Effects of Nitrite and Nitrate on DNA Damage Induced byUltraviolet Light

Toshinori Suzuki* and Michiyo Inukai

Department of Biological Pharmacy, School of Pharmacy, Shujitsu UniVersity, 1-6-1 Nishigawara,Okayama 703-8516, Japan

ReceiVed December 12, 2005

UV light is a major cause of human skin cancers. Nitrite and nitrate are well-known potential riskfactors for gastric cancer. Little attention has been paid to the relationship between UV light and nitriteor nitrate on cancer. We examined the effects of nitrite and nitrate on the damage to nucleosides andDNA induced by UV light from a mercury lamp and by sunlight at neutral pH. A biologically relevantdose of nitrite and nitrate increased the generation of nucleobases and malondialdehyde on the reactionof nucleosides and DNA with UV light. The efficiency of nitrite enhancing the reaction was higher thanthat of nitrate at low doses. The contribution of the hydroxyl radical as the reactive species was suggestedfrom the results of the inhibitory effects of hydroxyl radical scavengers. Nitrite and nitrate also enhancedthe formation of nucleobases and malondialdehyde from DNA induced by sunlight. In the presence of 5µM nitrite, the concentration in human skin cells, the product yields by sunlight were 5-10-fold greaterthan those in the absence of nitrite. The addition of 80µM NO3

-, a concentration in human skin cells,also increased the yields significantly. Nitrite and nitrate may play a role in enhancing the genotoxiceffects of UV light in humans.

Introduction

Nitrite (NO2-), a normal constituent in human biological

fluids, including saliva, is a well-known potential risk factorfor gastric cancer (1). When NO2

- is protonated under acidicconditions, reactive nitrous acid is formed. Because nitrous acidis a weak acid with a pKa of 3.4 (2), nitrous acid could begenerated in the human stomach. The nitrous acid formed canreact with DNA directly to form deaminated nucleobases,including xanthine and oxanine from guanine (Gua), hypoxan-thine from adenine (Ade), and uracil from cytosine (Cyt) (3,4). All of the alterations can induce mutations when DNApolymerases replicate the DNA through the regions (3, 5). Thenitrous acid can also react with secondary amines in foodstuffs,generatingN-nitrosamines (6). Several of them are carcinogenicsince they react with DNA as alkylating reagents, resulting inadducts. In addition to NO2-, nitrate (NO3

-) is also a normalconstituent in humans with a higher concentration than NO2

-.Nitrate-reducing microorganisms in the oral cavity reduce NO3

-

in the saliva to NO2- (7, 8). Thus, NO3- is also a potential risk

factor for gastric cancer (1). However, NO2- and NO3

-, perse, are less reactive species. In a neutral solution, neither NO2

-

nor NO3- reacts with biological molecules, such as protein and

DNA.On the other hand, human skin cancers are most likely caused

by DNA damage produced directly by UV light (9). Becausepyrimidines in the DNA are prime targets for UV light, theirphotoproducts, including pyrimidine-pyrimidine dimers andhydroxylated monomers, are well-characterized and assayed(10). Modifications of purines in the DNA by UV light havealso been studied and reported (10).

Reportedly, in the presence of NO2-, UV light induces the

hydroxyl radical (•OH), a powerfully reactive oxygen species,

in neutral solution (11-13). •OH is also generated in neutralNO3

- solution by irradiation with UV light, with an efficiencylower than that of NO2- (14-17). The •OH generated shouldharm skin cells and their DNA. However, little attention hasbeen paid to the effects of NO2- and NO3

- on the DNA damagecaused by UV light.

In the present study, we investigated the reaction of nucleo-sides and DNA with UV light in the presence of NO2

- or NO3-

at neutral pH and revealed that both NO2- and NO3

- enhancedthe generation of nucleobases and malondialdehyde (MDA) fromnucleosides and DNA. We discuss the possible reaction mech-anisms in the present system and the contribution of NO2

- andNO3

- on human skin cancers.

Experimental Procedures

Materials. dAdo (2′-deoxyadenosine), dGuo (2′-deoxygua-nosine), dCyd (2′-deoxycytidine), dThd (2′-deoxythymidine), Ade,Cyt, Thy (thymine), and calf thymus DNA were obtained fromSigma (St. Louis, MO). Gua was from Kohjin (Tokyo, Japan).Sodium nitrite and sodium nitrate (both 99.99+%) were purchasedfrom Aldrich (Milwaukee, WI). All other chemicals of reagent gradewere purchased from Sigma, Aldrich, Nacalai Tesque (Osaka,Japan), and Cica (Tokyo) and were used without further purification.Water was distilled and then purified with a Millipore Milli-Qdeionizer.

HPLC Conditions. The HPLC system consisted of ShimadzuLC-10ADvp pumps and an SCL-10Avp system controller. On-lineUV spectra were obtained with a Shimadzu SPD-M10Avp UV-vis photodiode array detector. For the reversed phase (RP) HPLC,an Inertsil ODS-3 octadecylsilane column of 4.6 mm× 250 mmand particle size 5µm (GL Science, Tokyo) was used. The eluentwas 20 mM ammonium acetate buffer (pH 7.0) containingmethanol. The methanol concentration was increased from 0 to 40%for 30 min in linear gradient mode. The column temperature was40 °C, and the flow rate was 1.0 mL/min.

Reaction Conditions.For UV light reactions, a solution (1 mL)of nucleosides (dAdo, dGuo, dCyd, and dThd; 100µM each) or

* To whom correspondence should be addressed. Tel:+81-86-271-8346.Fax: +81-86-271-8320. E-mail: tsuzuki@shujitsu.ac.jp.

457Chem. Res. Toxicol.2006,19, 457-462

10.1021/tx050347l CCC: $33.50 © 2006 American Chemical SocietyPublished on Web 02/22/2006

0.159 mg/mL calf thymus DNA containing various concentrationsof sodium nitrite or sodium nitrate and 100 mM potassiumphosphate buffer (pH 7.4, 37°C) in a glass vial (12 mm i.d.) withouta cap was irradiated for 10 min with high-intensity UV lightoriginating from a 200 W high-pressure mercury lamp through aliquid light guide (SC-200, Inflidge, Yokohama, Japan). Theintensity of radiation on the surface of a sample solution wasmeasured with a photometer (UIT-150, Ushio, Tokyo) equippedwith a sensor UVD-S254 or UVD-S365. The intensities of the UVlight were 83 mW/cm2 for 254 nm and 586 mW/cm2 for 365 nm.For sunlight reactions, a solution (4 mL) of nucleosides (dAdo,dGuo, dCyd, and dThd; 100µM each) or calf thymus DNA (0.159mg/mL) containing sodium nitrite or sodium nitrate and 100 mMpotassium phosphate buffer (pH 7.4) in a glass vial (15 mm i.d.)with a cap lying on its side, was irradiated with sunlight for 6 h ona sunny day on the roof of a building at Shujitsu University. Forthe experiment of the concentration dependence of NO2

- and NO3-

shown in Figure 5A,B, averages of the intensities of the sunlightin the glass vial measured every 1 h were 19µW/cm2 for 254 nmand 393µW/cm2 for 365 nm. The average temperature of thesolution was 22°C. For the experiment of the effects of 5µMNO2

- and 80µM NO3- shown in Table 3, averages of the intensities

of the sunlight were 21µW/cm2 for 254 nm and 1116µW/cm2 for365 nm. The average temperature of the solution was 30°C. Controlsamples (no light) were prepared by wrapping the tubes inaluminum foil and then also irradiating them with the same sunlight.For Fenton reaction, a solution (1 mL) of 100µM nucleosides or0.159 mg/mL calf thymus DNA containing 1 mM FeSO4, 2 mMH2O2, and 100 mM potassium phosphate buffer (pH 7.4) wasincubated at 37°C for 10 min. The reaction was terminated by theaddition of 1% ethanol. The H2O2 concentration was determinedfrom the UV absorbance at 240 nm, assumingε240 ) 39.4 M-1

cm-1 (18). All experiments were carried out in triplicate. Aconcentration of 0.159 mg/mL was used for the light reactions ofcalf thymus DNA, since the DNA solution contained similaramounts of nucleobases on molar basis to those in the nucleosidemixture used in this study. We confirmed that 100µM Ade wasdetected by RP-HPLC analysis when the DNA solution washydrolyzed with 1.1 M HCl at 90°C for 2 h.

Preparation of the Authentic MDA. The authentic MDA wasprepared according to a method reported previously (19). Shortlyafter, 1,1,3,3-tetramethoxypropane (4 mL, 16.7 mmol) and Dowexmonosphere650C (H) cation exchange resin (H form, 4.0 g) wereadded to 2 mL of water and stirred in a water bath at 25°C for 18h. The supernatant solution was neutralized with 5 M NaOH.Unreacted 1,1,3,3-tetramethoxypropane was removed by ethylacetate extraction. The aqueous phase was poured into acetone, andthe precipitate was collected. The sample was recrystallized fromH2O/acetone at-20 °C. Spectrometric data of MDA in aqueoussolution, where it exists as its tautomer,â-hydroxyacrolein, with apKa ) 4.6, are as follows (20). 1H NMR (500 MHz, in D2O at 25°C): δ (ppm/DSS) 8.63 (d,J ) 10.3 Hz, 2H), 5.29 (t,J ) 10.3Hz, 1H). 13C NMR (125 MHz, in D2O at 25°C): δ (ppm/DSS)195.6, 112.2. UV:λmax ) 266 (pH 7.0).ε266 ) 38100 M-1 cm-1

(pH 7.0).Quantitative Procedures. The concentrations of nucleosides,

nucleobases, and MDA in the reaction mixtures were evaluatedfrom integrated peak areas on HPLC chromatograms detected at260 nm, as compared with those of authentic standard solutions.

Results

A nucleoside mixture (dAdo, dGuo, dCyd, and dThd; 100µM each) in 100 mM potassium phosphate buffer was irradiatedwith high-intensity UV light originating from a mercury lampat pH 7.4 and 37°C for 10 min. The reaction was monitoredby RP-HPLC with detection at 260 nm. Nucleosides wereslightly consumed. Several product peaks, including traceamounts (<0.2 µM) of nucleobases (Ade, Gua, Cyt, and Thy),were observed in the chromatogram. However, when the

reaction was carried out in the presence of 100µM NO2-, the

consumption of nucleosides increased greatly and large amountsof the nucleobases were observed in the RP-HPLC chromato-gram (Figure 1). In addition to the nucleobases, a significantproduct peak was observed at an HPLC retention time of 3.4min with aλmax ) 266 nm in the on-line detected UV spectrum.The product was identified as MDA from the coincidence ofthe retention time and the UV spectrum with those of authenticMDA. Figure 2A,B shows the concentrations of the startingmaterials, the nucleosides, and the products, nucleobases andMDA, respectively, after the nucleoside mixture was irradiated

Figure 1. RP-HPLC chromatogram of a reaction solution of nucleosidemixture irradiated with UV light in the presence of NO2

-. A solution(1 mL) of nucleosides (dAdo, dGuo, dCyd, and dThd, 100µM each)containing 100µM NO2

- and 100 mM potassium phosphate buffer(pH 7.4) in a glass vial (12 mm i.d.) was irradiated with UV lightoriginating from a 200 W mercury lamp at 37°C for 10 min. TheRP-HPLC chromatogram was detected at 260 nm.

Figure 2. (A) NO2- dose dependence of the concentration of remaining

dAdo (open circle), dGuo (open square), dCyd (open rhombus), anddThd (open triangle). (B) NO2- dose dependence of the yields of MDA(open circle), Ade (closed circle), Gua (closed square), Cyt (closedrhombus), and Thy (closed triangle). (C) NO3

- dose dependence ofthe concentration of remaining dAdo (open circle), dGuo (open square),dCyd (open rhombus), and dThd (open triangle). (D) NO3

- dosedependence of the yields of MDA (open circle), Ade (closed circle),Gua (closed square), Cyt (closed rhombus), and Thy (closed triangle).A solution (1 mL) of nucleosides (dAdo, dGuo, dCyd, and dThd, 100µM each) containing 0-10 mM NO2

- or NO3- and 100 mM potassium

phosphate buffer (pH 7.4) in a glass vial (12 mm i.d.) was irradiatedwith UV light originating from a 200 W mercury lamp at 37°C for 10min. The intensities of the UV light were 83 mW/cm2 for 254 nm and586 mW/cm2 for 365 nm. The concentrations were determined by RP-HPLC analysis. Means( SD (n ) 3) are presented.

458 Chem. Res. Toxicol., Vol. 19, No. 3, 2006 Suzuki and Inukai

with the UV light in the presence of various concentrations ofNO2

-. The consumptions of nucleosides and the yields ofnucleobases and MDA increased with the increasing dose ofNO2

-. NO3- also increased the consumptions of nucleosides

and the product yields, as shown in Figure 2C,D, respectively.Although the efficiency of NO3- enhancing the UV reactionwas lower than that of NO2- at low doses, it was comparableto that of NO2

- at 10 mM. For both reactions with NO2- andNO3

-, the yield of each nucleobase was several times smallerthan the consumption of the corresponding nucleoside. Toevaluate the stability of nucleobases in the UV reaction system,we carried out the reaction of nucleobases with UV light in thepresence of various concentrations of NO2

- or NO3-. When a

nucleobase mixture (Ade, Gua, Cyt, and Thy; 10µM each) wasirradiated with the UV light at pH 7.4 and 37°C for 10 min,nucleobases were consumed slightly. Increasing the concentra-tions of NO2

- and NO3-, the consumptions of nucleobases were

increased, as shown in Figure 3A,B, respectively. Although theefficiency of NO2

- was greater than that of NO3- at lowconcentrations (<1 mM), they were inverted at higher concen-trations.

Similar UV reactions were carried out for double-strandedDNA monitoring with RP-HPLC. When 0.159 mg/mL of calfthymus DNA solution, which includes similar amounts ofnucleobases to the above nucleosides solution (see ExperimentalProcedures), in 100 mM potassium phosphate buffer wasirradiated with the UV light at pH 7.4 and 37°C for 10 min,trace amounts of nucleobases and MDA (<0.3 µM) wereobserved. However, in the presence of 100µM NO2

-, significantamounts of nucleobases and MDA were observed in an RP-HPLC chromatogram. The product yields increased with theincreasing dose of the NO2- and reached a plateau above 1 mM(Figure 4A). NO3

- increased the yields in a dose-dependentmanner (Figure 4B). The efficiency of NO3

- is lower than thatof NO2

- at lower concentrations (<1 mM). However, at higherconcentrations, NO3- is more effective than NO2-.

To obtain information about the reactive species in the UVreaction with DNA in the presence of NO2

- or NO3-, the

reaction was carried out with•OH scavengers. As shown inTable 1, the reaction was depressed by the addition of mannitol,formate, or thiourea. The efficiency of inhibition was in theorder of mannitol< formate< thiourea. The reaction was alsoinhibited by 1% ethanol. To compare the results with those ofthe •OH reaction, a Fenton reaction system was conducted forDNA. When 0.159 mg/mL calf thymus DNA was incubatedwith 1 mM FeSO4 and 2 mM H2O2 in 100 mM potassium

phosphate buffer at pH 7.4 and 37°C for 10 min, nucleobasesand MDA were generated. The scavengers inhibited the Fentonreaction in a manner similar to the UV reaction (Table 2).

To clarify whether the UV light-induced reactions in thepresence of NO2- or NO3

- have relevance in nature, similarexperiments were carried out using sunlight. The nucleosidemixture was irradiated with sunlight at pH 7.4 and ambienttemperature for 6 h. As shown in Figure 5A,B, the consumptionof nucleosides and the production of nucleobases increased withthe increasing dose of NO2-. NO3

- also increased the consump-tions of nucleosides and the yields of nucleobases and MDAbut with a lower efficiency (Figure 5C,D). For DNA, theproductions of nucleobases and MDA increased with theincreasing dose of NO2- (Figure 6A). The reaction was alsodepressed by the addition of the•OH scavengers in a mannersimilar to the result for UV light (data not shown). The additionof NO3

- also enhanced the reaction (Figure 6B). To assess thebiological importance of the acceleration of DNA damage byNO2

- and NO3-, another experiment was conducted for DNA

with sunlight. The calf thymus DNA solution, including 5µMNO2

-, a concentration comparable to that in human skin cells,was irradiated with sunlight at pH 7.4 and ambient temperaturefor 6 h. The results are shown in Table 3. The production ofMDA from DNA with 5 mM NO2

- was 8-fold greater thanthat without NO2

-. Although the yields of purine bases (Adeand Gua) from DNA with 5µM NO2

- were 5-fold greater thanthose without NO2-, the production of pyrimidine bases (Cytand Thy) was 10-fold greater than that without NO2

-. Theaddition of 80µM NO3

-, a concentration comparable to that inhuman skin cells, also increased the product yields significantly,although the efficiencies were lower than those for NO2

-.Without sunlight, no effects were observed for the yields bythe addition of both 5µM NO2

- and 80µM NO3-.

Discussion

In the present study, we revealed that NO2- and NO3

-

enhanced the reaction of nucleosides and DNA with UV light.The major products were nucleobases and MDA. The UVreaction of DNA was depressed by the•OH scavengers with anefficiency in the order of mannitol< formate< thiourea (Table1). The efficiency of the scavengers for the UV reactionaccorded with their reported reaction rates to•OH is asfollows: 1.0× 109 M-1 s-1 for mannitol, 2.7× 109 M-1 s-1

for formate, and 4.7× 109 M-1 s-1 for thiourea (21). The Fentonsystem also caused the formation of nucleobases and MDA from

Figure 3. (A) NO2- dose dependence of the yields of Ade (closed

circle), Gua (closed square), Cyt (closed rhombus), and Thy (closedtriangle). (B) NO3

- dose dependence of the yields of Ade (closed circle),Gua (closed square), Cyt (closed rhombus), and Thy (closed triangle).A solution (1 mL) of 0.159 mg/mL calf thymus DNA containing 0-10mM NO2

- or NO3- and 100 mM potassium phosphate buffer (pH 7.4)

in a glass vial (12 mm i.d.) was irradiated with UV light at 37°C for10 min. The concentrations were determined by RP-HPLC analysis.Means( SD (n ) 3) are presented.

Figure 4. (A) NO2- dose dependence of the yields of MDA (open

circle), Ade (closed circle), Gua (closed square), Cyt (closed rhombus),and Thy (closed triangle). (B) NO3- dose dependence of the yields ofMDA (open circle), Ade (closed circle), Gua (closed square), Cyt(closed rhombus), and Thy (closed triangle). A solution (1 mL) of 0.159mg/mL calf thymus DNA containing 0-10 mM NO2

- or NO3- and

100 mM potassium phosphate buffer (pH 7.4) in a glass vial (12 mmi.d.) was irradiated with the UV light at 37°C for 10 min. Theconcentrations were determined by RP-HPLC analysis. Means( SD(n ) 3) are presented.

DNA Damage by UV Light with NO2- or NO3- Chem. Res. Toxicol., Vol. 19, No. 3, 2006459

DNA. The •OH scavengers depressed the Fenton reaction in amanner similar to the UV reaction (Table 2). The results stronglysuggest the involvement of•OH as the reactive species in thepresent UV reaction systems with NO2

- or NO3-. It has been

reported that•OH is produced from NO2- by the primaryphotolytic process via the formation of the oxygen anion radicalO•- and subsequent hydrolysis (eqs 1 and 2) (11-13).

In our reaction systems,•OH would be formed from NO2- bythe above reaction.•OH production from NO3- by UV irradia-tion has been reported with complexity (14-17). Three distinctroutes are proposed for the•OH production. The first route isvia peroxynitrite (eqs 3 and 4).

However, 8-nitroguanine, a specific product from Gua residuesof nucleoside and DNA caused by the reaction with ONOO-

(22, 23), was not detected in our UV reaction systems (datanot shown). Thus, the route via ONOO- is not likely to be amajor one. The second route is via the formation of NO2

- (eq5). The NO2

- generated is converted into•OH by further reactionwith UV light (eqs 1 and 2).

Table 1. Effects of Scavengers on the Reaction of DNA with UV Light in the Presence of 100µM NO2- a

additives MDA (µM) Ade (µM) Gua (µM) Cyt (µM) Thy (µM)

none 0.36( 0.01 1.30( 0.03 0.28( 0.01 1.10( 0.04 0.75( 0.011 mM mannitol 0.14( 0.01 0.51( 0.01 0.13( 0.01 0.50( 0.03 0.35( 0.011 mM formate 0.10( 0.01 0.35( 0.02 0.08( 0.01 0.36( 0.01 0.25( 0.011 mM thiourea n.d.b 0.16( 0.01 0.09( 0.01 0.14( 0.01 0.13( 0.011% ethanol 0.01( 0.00 0.04( 0.00 0.01( 0.00 0.04( 0.00 0.03( 0.00

a A solution (1 mL) of 0.159 mg/mL calf thymus DNA containing 100µM NO2- was incubated in 100 mM potassium phosphate buffer (pH 7.4) at 37

°C and irradiated with UV light for 10 min in the presence of each additive. Means( SD (n ) 3) are presented.b Not determined: The concentration ofMDA could not be determined due to interference peaks of other compounds on the RP-HPLC chromatogram.

Table 2. Effects of Scavengers on the Reaction of Calf Thymus DNA with Fenton Reagenta

additives MDA (µM) Ade (µM) Gua (µM) Cyt (µM) Thy (µM)

none 0.07( 0.00 1.20( 0.06 0.40( 0.04 1.09( 0.07 0.83( 0.061 mM mannitol 0.05( 0.00 0.29( 0.01 0.12( 0.01 0.35( 0.01 0.23( 0.011 mM formate 0.03( 0.00 0.20( 0.00 0.08( 0.00 0.26( 0.00 0.17( 0.021 mM thiourea n.d.b 0.09( 0.01 0.06( 0.00 0.10( 0.01 0.07( 0.011% ethanol 0.01( 0.00 0.08( 0.01 0.02( 0.01 0.10( 0.01 0.03( 0.01

a A solution (1 mL) of 0.159 mg/mL calf thymus DNA containing 1 mM FeSO4, 2 mM H2O2, and 100 mM potassium phosphate buffer (pH 7.4) wasincubated at 37°C for 10 min in the presence of the additives. The reaction was terminated by the addition of 1% ethanol. Means( SD (n ) 3) arepresented.b Not determined: The concentration of MDA could not be determined due to interference peaks of other compounds on the RP-HPLC chromatogram.

Figure 5. (A) NO2- dose dependence of the concentrations of

remaining dAdo (open circle), dGuo (open square), dCyd (openrhombus), and dThd (open triangle). (B) NO2

- dose dependence ofthe yields of MDA (open circle), Ade (closed circle), Gua (closedsquare), Cyt (closed rhombus), and Thy (closed triangle). (C) NO3

-

dose dependence of the concentration of remaining dAdo (open circle),dGuo (open square), dCyd (open rhombus), and dThd (open triangle).(D) NO3

- dose dependence of the yields of MDA (open circle), Ade(closed circle), Gua (closed square), Cyt (closed rhombus), and Thy(closed triangle). A solution (4 mL) of nucleosides (dAdo, dGuo, dCyd,and dThd, 100µM each) containing 0-10 mM NO2

- or NO3- and

100 mM potassium phosphate buffer (pH 7.4) in a glass vial (15 mmi.d.) was irradiated with sunlight and ambient temperature for 6 h. Theaverage intensities of the sunlight measured every 1 h were 19µW/cm2 for 254 nm and 393µW/cm2 for 365 nm in the glass vial. Theaverage temperature of the solution was 22°C. The concentrations weredetermined by RP-HPLC analysis. Means( SD (n ) 3) are presented.

Figure 6. (A) NO2- dose dependence of the yields of MDA (open

circle), Ade (closed circle), Gua (closed square), Cyt (closed rhombus),and Thy (closed triangle). (B) NO3- dose dependence of the yields ofMDA (open circle), Ade (closed circle), Gua (closed square), Cyt(closed rhombus), and Thy (closed triangle). A solution (4 mL) ofnucleosides (dAdo, dGuo, dCyd, and dThd, 100µM each) containing0-10 mM NO2

- or NO3- and 100 mM potassium phosphate buffer

(pH 7.4) in a glass vial (15 mm i.d.) was irradiated with sunlight andambient temperature for 6 h. The average intensities of the sunlightmeasured every 1 h were 19µW/cm2 for 254 nm and 393µW/cm2 for365 nm in the glass vial. The average temperature of the solution was22 °C. The concentrations were determined by RP-HPLC analysis.Means( SD (n ) 3) are presented.

NO2- + hν f •NO + O•- (1)

O•- + H2O f •OH + OH- (2)

NO3- + hν f ONOO- (3)

ONOO- + H2O f •NO2 + •OH + OH- (4)

460 Chem. Res. Toxicol., Vol. 19, No. 3, 2006 Suzuki and Inukai

However, the profiles for the concentration dependence of NO3-

were vastly different from those of NO2- (Figures 2 and 4-6).Thus, this pathway is not likely to be a major route for theformation of •OH. The third route involves the formation ofO•- (eq 6) and its subsequent hydrolysis (eq 2).

This route would be the most likely for the present NO3- system.

Reportedly, the efficiency of NO2- is greater than that of NO3-

for •OH generation by UV light irradiation in neutral solution(15). Our results show that the efficiency of NO2

- for nucleobaseproduction was greater than that of NO3

- at a lower concentra-tion (<1 mM) (Figure 2). At higher concentrations, theefficiency of NO2

- became, however, comparable to or smallerthan that of NO3

- because the efficiency of NO2- decreasedwith the increase in NO2- concentration and reached a plateau.Reportedly, NO2- acts as an•OH scavenger (eq 7) with a rateconstant of 1.0× 1010 M-1 s-1 (11).

A high concentration of NO2- would efficiently remove the•OHformed in the present system by the scavenging reaction.

A reaction mechanism, including a peroxyl radical intermedi-ate at C4′ of the deoxyribose moiety and base propenals togenerate nucleobases and MDA, has been proposed for thereaction of•OH with nucleosides and DNA (Scheme 1) (24-27). Although nucleobases and MDA were formed in the presentUV reaction with NO2

- or NO3-, the yield of each nucleobase

formed from each nucleoside was several times smaller thanthe consumption of the corresponding nucleoside in the reactionof nucleosides with both UV light and sunlight (Figures 2 and5). The present study also showed that substantial amounts ofnucleobases were decomposed by the UV reaction in thepresence of NO2- and NO3

- (Figure 3). Thus, the considerableamounts of nucleobases formed in the UV reaction of nucleo-sides and DNA should also be consumed by the further reaction.A major path of the UV reaction would be shown in Scheme 1.As a consequence of the reaction forming nucleobases andMDA, single strand breaks take place in DNA. Unless thedamage is repaired perfectly, cell death or mutation will occur.Secondary DNA damage should also be taken into account forthe UV reaction, since MDA is a reactive species to DNA. MDAis a well-characterized compound as a major product of lipidperoxidation (20). MDA is also generated from 2-deoxyribose,nucleosides, and DNA by reaction with•OH formed byγ-irradiation or a Fenton system (24, 25). MDA is mutagenicand carcinogenic (28, 29). MDA can react with nucleobasemoieties in DNA forming adducts. A major product of thereaction of MDA with DNA is a tricyclic Gua adduct, pyrimido-(1,2-R)-purin-10(3H)one, while MDA can also react with Ade

and Cyt, forming the corresponding adducts (20, 30, 31). TheGua adduct results in G to A and G to T mutations (32). MDAgenerated from free nucleosides or DNA by UV light may reactwith the intact moiety of DNA forming adducts in human skincells. Unless perfect repair of the adducts occurs, mutation willoccur.

NO2- and NO3

- are ubiquitous anions in humans. Theirconcentrations have been reported for various biological fluidsin healthy humans (33-36). In sweat, the concentrations were3.4 ( 0.4 µM for NO2

- and 39.7( 4.3 µM for NO3- (36).

Recently, the concentrations were determined to be 5.1( 1.6µM for NO2

- and 82.4( 33.6 µM for NO3- in human skin

cells derived from mammoplastic surgery (37). We examinedthe effects of NO2- and NO3

- at the human skin cell level tothe photoreaction of DNA by sunlight (Table 3). The additionof 5 µM NO2

- increased the formation of nucleobases and MDA5-10-fold greater than that without the addition of NO2

-. Theaddition of 80 µM NO3

- also increased the formation ofnucleobases and MDA significantly. These data suggest thatbiologically relevant doses of NO2- and NO3

- for the skin areeffective for the generation of•OH by sunlight. The•OHgenerated on the skin surface may play a role in the host defensemechanism killing bacteria attached to the skin. On the otherhand, the•OH generated in skin cells would harm the DNAboth directly and indirectly, resulting in mutation or cell death.

In conclusion, we investigated the effects of NO2- and NO3

-

in the reaction of nucleoside mixture and DNA with UV lightand sunlight in a neutral solution and revealed that biologicallyrelevant doses of NO2- and NO3

- accelerate DNA damage,probably due to•OH formation. The present results drawattention to the contribution of NO2- and NO3

- in human skincancers.

Acknowledgment. This work was supported by a Grant-in-Aid for Cancer Research (16-7) from the Ministry of Health,Labor and Welfare of Japan.

Table 3. Effects of NO2- or NO3

- on the Reaction of Calf Thymus DNA with Sunlighta

additives sunlight MDA (nM) Ade (nM) Gua (nM) Cyt (nM) Thy (nM)

none - 9.7( 1.6 13.2( 2.6 9.7( 2.7 2.2( 0.6 2.0( 0.5+ 36.1( 4.1 41.8( 3.7 15.0( 1.1 17.8( 1.7 12.2( 1.3

5 µM NO2- - 10.5( 0.9 14.2( 3.1 8.0( 1.1 2.8( 0.6 1.8( 0.4

+ 287.8( 8.8 203.0( 5.8 72.0( 1.8 170.6( 5.7 127.0( 5.980 µM NO3

- - 10.4( 1.6 12.4( 1.2 5.9( 3.4 3.5( 0.4 1.8( 0.3+ 80.5( 3.4 46.5( 0.8 24.4( 3.8 39.2( 3.0 27.8( 1.7

a A solution (4 mL) of 0.159 mg/mL calf thymus DNA containing 5µM NO2- or 80 µM NO3

- and 100 mM potassium phosphate buffer (pH 7.4) wasirradiated with sunlight for 6 h. Average intensities of the sunlight were 21µW/cm2 for 254 nm and 1116µW/cm2 for 365 nm in the glass vial. The averagetemperature of the solution was 30°C. Means( SD (n ) 3) are presented.

NO3- + hν f NO2

- + O (5)

NO3- + hν f •NO2 + O•- (6)

•OH + NO2- f •NO2 + OH- (7)

Scheme 1

DNA Damage by UV Light with NO2- or NO3- Chem. Res. Toxicol., Vol. 19, No. 3, 2006461

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