests that the nitroxides protect even by other means, forwhich we are unable to give explanations at present. Apossible explanation to interpret the finding that 2 mMnitroxides did not protect DNA damage induced by 10mM Cu(I) (Fig. 6B), whereas they did in the case of 10mM Fe(II), could be found in the redox potentials of themetal species involved [Cu(I)/Cu(II)5 10.15 V vs.NHE in H2O; Fe(II)/Fe(III) 5 10.77 V vs. NHE inH2O]: hence Cu(I) is a more potent reducing agent thanFe(II). Bearing this in mind, when the concentration ofthe metal is higher than that of the nitroxides, Cu(I) couldmaintain the nitroxides in a reduced state for a longerperiod of time than can Fe(II) during the 1 h time course


of the experiments. Therefore, less nitroxide is availablefor participation in antioxidant reactions.

Because nitroxides protect DNA from scission ex-erted by metals alone, where it is assumed that thehydroxyl radical is generated indirectly via superoxideradical as described above, one would expect nitroxidesto also protect DNA against damage caused by hydroxylradicals generated directly by the Fenton system usingboth copper or iron ions in the presence of hydrogenperoxide. However, this was not the case, because whenthese compounds were incubated with the Fenton sys-tems under the experimental conditions reported in Fig.4, no inhibition of DNA damage was observed (resultsnot shown). A possible explanation for this result may bebased on the fact that, during the time course of theexperiment (1 h), the formation of hydroxyl radicalsfrom the metals alone, generated by the multistage path-way described previously, could be much slower than inthe second case. Therefore, the antioxidant properties ofthe nitroxides may be fully expressed during this timecourse. However, when the metal ions are added directlyto the incubation mixture already containing hydrogenperoxide, we presume that the hydroxyl radicals or otheroxidizing species that could be formed in the Fentonreaction are generated locally at very high concentrationsleading very rapidly to “site-specific” damage in a veryshort time. In this case, the nitroxide compounds testedusing our experimental system have no ability to protectagainst this highly reactive species. This is not unusual,because it has been demonstrated in the past that evenrelatively high concentrations of hydroxyl radical scav-engers do not always protect the systems under study.The metal ions can bind near the biological target, i.e.,DNA, so that hydroxyl radicals react with DNA basesimmediately after its formation rather than with the scav-engers [39,52].

Because of their versatility and favorable properties,in addition to their relative nontoxicity in both in vitroand in vivo systems [53,54], it is likely that nitroxideswill find increasing use in viable biological systems. Tothis end, a thorough understanding of their chemical andbiological interactions is of key importance. The resultsobtained overall in this in vitro study of oxidative DNAdamage and the effect of nitroxides shed more light onthe antioxidant activity of this particular class of com-pounds and lead to a better understanding of the fullexploitation of these powerful tools.

Acknowledgements— The authors thank the Italian MURST (Minis-tero dell’Universitae della Ricerca Scientifica e Tecnologica) and theUniversity of Ancona for financial support.

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