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Address correspondence to Kenneth P. Blemings, Ph.D., WestVirginia University, P.O. Box 6108, Morgantown, WV 26506,USA; tel 304 293 2631; fax 304 293 2232; e-mailkbleming@wvu.edu.

Free Radical Scavenging, DNA Protection, and Inhibitionof Lipid Peroxidation Mediated by Uric Acid

Beth Stinefelt,1 Stephen S. Leonard,2 Kenneth P. Blemings,1 Xianglin Shi,2 and Hillar Klandorf 11 Division of Animal and Veterinary Science, West Virginia University, Morgantown, West Virginia2 Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute forOccupational Safety and Health, Morgantown, West Virginia

Abstract. Uric acid (UA) has been proposed to be the dominant antioxidant in birds. The objective of thisstudy was to investigate the quenching effect of varying concentrations of UA, including those found inavian plasma, on specific reactive oxygen species (ROS) and to determine the ability of UA to protect DNAand cellular membranes from ROS-mediated damage. Hydroxyl (•OH) and superoxide (O2

•-) radicalswere detected by electron spin resonance (ESR) and their presence was reduced following addition of UA (p<0.05) in a concentration-dependent manner. UA inhibited hydroxyl-mediated DNA damage, indicatedby the presence of more precise, dense bands of λ Hind III DNA after agarose gel electrophoresis andethidium bromide staining (p <0.05). Lipid peroxidation of silica-exposed RAW 264.7 cell membraneswas diminished (p <0.02) after addition of UA to the cell incubation mixture. These studies demonstratethat UA scavenges hydroxyl and superoxide radicals and protects against DNA damage and lipid peroxidation.These results indicate specific antioxidant protection that UA may afford birds against ROS-mediateddamage. (received 6 October 2004; accepted 21 October 2004)

Keywords: uric acid, oxidative stress, electron spin resonance, lipid peroxidation, DNA oxidation

Introduction

Molecular oxygen is required for indispensablemechanisms in aerobic organisms. However, someoxygen and nitrogen metabolites, termed reactiveoxygen or nitrogen species, are toxic and the bodyrequires defense mechanisms against these highlyreactive molecules. Oxidative stress occurs whenreactive oxygen/nitrogen species overwhelm theantioxidant defense system. This is observed as achange in the organism’s redox status that favors adisproportionate increase in reactive species or adecrease in the antioxidant defense [1]. If reactivespecies are not scavenged by antioxidants, they reactwith other cellular components [2]. The conse-quences of such detrimental reactions include lipid

peroxidation [3], protein modification [4], andDNA oxidation [5].

The synergistic theory of aging proposes thatage-related deterioration of tissues is, in part, relatedto the accumulation of glycosylation end-productsand their interactions with Maillard products andfree radicals [6], which indicate and cause oxidativestress and oxidative damage to proteins [7], DNA[8], and lipids [9]. Avian species have a metabolicrate (oxygen consumption) that is approximately 2to 2.5 times higher than that of mammals ofcomparable body size [10]. In mammals, mito-chondrial electron leak, which is a major source ofhydrogen peroxide and superoxide, is estimated tobe 1 to 2% of the oxygen consumption rate [11].Hence, considering avian metabolism and theincreased potential for reactive species production,birds should age faster than mammals of comparablesize. However, the opposite actually occurs; birdslive much longer than mammals of similar size [10].

0091-7370/05/0100-0037. $2.25. 2005 by the Association of Clinical Scientists, Inc.

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A combination of oxidative stress-reducingfactors characteristic of avian species, when coupledwith existing theories of aging, may explain avianlongevity. One factor is that birds have a lower rateof heart, brain, kidney, and lung mitochondrialreactive oxygen species production, resulting froma reduced free electron leak relative to mammals ofsimilar body size [12]. Another contributing factoris that birds have higher concentrations of plasmauric acid than mammals of similar size [13-15],which protects tissues from reactive species-mediateddamage. Positive correlations have been reportedbetween the maximum life span potential (MLSP)and the uric acid concentrations in brain and plasmaper specific metabolic rate [16].

The plasma concentration of uric acid in broilerchickens ranges from 0.2 to 0.8 mM [17,18].Decreasing uric acid production in birds by approx-imately 33% causes an increase in oxidative stress,as evidenced by increased accumulation of markersof reactive species mediated tissue damage [19]. Skinpentosidine, an intramolecular tissue glycoxidationproduct that accumulates in collagen, and breastmuscle shear force, which are both markers of agingor oxidative stress, were also increased in birds withlow plasma uric acid concentrations [20]. Treatingbroiler chickens with inosine, a uric acid precursor,increases plasma uric acid concentrations, and isassociated with a reduction of oxidative stress andsome reactive species mediated markers of aging[18]. These studies are consistent with the birdsusing uric acid as a defense to prevent prematureaging and tissue damage caused by reactive species.

Uric acid is an antioxidant because it caninactivate an oxidant by an electron transfer beforethe oxidant can react with the targeted biologicalmolecule [21]. Evidence suggests that uric acidquenches hydroxyl radical generation by the Fentonreaction [22]. Uric acid is unreactive with diatomicatmospheric oxygen [21]. The ability of uric acidto quench the diatomic superoxide radical isquestionable. Although uric acid is unable to protectalcohol dehydrogenase from inactivation whenexposed to superoxide [23], uric acid can inhibitsuperoxide-mediated DNA damage [5].

The objective of this study was to determinethe ability of uric acid to scavenge specific free

radicals and to protect DNA from oxidation by thehydroxyl radical. The ability of uric acid to protectmonocyte cell membranes from lipid peroxidationmediated by reactive species released during anoxidative burst was also determined. The uric acidconcentrations utilized in these experiments includeand exceed the plasma concentrations of uric acidin birds.

Materials and Methods

Free radical measurements. Electron spin resonance(ESR) spin trapping was used to detect short-livedfree radical intermediates using a modified technique[24]. ESR involves the addition-type reaction of ashort-lived radical with a spin-trap to form arelatively long-lived free radical product (spinadduct) that can be detected by conventional ESR.The intensity of the signal is used to quantify theamount of free radicals produced in the reaction;hyperfine splittings of the spin-adduct are used toidentify the trapped radicals. All ESR measurementswere conducted using a Bruker EMX spectrometer(Bruker Instruments, Billerica, MA) and a flat cellassembly. The Acquisit program was used for dataacquisition and analysis.

The hydroxyl radical was generated from aFenton reaction with a final concentration of 0.5mM iron sulfate (FeSO4), 0.5 mM hydrogenperoxide (H2O2), and 10.0 mM 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin-trap. Uric acidsolution was added to the reaction mixture to finalconcentrations of 0.0, 0.5, 1.5, 2.5, and 3.5 mM.Sodium formate (50 mM) was added to anadditional Fenton reaction along with uric acid. Thereaction mixtures were brought to a 1 ml volumewith phosphate buffered saline (PBS).

The superoxide radical was generated with 3.5mM xanthine and 4 units xanthine oxidase, with100 mM DMPO as a spin trap. All reagents werekept on ice to retard decomposition of superoxideradical adducts. Uric acid was added to the reactionmixture to final concentrations of 0.0, 0.125, 0.25,0.5, and 1.0 mM. The reaction mixtures werebrought to a volume of 1 ml with PBS.

The PBS (pH 7.4) was treated with Chelex 100to remove transition metal ion contaminants.

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Fig. 1: ESR spectra of hydroxyl radical adducts. Spectra indicateradical adducts created in a PBS buffered system containing10.0 mM DMPO only (A) and 10.0 mM DMPO, 0.5 mMFeSO4, and 0.5 mM H2O2 with 0.0 mM uric acid (B), 0.5mM uric acid (C), 1.5 mM uric acid (D), 2.5 mM uric acid(E), 3.5 mM uric acid (F), and 3.5 mM uric acid and 50 mMsodium formate (G). ESR spectrometer settings were: receivergain, 6.32 x 104; time constant, 20.48 msec; modulationamplitude, 1.00 G; scan time, 20.97 sec; magnetic field 3480± 100 G.

Fig. 2. ESR spectra of superoxide radical adducts. Reactionmixtures in a PBS buffered system containing 100 mM DMPOonly (A) and 100 mM DMPO, 3.5 mM xanthine, 4 unitsxanthine oxidase and 0.0 mM uric acid (B) 0.125 mM uricacid (C), 0.25 mM uric acid (D), 0.5 mM uric acid (E), and1.0 mM uric acid (F). ESR spectrometer settings were: receivergain, 6.32 x 104; time constant, 20.48 msec; modulationamplitude, 0.50 G; scan time, 20.97 sec; magnetic field 3480± 100 G.

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DMPO was purified by charcoal decolorization. Allreactions were initiated by mixing in a test tube andeach reaction mixture was transferred to a flat cellfor ESR measurement. Experiments were performedunder ambient laboratory conditions. Signalintensity was quantified by averaging the twoindividual heights (mm) from the valley to the peakof the two highest signals.

DNA strand breakage assay. The DNA strandbreakage assay was carried out by an establishedmethod [25]. Briefly, each reaction mixturecontained 10 mg DNA (λ Hind III fragments), 1mM FeSO4, 10 mM H2O2, and 0.0, 0.5, 1.0, or2.5 mM uric acid; the volume was brought to 100µl with PBS. The mixtures were incubated for 20min in a test tube at 37°C. Five µl of the reactionmixtures were mixed with 2 µl of gel loading solution(0.1 M EDTA, 0.5% sodium dodecyl sulfate, 40%sucrose, and 0.5% bromophenol blue) and loadedinto individual wells in a 0.7% agarose gel forelectrophoresis at 1 to 2 V/cm in TBE buffer (0.1M tris; 0.09 M boric acid; 0.001 M EDTA). Gelswere stained with Vistra Green (Amersham Bio-sciences, San Francisco, CA) for 30 min and werephotographed under ultraviolet light using aStratagene Eagle Eye II camera (Stratagene Inc, LaJolla, CA). DNA damage was assessed byquantitative analysis of the first smeared band, whichindicates fragmentation resulting from hydroxyl-mediated strand cleavage. Densitometry wascompleted in the immediate region surrounding thefirst band in each lane.

Lipid peroxidation assay. Lipid peroxidation ofsilica-exposed RAW 264.7 mouse peritoneal mono-cytes was measured with a colorimetric assay for lipid

peroxidation products (Bioxytech MDA-586 kit,Oxis International, Portland, OR). The reactionmixture included 1 x 107 cells, 100 µl of 1 mg/mlMin-U-Sil (crystalline silica) or 5 mg/ml Min-U-Sil, with final concentrations of 0.0, 0.5, 1.0, 2.0,or 2.5 mM uric acid. Reactions were brought to afinal volume of 1 ml with PBS. The mixture wasincubated 1 hr in a shaking water bath at 37°C.Measurement of lipid peroxidation was based on thereaction of a chromogenic reagent with malondialde-hyde (MDA) and 4-hydroxyalkenals after a furtherincubation at 45°C for 60 min. Standards andreagent blanks were used to generate a standardcurve. After cells were removed by centrifugation(500 x g for 5 min), the absorbance of the super-natant was measured at 586 nm.

Statistical analysis. Data were analyzed by analysisof variance with the PC-SAS general linear modelsprocedure for significant differences among treat-ment means. In the event of a significant F value,the LSD procedure was used for means comparisons.Differences were considered significant at p 0.05.

Results

Amelioration of hydroxyl and superoxide radicals.Representative ESR spectra generated from theFenton reaction using DMPO as a spin trap areshown in Fig. 1. This spectrum consists of a 1:2:2:1quartet with splittings of aH = aN = 14.9 G [26].Based on these splitting constants the quartet wasassigned to a DMPO/•OH adduct. The Fentonreaction was also exposed to sodium formate, whichserves as a hydroxyl radical scavenger to confirm thepresence of the hydroxyl radical adduct. In thisreaction, a new radical is formed that can be trapped

Table 1. Concentration-dependent effects of uric acid on spectral intensities of the hydroxyl and superoxide ESR signals (see text).

Hydroxyl ESR signal (Fig. 1) Superoxide ESR signal (Fig. 2)Uric acid conc. (mM) % reduction Uric acid conc. (mM) % reduction

0.0 0.0 0.0 0.00.5 31.6 0.125 18.91.5 36.1 0.25 37.82.5 49.6 0.5 54.33.5 51.1 1.0 70.1

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by DMPO, which shows a new adduct signal withhyperfine splittings of aH = 15.8 G and aN = 18.8 Gthat are typical of a DMPO/•CO2- adduct. Theintensity of the ESR signal generated from theFenton reaction without any uric acid serves as thecontrol and is set at 0% inhibition. Adding uricacid to the Fenton reaction was effective in reducingspin adduct signal intensity over 50% with 3.5 mMuric acid (Fig. 1F). Table 1 shows the concentration-dependent effect of uric acid on the ESR signalintensity (r2 = 0.77, p = 0.05). Spectra are shown inFig. 1.

Fig. 2 displays the spin adduct generated by thexanthine and xanthine oxidase system in the absenceor presence of uric acid. The superoxide-DMPOadducts produced from this reaction are representedas the hyperfine splittings in Fig. 2 [26]. A reduction

(70%) of the signal is observed when the superoxidegenerating reaction occurs in 1.0 mM uric acid (Fig.2E). Table 1 shows the concentration-dependenteffect of uric acid on the superoxide adduct intensity(r2 = 0.88, p <0.02) that is observed in the ESRspectra of Fig. 2.

Reduction of DNA fragmentation. Hydroxyl radicalsgenerated by the Fenton reaction are capable ofoxidizing DNA and causing strand breaks, resultingin DNA fragmentation. After gel electrophoresisof DNA exposed to hydroxyl radicals, thefragmented DNA looks smeared throughout the lanedue to the different migration rates of the variousfragment sizes (Fig. 3). DNA exposed to the Fentonreaction is protected from hydroxyl radical-mediatedfragmentation when incubated with uric acid, as

Fig. 3. Agarose gel electrophoresis of λ Hind III DNA fragments. Incubation mixtures include 10 mg DNA only (lane 1), and 10mg DNA, 1 mM FeSO4, 10 mM H2O2 and 0.0 mM uric acid (lane 2), 0.5 mM uric acid (lane 3), 1.0 mM uric acid (lane 4), and2.5 mM uric acid (lane 5). % density = top band density/control band density x 100. Control band density is set at 100%.

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observed by more precise bands with reducedsmearing (r2 = 0.97, p <0.02). In Fig. 3, lanes 2through 5 represent DNA exposed to the Fentonreaction and uric acid at specified concentrations.The % density of the first band, scaled to the firstband in lane 1, declines as the concentration of uricacid decreases. Therefore, uric acid protects DNAfrom hydroxyl-mediated fragmentation in a concen-tration-dependent manner, as shown by a densityreduction and visually less precise bands with moresmearing as concentrations of uric acid decrease.

Lipid peroxidation. Min-U-Sil is a stimulant ofoxidative burst in phagocytes. Incubation of RAW264.7 mouse peritoneal monocytes with Min-U-Silcauses an oxidative burst that damages cellmembranes, as evidenced by lipid peroxidationproducts. Incubation of the cells with uric acidreduced lipid peroxidation (p <0.02) in a concen-tration-dependent manner (Fig. 4). However, lipidperoxidation increased when reaction mixtures wereincubated with 2.5 mM uric acid (p < 0.05), perhapsdue to generation of a potent radical from interactingreactive species (see Discussion).

Discussion

The range of uric acid concentrations used in thepresent study includes and exceeds the plasmaconcentrations of uric acid in broiler chickens. Theconcentration-dependent decrease in the hydroxylradical adduct ESR signal indicates that uric acidcan scavenge the hydroxyl radical. Therefore, birdsand other species with high uric acid concentrationsmay have an enhanced defense against the highlyreactive, nonspecific hydroxyl radical. Thisenhanced ability to scavenge hydroxyl radicals maycontribute to the reduction in markers of reactivespecies-mediated tissue injury observed in broilerchickens with elevated plasma uric acid concen-trations [18]. Hydroxyl radicals are highly unstable,reacting nonspecifically with substrates. The efficacyof uric acid to scavenge these radicals is crucial forbroiler health and longevity. Consequently, birdswith low uric acid concentrations may be morevulnerable to hydroxyl-mediated tissue damage andshould exhibit an increase in the age-related markersof oxidative stress.

Fig. 4. Effect of uric acid on lipid peroxidation of RAW 264.7 mouse perintoneal monocytes. Different letters represent significantdifferences within a Min-U-Sil level, p < 0.05. Values represent averages between 3 replicates ± SEM. Regression of lipidperoxidation with uric acid concentrations resulted in r2 = 0.67 and p = 0.018 for 1 mg/mL Min-U-Sil and r2 = 0.79, p = 0.020for 5 mg/mL Min-U-Sil.

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Results from the superoxide experimentsindicate that the superoxide radical can be scavengedby uric acid concentrations from 0.0 to 1.0 mM.Superoxide is a free radical produced from electronleak in mitochondria, and its rate of production thereis inversely related to mammalian MLSP [27]. Theability to scavenge superoxide at its production siteis crucial for the protection of mitochondrialcomponents. Considering the average plasma uricacid concentration of a bird, it is likely that themitochondrial uric acid concentration falls withinthe range utilized in the present experiment.Therefore, uric acid scavenging of the superoxideradical in mitochondria is likely to occur, which, inaddition to the superoxide scavenging enzymesalready in place, may afford the bird additional freeradical protection and help to extend its longevity.

There is conflicting evidence regarding theability of uric acid to protect cellular componentsfrom superoxide-mediated oxidation. By examiningthe chemistry of this reaction, it appears that uricacid can scavenge the superoxide radical only ifsecondary generation of the hydroxyl radical canoccur [28]. In other studies, uric acid was able toprotect DNA from superoxide-mediated damagegenerated by a xanthine oxidase system [5]. Theability of uric acid to react with the superoxideradical may be attributed to the reactivity of the urateradical with superoxide. Generation of the urateradical can only occur if uric acid is oxidized bycertain reactive species such as the hydroxyl radical,hypochlorous acid, or peroxynitrite [29], which arethe short-lived reactive species that uric acid has beenfound to be most effective at scavenging [30].

The results of the DNA protection assay indicatethat uric acid protects DNA against hydroxylmediated damage generated by Fenton chemistry.These results agree with observations that uric acidcan protect DNA from damage mediated by otherfree-radical generating systems [5,31]. The abilityof uric acid to protect DNA can be attributed to itsability to scavenge hydroxyl radicals before theradicals oxidize DNA. Although the intranuclearuric acid concentration of avian cells is unknown,presumably the uric acid found in the nucleus canprotect DNA from oxidation by the hydroxyl radical.Mitochondrial DNA is the most vulnerable to

oxidation due to its proximity to the mitochondrialmembrane and lack of protective histones [32,33].Therefore the ability of uric acid to scavengesuperoxide to prevent DNA oxidation is mostsignificant at this location. Uric acid may contributeto the repair of mitochondrial DNA as it has beenfound to restore guanine from the guanyl radicalgenerated by pulse radiolysis [21]. The quantity ofoxidized DNA, measured as 8-hydroxy-2’-deoxy-guanosine (8-oxoG) in heart mitochondria, is higherin rats than in pigeons [34] and correlates negativelywith lifespan across mammalian species [35].Mitochondrial 8-oxoG increases with age in variousrat [36] and human tissues [37], which is consistentwith a link between reactive species production andaging. In birds, the effect of both a lower rate ofreactive oxygen species production in mitochondriaand a higher concentration of plasma uric acid maybe the reduction of mitochondrial DNA oxidationand subsequent retardation of the aging process.

Uric acid protects erythrocyte ghosts from lipidperoxidation induced by oxo-heme oxidants [38]and t-butylhydroperoxide [3]. In the present experi-ment, RAW 264.7 cells were exposed to silica tostimulate an oxidative burst of reactive species.Reactive species released during oxidative burstinclude superoxide, hydrogen peroxide, and thehydroxyl radical, each of which has the ability todamage cellular membranes, causing lipidperoxidation [39]. Unsaturated fatty acids in tissueare more sensitive to peroxidation than saturatedfatty acids and this sensitivity increases as the numberof double bonds increases. It would seem advan-tageous to have relatively lower polyunsaturated fattyacids to reduce sensitivity to lipid peroxidation.Birds seem to have developed a non-antioxidantdefense against lipid peroxidation. Compared torats, pigeons have a lower abundance of unsaturatedfatty acids in heart and liver mitochondria, and, asexpected, pigeons are more resistant to lipidperoxidation [40,41]. This increased resistance toperoxidation presumably contributes to avian long-evity. Also contributing to the reduced lipidperoxidation in avian cells may be a higherconcentration of uric acid compared to that of othershort-lived mammals.

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In the present experiment, uric acid reducedlipid peroxidation caused by reactive species releasedupon silica-induced oxidative burst. The observedincrease in lipid peroxidation in 2.5 mM uric acidis consistent with a report that uric acid may have astimulatory effect on lipid peroxidation at higherconcentrations [42]. This may occur by free radicalproduction from uric acid decomposition products,such as the urate radical, that can further react withreactive nitrogen species to create a strong reducingagent. The 2.5 mM concentration of uric acid thatresulted in increased lipid peroxidation exceeds therange of plasma uric acid found in birds. Therefore,the conditions that favor a pro-oxidative action ofuric acid are unlikely to occur in vivo.

Results of the present study indicate that uricacid has a concentration-dependent effect onscavenging hydroxyl radicals and superoxide andinhibits oxidation of DNA and of lipids in cellularmembranes. The ability of uric acid to preventreactive species oxidation of biological componentsgives it a crucial antioxidant role in vivo, slowingthe accumulation of reactive species-mediatedmarkers of tissue injury. Consistent with thesynergistic theory of aging, uric acid is important asan antioxidant for its contribution to decelerationof aging, which is supported by positive correlationbetween uric acid concentrations and MLSP. Areduced mitochondrial reactive oxygen speciesproduction, a lower proportion of polyunsaturatedfatty acids, and a higher plasma uric acidconcentration may act in combination to reduce age-related tissue damage caused by reactive species andcontribute to avian longevity.

Acknowledgements

This research was supported by the West VirginiaAgriculture and Forestry Experiment Station(H393). This is paper 2899 of the West VirginiaAgriculture and Forestry Experiment Station.

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