reactive oxygen species 
 ( ros ) and reactive nitrogen species ( rns ) , 
 e.g. , hydrogen peroxide , hydroxyl radical , superoxide , peroxynitrite , 
 and singlet oxygen , are produced physiologically as a result of normal 
 aerobic metabolism , immune response , and inflammation . ros and rns play a dual role in a living cell : while required for 
 certain cellular processes such as signal transduction and protection 
 against pathogens , they can induce oxidative stress and degradation 
 of cellular biomolecules when formed in excess . ros - mediated oxidation of dna nucleobases has received 
 significant 
 attention in the literature due to its important role in aging , cancer , 
 and neurodegenerative diseases . guanine bases within dna are preferentially targeted by oxidants 
 due to their relatively low redox potential as compared to the other 
 three nucleobases . one - electron oxidation of guanine 
 produces guanine radical cations , which migrate along the -stack 
 of base pairs within dna duplexes until being trapped via irreversible 
 reactions with water and/or molecular oxygen to form stable nucleobase 
 adducts ( scheme 1 ) . guanine oxidation 
 gives rise to more than 10 different dna lesions , 
 including 8-oxo-7,8-dihydro-2-deoxyguanosine ( og ) , spiroiminodihydantoin 
 ( sp ) , 5-guanidinohydantoin ( gh ) , and 2,5-diaminoimidazol-4-one ( iz ) 
 and its hydrolysis product , 2,2,4-triamino-2h - oxal-5-one 
 ( z ) ( scheme 1 ) . under aerobic conditions , og and z are produced in the highest yield , 
 with typical in vivo concentrations in living cells of 110 
 adducts per 10 normal nucleotides . og 
 is prone to further oxidation in the presence of ros , while z is resistant to further oxidation . because electron  hole pausing at the sites 
 of the 
 lowest ionization potential ( ip ) increases the probability of stable 
 adduct formation , dna oxidation tends to be sequence - dependent . high resolution gel electrophoresis 
 ( page ) analysis of dna strand breaks generated at the sites of nucleobase 
 damage by the action of repair endonucleases or hot piperidine treatment 
 reveals characteristic oxidation patterns . sites containing multiple adjacent guanines , e.g. , 5g < 
 5gg < 5ggg , are preferentially targeted for oxidative 
 adduct formation upon photooxidation , supposedly a result of  
 stacking and orbital interactions , which lower the ips of the 5-guanines 
 in runs of several gs . preferential oxidation of two- and three - base 
 runs of gs have been also reported for other oxidants such as co / benzoyl peroxide , carbonate radicals , -radiation , 
 and hydroxyl radicals generated by fenton chemistry ( fe  edta / h2o2 ) . however , a different sequence specificity 
 was reported for nitrosoperoxycarbonate ( onoo ) , 
 which reacted preferentially at sites of high ip values , e.g. , tgc , 
 agc , cgc . in addition to nucleotide sequence 
 context , guanine reactivity 
 toward oxidants may be affected by epigenetic dna modifications , e.g. , 
 cytosine methylation to 5-methylcytosine ( c).c bases are formed endogenously by enzymatic methylation 
 of the c5 position of cytosine in cg dinucleotides ( cpg sites ) and 
 represent about 1% of total bases in the mammalian genome , playing 
 a critical role in gene expression , cell differentiation , host defense , 
 genomic imprinting , and x chromosome inactivation . cytosine methylation has been shown to increase the reactivity of 
 guanine bases in cg dinucleotides toward electrophiles 
 and oxidants . for example , kawai et al . reported that the rate of 
 electron - transfer quenching of triplet n , n - dibutylnaphthaldiimide was increased from 6.8  10 m s for c : g base 
 pair to 8.1  10 m s for c : g base pair , likely due to the transmission of 
 an electronic effect from the c to its partner guanine 
 through hydrogen bonding within the c : g base pair . preferential oxidation of guanine adjacent to c may contribute to mutational  hot spots  
 at cg dinucleotides of the p53 tumor 
 suppressor gene ( figure s1 in the supporting information ) . however , a more recent study by kanvah 
 et al . did not detect any effect of c on the migration 
 of radical cations along dna duplexes . a major limitation of the earlier studies is that oxidative 
 adduct 
 formation was detected indirectly by gel electrophoresis of alkali - generated 
 dna fragments . this approach provides 
 little structural information about the lesions formed and suffers 
 from a high background due to the direct oxidation of the dna backbone , 
 which constitutes up to 50% of total damage following treatment with 
 strong oxidants such as -radiation . a more recent methodology developed by margolin et al . reduces the 
 background using escherichia coli exonuclease iii 
 to remove direct fragments containing direct strand breaks , but does not allow for adduct identification . the resulting oxidative adducts have been classified as  fpg 
 sensitive lesions  and  piperidine sensitive lesions  , but their actual identities could 
 not be established . this is important because each of the various 
 guanine oxidation products is associated with different biological 
 outcomes in terms of their mutagenic potential and the ability to 
 be removed by repair enzymes . for example , secondary 
 oxidation products such as z , sp , gh , and oxaluric acid are significantly 
 more mispairing than og . a qtof ms method was 
 recently employed to examine the effects of dna sequence on the susceptibility 
 of og in dna toward further oxidation , unfortunately , 
 this approach is not suitable for oxidation studies of dna containing 
 standard dna bases . in the present work , stable isotope labeling 
 of dna  mass 
 spectrometry methodology developed in our laboratory ( ild  ms ) was employed to accurately quantify og and z adducts originating 
 from specific guanine bases within dna duplexes derived from the p53 tumor suppressor gene . our results reveal characteristic 
 patterns of distribution of the two oxidative guanine lesions along 
 dna duplexes . adduct yields are influenced by the by the presence 
 of neighboring 5-methylcytosines , flanking sequence , and solvent exposure , 
 with a strong preference for cgg trinucleotides . these results are important because they may provide 
 insight into the origins of mutational hotspots at endogenously methylated 
 cpg sites within the human genome . ammonium acetate , ammonium formate , sodium 
 cacodylic acid , sodium chloride , desferrioxamine , acetonitrile , zinc 
 chloride , trizma base , methanol , sodium bicarbonate , nuclease p1 , 
 alkaline phosphatase , and riboflavin were purchased from sigma - aldrich 
 ( milwaukee , wi ) . triethylamine and hydrochloric acid were obtained 
 from fisher scientific ( hanover park , il ) . n3,c1-dg and oxazolone , 
 8-oxo - dg , and their n , c - labeled analogues 
 were prepared from dg and n3,c1-dg as described elsewhere . synthetic oligodeoxynucleotides 
 were prepared by standard phosphoramidite chemistry using an applied 
 biosystems abi 394 dna synthesizer ( foster city , ca ) . n3,c1-dg was introduced at specified 
 positions by solid phase synthesis using 1,7,nh2-n-2-c - dg phosphoramidite . all dna oligomers were purified by hplc to 99+% purity ( figure s2 
 in the supporting information ) and characterized 
 by uv spectroscopy and maldi mass spectrometry as described previously . the identity and the purity of each dna strand esi - ms ( table 1 , figures s3s8 in the supporting information ) ( esi : electrospray ionization ) . dna 
 quantification was based on hplc  uv analysis of dg in enzymatic 
 digests . dna oligomers were annealed 
 to equimolar amounts of the corresponding complementary strands in 
 the presence of 100 mm nacl to obtain double - stranded dna . dna duplexes 
 were characterized by uv melting and native gel electrophoresis to 
 confirm their purity and stability . all duplexes were characterized 
 by well - defined cooperative melting curves ( see example in figure 
 s10 in the supporting information ) . a 62.5 m 
 solution of riboflavin in 20 mm sodium cacodylate ( ph 7 ) and 10 mm 
 nacl was placed on ice and vigorously bubbled with oxygen gas for 
 1 min . dna duplexes ( 3 nmol , in triplicate ) were combined with the 
 oxygenated riboflavin solution to reach the final concentration of 
 50 m riboflavin and 40 m dna . samples were transferred 
 to glass vials and suspended in an ice - cold water bath . the solution 
 was irradiated for 20 min with a 60 w tungsten bulb positioned 2 cm 
 from the vial . following oxidation , samples were promptly transferred 
 to low actinic vials and immediately frozen on dry ice , followed by 
 storage at 80 c until further processing . dna duplexes ( 2 nmol 
 in 10 l of water , in triplicate ) were mixed with 3.3 l 
 of 125 mm sodium bicarbonate/750 mm sodium phosphate buffer , ph 6.9 . following the addition of a 3.3 l aliquot of 5 mm peroxynitrite 
 solution ( cayman chemical , ann arbor , mi ) , the solutions were vigorously 
 mixed by vortexing for 30 s , and the reaction mixtures were left at 
 25 c for 30 min prior to analysis . isotopically tagged 
 dna duplexes ( 2 nmol in 10 l of water , in triplicate ) were 
 mixed with 35 l of 10 mm sodium cacodylic acid , ph 7 . oxidation 
 was intitiated by adding a 20 l aliquot of cobalt dichloride 
 solution ( 50 m ) and 5 l of benzoyl peroxide solution 
 ( 1 mm in acetonitrile ) . samples were incubated at 37 c for 5 
 min and frozen until further processing . ros - treated dna ( 4.5 nmol ) 
 was dissolved in a buffer containing 25 mm ammonium acetate , 2.5 mm 
 zinc chloride , and 5 mm desferrioxamine ( dfo ) ( ph 5.3 , total volume 
 450 l ) . dna was digested to 2-deoxynucleosides in the 
 presence of nuclease p1 ( 18 u ) and alkaline phosphatase ( 60 u ) for 
 2 h at 37 c . samples were split into two equal aliquots for separate analyses 
 of oxazolone and og and immediately fractionated by hplc or frozen 
 until further analysis . dna hydrolysates obtained 
 from 4.5 nmol of dna were subjected to offline hplc to enrich for 
 og . this step was necessary to prevent spontaneous oxidation of dg 
 to og in dna hydrolysates , which would interfere with isotope labeling 
 results . a synergi hydro - rp hplc column ( 4.6  250 mm , 4 um , 
 phenomenex , torrance , ca ) was eluted at a flow rate of 1 ml / min , at 
 25 c , with a gradient of 6% methanol in 10 mm ammonium formate , 
 ph 4.2 ( solvent a ) and acetonitrile ( solvent b ) . solvent composition 
 was maintained at 0% b from 0 to 32 min , increased to 50% b from 32 
 to 36 min , kept at 50% b for 4 min , and brought back to 0% b by 43 
 min , and finally equilibrated at 0% b for 17 min . hplc fractions containing 
 og and n3,c1-og ( 32.635.2 
 min ) were collected using an agilent 1100 fraction collector , immediately 
 frozen on dry ice , and concentrated under vacuum . enzymatic 
 dna hydrolysates obtained from 4.5 nmol of dna were incubated at 25 
 c for 18 h to convert imidazolone to oxazolone ( scheme 1 ) . oxazolone and its n2,c1-labeled analogue 
 were isolated by solid phase extraction using extract - clean carbograph 
 solid phase extraction cartridges ( 150 mg/4 ml , grace , deerfield , 
 il ) . the cartridges were equilibrated with methanol and water , followed 
 by sample loading in 1 ml of water . cartridges were washed with 1 
 ml of water , and the analyte was eluted with 3 ml of 20% methanol 
 in water . solid phase extraction ( spe ) fractions containing oxazolone 
 and n2,c1-oxazolone 
 were dried under reduced pressure and redissolved in 20 l for 
 analysis by capillary hplc  esi - ms / ms . quantitative analyses of og and 
 z in dna hydrolysates were performed 
 using a thermo tsq quantum ultra mass spectrometer ( palo alto , ca ) 
 interfaced with a waters nanoacquity uplc system ( milford , ma ) . for analyses of oxazolone , a thermo - finnigan hypercarb column ( 0.5 
  100 mm , 5 m ) was eluted at a flow rate of 12 l / min 
 with a gradient of 0.05% acetic acid in water ( solvent a ) and a 3:1 
 mixture of isopropanol : acetonitrile ( solvent b ) . hplc solvent composition 
 was gradually changed as follows : 0 min , 1.5% b ; 7.1 min , 9.5% b ; 
 7.6 min , 1.5% b ; 16 min , 1.5% b. using this gradient , oxazolone eluted 
 at 9.4 min . the thermo tsq quantum ultra triple quadrupole 
 mass spectrometer was operated in the electrospray ionization ( esi ) 
 mode , with a spray voltage typically maintained at 2.8 kv and a capillary 
 temperature at 250 c . the first quadrupole 
 was set to isolate the protonated molecules ( [ m + h ] ) 
 of oxazolone ( m / z 247.1 ) and n2,c1-oxazolone ( m / z 250.1 ) , and their fragmentation was 
 induced in the second quadrupole serving as a collision cell . collision - induced 
 dissociation ( cid ) was performed with ar as a collision gas ( 1.0 mtorr ) 
 at a collision energy of 14 v. the third quadrupole was set to detect 
 the product ions corresponding to the neutral loss of deoxyribose 
 and co2 ( [ m + 2h  dr  co2 ] ) : m / z 87.1 for oxazolone 
 and m / z 90.1 for n2,c1-oxazolone . the lower limit of 
 detection for oxazolone was estimated as 5 fmol ( s / n = 10 ) . ms parameters 
 were optimized for maximum response during the infusion of standard 
 solutions . for analysis of og , an agilent extend - c18 column 
 ( 0.5  150 
 mm , 3.5 um , agilent , santa clara , ca ) was eluted isocratically with 
 13% methanol in 10 mm ammonium formate , ph 4.2 , at a flow rate of 
 11 l / min . typically , esi was achieved at a spray voltage of 3.2 kv and a capillary 
 temperature of 250 c . quantitative analyses were performed in 
 the selected reaction monitoring mode . the first quadrupole was set 
 to isolate the protonated molecules ( [ m + h ] ) of og ( m / z 284.1 ) and n3,c1-og ( m / z 288.1 ) , and their fragmentation was induced in the second quadrupole 
 serving as a collision cell . cid was performed with ar as a collision 
 gas ( 1.0 mtorr ) at the collision energy of 14 v. the third quadrupole 
 was set to detect the product ions corresponding to the neutral loss 
 of deoxyribose ( [ m + 2h  dr ] ) : m / z 168.1 for og and m / z 172.1 for n3,c1 - 8-oxo - dg . the lower limit of detection for og was 3 fmol ( s / n = 10 ) . ms parameters 
 were optimized for maximum response during the infusion of standard 
 solutions . initial experiments 
 have established optimal photooxidation conditions that ensured sequence 
 selectivity for guanine oxidation , but produced sufficient numbers 
 of oxazolone and og adducts at each guanine to be detected by hplc following 20 min photooxidation , 4% of total 
 dna strands contained og and 2% of strands contained z ( figure 
 s12 in the supporting information ) . given 
 the hplc  esi - ms / ms detection limits for og - dg and z at 35 fmol , we estimated that photooxidation of 3 nmol of 
 dna should produce sufficient amounts of og and z at each guanine 
 to be accurately quantified by our methodology . similar preliminary 
 experiments have established that dna treatment with 1 mm nitrosoperoxycarbonate 
 for 30 min and 50 m co / benzoyl peroxide treatment for 5 min 
 were optimal for stable isotope labeling experiments ( figure s13 in 
 the supporting information ) . the extent of adduct formation at the 
 isotopically tagged guanines ( gx ) was 
 calculated from the amounts of n , c - labeled 
 and unlabeled adducts in dna hydrolysates using the following equation : where alabeled and aunlabeled are the areas under the 
 hplc  esi - ms / ms peaks corresponding to the n , c - labeled and unlabeled adducts , respectively . this was repeated for each guanine of interest 
 to determine the distribution of og and z along dna duplexes . one - way analysis of variance ( anova ) was used to examine the mean 
 reactivity differences among groups . in cases in which statistically 
 significant differences were revealed by the overall f - test , pairwise group comparisons were conducted using two - sided , 
 two - sample t tests , using the bonferroni correction 
 for multiple comparisons . each group s mean percent reactivity 
 was also compared to a theoretical  random reactivity 
 value , using a two - sided , one - sample t test , with 
 bonferroni - adjusted p - values . the theoretical value 
 is given by 1/n , where n is the 
 total number of groups in each set . for all t tests , 
 standard deviation was estimated by the square root of mean squared 
 error ( mse ) from anova . all statistical analyses were conducted in 
 sas ( statistical analysis software ) version 9.2 . in the present work , we 
 re - examined the influence of cytosine methylation and local dna sequence 
 on guanine oxidation using a novel mass spectrometry based approach 
 developed in our laboratory ( stable isotope labeling of dna mass 
 spectrometry ( ild  ms ) ( scheme 2 ) . in this method , guanine bases 
 within a sequence of interest are replaced with stable isotope - tagged 
 guanine ( n3,c1-g ) . 
 following oxidative treatment , dna duplexes are enzymatically digested 
 to deoxyribonucleosides , and the relative amounts of og and z produced 
 at the n3,c1-labeled 
 guanine as compared to other guanine bases are determined by hplc since any og and z adducts originating from n3,c1-g contain the n , c isotope tag , they can be distinguished from lesions 
 formed at other sites by their molecular weight ( scheme 2 ) . we chose three types of oxidants : photoactivated riboflavin 
 ( a type i photosensitizer ) , nitrosoperoxycarbonate ( a chemical mediator 
 of inflammation that decomposes to no2 and co3 radicals ) , and co / benzoyl peroxide , which was used in classical studies of sequence - specific 
 dna oxidation . figures 1 and 2 illustrate 
 hplc  esi - ms / ms analysis of site - specific og and z adducts originating 
 from a double stranded dna 19-mer subjected to photooxidation in the 
 presence of riboflavin . signals specific to og and n3,c1-og are observed in the corresponding 
 ion channels ( m / z 284.1 [ m + h] m / z 168.0 [ m + 
 2h  dr ] ) and m / z 288.1 [ m + h] m / z 172.0 , respectively ; see figure 1 ) . shaded 
 peaks in figure 1b represent n3,c1-og adducts originating from n3,c1-g , while the top 
 panel ( figure 1a ) corresponds to unlabeled 
 og that derive from guanines elsewhere in the sequence . the extent 
 of og and z formation at the n3,c1-tagged guanine was calculated directly from the hplc  esi - ms / ms areas corresponding to the labeled and unlabeled adducts 
 according to the equationwhere alabeled and aunlabeled are the areas under the 
 hplc  esi - ms / ms peaks corresponding to the n , c - labeled and unlabeled adducts , respectively . z formation 
 at the guanine of interest was determined analogously using the transitions m / z 247.1  87.1 and m / z 250.1 [ m + h] m / z 87.2 [ m + 2h  dr  co2 ] , respectively ( figure 2 ) . it 
 should be noted that a + 3 rather than + 4 mass shift is observed for 
 z adducts originating from isotopically labeled dg due to a loss of 
 the n - nitrogen from the n-2 position of labeled dg during 
 adduct formation ( scheme 1 ) . esi - ms / ms analysis of og ( a ) and n3,c1-og ( b ) in enzymatic hydrolysates 
 of a n3,c1-dg containing 
 dna duplex following photooxidation in the presence of riboflavin . riboflavin concentration was 62.5 m , and samples were purged 
 with oxygen for 1 min , followed by irradiation for 20 min at 0 c . esi - ms / ms analysis of 
 z ( a ) and n2,c1z 
 ( b ) in enzymatic 
 hydrolysates of a n3,c1dg containing dna duplex subjected to photooxidation in the 
 presence of riboflavin . see figure 1 for photooxidation 
 conditions . endogenously methylated 
 cg dinucleotides contain two c bases ( one per strand ) . 
 in order to independently examine the effects 
 of base paired c and 5 neighboring c on guanine reactivity toward oxidants , methylated cytosine was 
 placed in either one or both strands of synthetic dna duplexes ( table 2 ) . these sequences were derived from the p53 tumor suppressor gene and represent endogenously methylated 
 cg dinucleotides frequently mutated in lung cancer and surrounding 
 sequence . uv melting experiments confirmed that dna was completely 
 in the duplex form ( example in figure s10 in the supporting information ) . the extent of adduct formation at 
 the n3-labeled guanine was normalized to the 
 adduct yields at the same guanine in the absence of c , 
 which was set to 100% . for dna sequences derived from p53 codons 245 
 and 248 , introduction of methyl group on cytosine immediately 5 
 of the target g ( cg ) or at the 
 base paired cytosine ( c : g ) facilitated 
 riboflavin - mediated photooxidation , with the highest adduct yields 
 observed in fully methylated dinucleotides ( both 5 and base 
 paired c , see figures 3a and 3b ) . specifically , og adduct formation at the n3,c1-labeled g in codon 
 245 nearly doubled when c was placed immediately preceding 
 the target guanine ( 192% , p < 0.0001 ) , increased 
 by 70% in the presence of methyl group at the base paired cytosine 
 ( 177.5% , p < 0.0001 ) , and more than doubled in 
 fully methylated dinucleotide ( 212% , p < 0.0001 ) 
 ( figure 3a , white bars ) . a much 
 more pronounced effect of c was observed for c located within the context of p53 codon 248 . in 
 this case , the introduction of c opposite target g led 
 to a 9-fold increase of z adduct yields , while the 5-neighboring c caused a 4-fold increase in adduct formation ( figure 3b ) . in contrast , og yields were only increased by 
 2030% relative to the corresponding unmethylated p53 codon 248 . influence of cytosine methylation within cg dinucleotides 
 on the 
 formation of og ( white bars ) and z adducts ( black bars ) upon riboflavin - mediated 
 photooxidation : p53 codon 245 ( a ) and p53 codon 248 ( b ) . panel c shows the relative yields of z adducts at 
 guanine paired with c and phenylpyrrolo - c . isotopically 
 tagged g was placed in a dna duplex opposite dc , 5-methylcytosine , 
 or phenylpyrrolo - c , followed by photooxidation in the presence of 
 riboflavin . z adduct formation at the labeled g was quantified by 
 hplc  esi - ms / ms as shown in scheme 2 . these results suggest that while 
 cytosine methylation facilitates 
 guanine oxidation in both cpg dinucleotides examined , the extent of 
 methylation - mediated increase and the relative quantities of the oxidation 
 products are dependent upon surrounding dna sequence . we hypothesize 
 that the fate of riboflavin - induced guanine radical cation intermediate 
 ( scheme 1 ) is influenced by the local dna structure , 
 leading to different amounts of individual oxidation products in different 
 sequence contexts . it is possible that neighboring bases alter solvent 
 accessibility of the guanine radical cation intermediate ( g , scheme 1 ) and/or facilitate 
 its deprotonation , leading to different fractions of guanine radical 
 cations being ultimately converted to 8-oxo - dg vs oxazolone . in theory , c may accelerate the rate of photooxidation 
 of neighboring guanine bases in the presence of riboflavin - mediated 
 oxidants by affecting the local geometry and the electronics of c : g 
 pairs or by facilitating the intercalation of riboflavin photosensitizer 
 adjacent to c : g base pairs . to evaluate potential role 
 of riboflavin intercalation to the increased oxidative lesion yields 
 at the at cg sites , c was replaced with 
 phenylpyrroloc , an unnatural cytosine analogue known to facilitate 
  stacking interactions with flat aromatic molecules 
 such as riboflavin . we found that z yields at phenylpyrroloc - g 
 base pairs were even greater than in the presence of c ( figure 3c ) . this is consistent with the 
 idea that riboflavin intercalation at endogenously methylated cg dinucleotides 
 contributes to the increased og and z adduct formation at cg sites within the p53 gene . to determine 
 whether c - mediated increase in guanine 
 oxidation was specific for riboflavin - mediated reactions , ild ms 
 experiments were repeated with two additional oxidants , chemical mediator 
 of inflammation nitrosoperoxycarbonate and co / benzoyl 
 peroxide . unlike riboflavin , nitrosoperoxycarbonate and cobalt are 
 unable to participate in stacking interactions with c , 
 therefore any reactivity changes observed upon the introduction of 
 methylated cytosine are likely attributed to the electronic effects . we found that the presence of neighboring c had a relatively 
 small effect on co / benzoyl peroxide - mediated guanine 
 oxidation ( black bars in figure 4 ) , but significantly 
 increased the formation of z adducts upon treatment with nitrosoperoxycarbonate 
 ( striped bars in figure 4 ) . influence of cytosine 
 methylation within p53 codon 
 245 on oxidative adduct yields ( z ) on isotopically labeled guanine 
 ( g ) following oxidation of dna duplexes in 
 the presence of photosensitized riboflavin ( white bars ) , nitrosoperoxycarbonate 
 ( striped bars ) , and co / benzoyl peroxide ( black bars ) . the observed differences between 
 the effects of cytosine methylation 
 on nitrosoperoxycarbonate - mediated guanine oxidation as compared to 
 riboflavin - induced photooxidation ( figure 4 ) could be explained by different nature of the reactive oxygen species 
 participating in reaction with the target guanine . both co3 and triplet riboflavin are strong oxidants , with 
 reduction potentials of 1.59 and 1.7 v versus nhe , respectively . however , photoactivated riboflavin - mediated 
 oxidation of dna involves photosensitizer intercalation into the dna 
 duplex , followed by a direct electron transfer from dg to riboflavin , 
 producing guanine radical cation ( scheme 1 ) . in contrast , onooco2-mediated oxidation of guanine is mediated by co3 radicals and does not require intercalation . the photosensitizer intercalation step is likely 
 to be differentially affected by 5-methylc adjacent to the target 
 g versus the base paired 5-methylc , leading to different oxidative 
 adduct yields in these two duplexes . taken together , our results 
 suggest that endogenous cytosine methylation targets neighboring guanine 
 bases for the preferential oxidation by a combination of two mechanisms : 
 electronic effects that lower the ip of guanine bases in cg dinucleotides and stacking interactions 
 that facilitate the intercalation of riboflavin photosensitizer adjacent 
 to c : g base pairs . in humans , c is found predominantly at cg dinucleotides , with about 
 80% of all cg sites endogenously methylated in somatic cells . while many of the cg sequences 
 in the human genome are found at cpg islands  of regulatory 
 sequences that control the levels of gene expression , some of them 
 are located within coding sequences of genes . specifically , all cg dinucleotides of the p53 tumor 
 suppressor gene are endogenously methylated . the same sites , e.g. , p53 codons 158 , 245 , 248 , 
 and 273 , are the major  hotspots  for mutations in smoking - induced 
 lung cancer ( figure s1 in the supporting information ) , leading to the hypothesis that tobacco carcinogens may preferentially 
 modify endogenously methylated cg dinucleotides . we and others have previously reported that n - bpde - dg adducts of carcinogenic polycyclic 
 aromatic hydrocarbons such as benzo[a]pyrene are 
 preferentially formed at cg dinucleotides of the p53 tumor suppressor gene , specifically codons 157 , 158 , 
 245 , 248 , and 273 . to examine whether cytosine methylation preferentially 
 targets endogenously methylated cg dinucleotides of the p53 gene for oxidative degradation , a series of isotopically tagged 
 oligodeoxynucleotides were synthesized representing specific regions 
 of the p53 gene derived from exons 5 , 7 , and 8 ( table 1 ) . these were selected because they contain codons 
 157 , 158 , 245 , 248 , and 273 frequently mutated in lung cancer ( figure 
 s1 in the supporting information ) . c was inserted in both strands at all endogenously methylated 
 cg sites as observed physiologically ( table 1 ) . isotopically tagged strands were annealed to the complementary 
 strands , and duplex formation was confirmed by uv melting experiments . following riboflavin - mediated photooxidation and dna hydrolysis , the 
 extent of og and z formation at each location of interest along this 
 duplex was calculated based on isotope ratio hplc  esi - ms / ms as shown in scheme 2 and figures 1 and 2 . our initial isotope labeling 
 experiments were conducted with dna duplexes derived from p53 codons 243250 ( 5-atg1 g2g3c g4g5c atg6 aac cg7g8 ag9g10 ccc a-3 ) where c = endogenous 5-methylcytosine ) ( table 1 ) . n3,c1-dg was introduced 
 at one of the highlighted positions ( g4 , g5 , g6 , g7 , g8 , or g9 , table 1 ) , and c was inserted 
 at the two endogenously methylated sites ( c g4 and cg7 ) . since the p53 exon-7-derived 
 duplex contains a total of 17 guanines , a sequence - independent ( random ) 
 oxidation would lead to 5.88% of oxidative lesion occurring at each 
 individual guanine ( 100%/17 = 5.88% ) . however , og adduct numbers are 
 significantly above the average value at two distinct sites , g4 ( codon 245 ) and g7 ( codon 248 ) , which give rise to 18.7 and 14.2% 
 of total og adducts , respectively ( white bars in figure 5a ) . similar results are observed for z , with 15.9% of adducts 
 originating from g4 and 18.1% 
 coming from g7 ( black bars in 
 figure 5a ) . importantly , both sites of the 
 highest reactivity ( g4 and g7 ) have an identical sequence context 
 ( cgg ) , with the target g flanked by another 
 guanine at the 3 side and a methylated cytosine on the 5 
 side ( figure 5a ) . the same two sites correspond 
 to prominent lung cancer mutational hotspots at p53 codons 245 and 248 , respectively ( figure s1 in the supporting information ) . in contrast , the remaining guanine bases exhibited 
 below average reactivity ( figure 5a ) and are 
 infrequently mutated in lung cancer . studies with the corresponding single stranded oligonucleotides 
 revealed essentially random reactivity ( figure s14 in the supporting information ) , confirming that the 
 presence of intact double stranded dna is required for the observed 
 sequence selectivity . overall , our results for fully methylated p53 exon 7 duplex ( figure 5a ) are 
 consistent with the data for short duplexes containing single methylated 
 cpg sites ( figure 3a , b ) . however , the magnitude 
 of the effect observed upon cytosine methylation differs between the 
 two experiments , probably due to the presence of other neighboring 
 methylated cpg sites in the fully methylated duplex . distribution of og ( white 
 bars ) and z lesions ( black bars ) along 
 dna duplexes derived from p53 exon 7 ( a ) , p53 exon 5 ( b ) , and p53 exon 8 ( c ) following 
 oxidation in the presence of photosensitized riboflavin as determined 
 by stable isotope labeling . error bars correspond to standard deviation 
 of 35 independent measurements . in order to further investigate 
 the effects of dna sequence on the formation of oxidative guanine 
 lesions , this region of the p53 gene contains 
 several additional mutational  hotspots  characteristic 
 for smoking - induced lung cancer , e.g. , codons 154 ( ggc ) , 157 ( gtc ) , and 158 ( cgc ) , all containing endogenous 5-methylcytosine 
 ( cg ) . in our experiments , 
 the highlighted guanines ( g1 , g2 , g3 , g4 , or g5 ) were sequentially replaced with n3,c1-dg ( table 1 ) , and c was incorporated at all physiologically 
 methylated 5-cg sites : 5-ccc g1g2c 
 acc cg3c g4tc cg5c g6 [ ( + ) strand ] 
 ( table 1 ) . following 
 riboflavin - mediated photooxidation and dna hydrolysis of the isotopically 
 labeled duplexes , the amounts of og and z originating from each location were calculated from hplc  esi - ms / ms isotope ratios 
 as described above ( scheme 2 ) . unlike 
 our results for exon 7 derived duplexes , which showed similar distribution 
 patterns of both oxidative adducts ( figure 5a ) , the patterns of z and og formation along the duplexes derived 
 from exon 5 were quite different from each other ( figure 5b ) . while og was preferentially formed at g1 , g3 , and g5 , z yields were 
 below 5% for all guanines tested within ( + ) strand of dna ( less than 
 the predicted random reactivity value of 5.88% , see figure 5b ) . in general , the formation of og followed the 
 following order : g1 ( cg1g2 ) > g3 ( cg3c ) 
  g5 ( cg5c ) 
  g2 ( g1g2c )  g4 ( cg4 t ) ( figure 5b , white 
 bars ) . the sites of the highest og formation included a guanine flanked 
 by 5-g and a 3-c ( cg1 g ) and two sites flanked by two 
 5-methylcytosines ( cgc , g3 and g5 ) . to identify the sites of preferential z formation 
 in this duplex , 
 we conducted an additional isotope labeling experiment for guanine 
 9 of the (  ) strand ( figure 5b ) . we 
 found that (  ) g9 ( ggc 
 sequence context ) was highly susceptible to photooxidation , giving 
 rise to over 13% of total z adducts ( figure 5b ) . the same sequence was identified as the main target for photooxidation 
 within p53 exon 7 ( figure 5a ) . taken together , our results for p53 exon 7 indicate 
 that the two oxidative lesions ( og and z ) have distinct distribution 
 patterns within this region of the genome , but share one target sequence 
 ( ggc ) . furthermore , one of the sites favored 
 for og production ( g5 ) coincides 
 with a known p53 lung cancer mutational hotspot at 
 codon 158 , while the other two frequently adducted sites ( g1 and g3 ) do not . the third p53-derived sequence 
 examined ( 5-g1ct ttg2 ag3g4 tg5c g6tg7 ttt 
 g8tg9 ) corresponds to a frequently mutated region 
 of p53 exon 8 and contains an important smoking - associated 
 lung cancer mutational  hotspot  at codon 273 ( cgt  
 ctt ) ( figure s1 in the supporting information ) . initial isotope labeling studies conducted 
 with p53 exon 8-derived duplexes have revealed that 
 og formation was the highest at g3 ( ag3g4 ) and g2 ( tg2a ) 
 ( figure 5c ) . in contrast , z yields were well 
 below the theoretical random reactivity value ( 7.7% ) at all gs examined , 
 with the exception of the terminal guanine ( g1 ) , which gave rise to over 30% of total z adducts ( figure 5c , black bars ) . these results suggest that z , but 
 not og adducts , are overproduced at solvent exposed sites such as 
 the ends of dna duplexes . interestingly , lee et al . have previously 
 reported that the formation of photooxidation - induced alkali - labile 
 lesions ( such as z ) was facilitated at guanines adjacent to an abasic 
 site as a result of increased solvent exposure . this may facilitate the formation of tandem dna lesions , 
 which are more difficult to repair and may add to the mutagenic and 
 toxic effects of oxidative stress . oxidative 
 dna damage is a key contributor to the pathogenesis of 
 smoking - induced lung cancer . tobacco smoke contains 
 high concentrations of nitric oxide ( no ) , which can be oxidized to 
 the nitrating and oxidizing agent , nitrogen dioxide . furthermore , phenolic and polyphenolic species present in cigarette 
 tar and formed during carcinogen metabolism can undergo redox cycling 
 yielding superoxide anion radicals , o2 . superoxide combines with no at a diffusion 
 controlled rate to yield peroxynitrite , onoo , which 
 conjugates with carbon dioxide to form a strong oxidant , nitrosoperoxycarbonate 
 onooco2 . o2 is also subject to spontaneous 
 or enzymatic dismutation to hydrogen peroxide ( h2o2 ) , which can undergo the fenton reaction with fe to produce highly reactive hydroxyl radicals ( ho ) . cigarette smokers excrete elevated 
 amounts of the oxidative dna lesion , og-7,8-dihydro-2-deoxyguanosine 
 ( og ) in urine , which is reversed upon smoking cessation . og levels are also increased in leukocyte dna 
 of smokers as compared with nonsmoking controls . the increased cellular load of oxidative dna lesions in 
 the tissues of smokers is likely to play a role in lung tumor induction . for example , knockout mice deficient at repairing oxidative lesions 
 are predisposed to the development of lung adenocarcinoma . it has been shown that smokers who exhibit low 
 levels of hogg1 , a repair protein responsible for the removal of og 
 and other oxidative lesions , are at an increased risk of lung cancer . if unrepaired , these oxidized bases are strongly 
 mispairing , inducing large numbers of g  t and g  
 c transversions . of the multiple 
 genes mutated in lung cancer , the p53 tumor suppressor 
 gene is among the most important . p53 protein is 
 involved in multiple important cellular processes , including cell 
 cycling , gene transcription , chromosomal segregation , dna repair , 
 and apoptosis . the p53 gene 
 is frequently mutated in smoking - induced lung cancer , with the majority of genetic changes being g  t 
 transversions at p53 exons 5 , 7 , and 8 ( figure s1 
 in the supporting information ) . prominent p53 mutational  hotspots  include codons 154 
 ( ggc  gtc ) , 157 ( gtc  ttc ) , 158 ( cgc  ctc ) , 245 ( ggc  tgc ) , 
 248 ( cgg  ctg ) , 249 ( agg  atg ) , and 273 ( cgt  ctt ) ( figure s1 in the supporting information ) . most of these sites correspond to guanines within endogenously methylated 
 dinucleotides ( cg , where c = 5-methycytosine ) . because p53 exons 58 encode the dna binding 
 domain of the p53 protein , mutations within these 
 regions inactivate the protein , contributing to cancer initiation 
 and progression . however , the chemical 
 origins of the p53 gene mutations observed in lung 
 cancer have not been established , although several studies have implicated 
 dna damage induced by polycyclic aromatic hydrocarbons and aldehydes 
 present in tobacco smoke . in the present 
 work , an isotope labeling of dna  mass spectrometry based approach developed in our laboratory was used to quantify the formation of og and z lesions at specific 
 guanine bases within dna duplexes derived from the p53 tumor suppressor gene . these lesions are among the major guanine 
 oxidation products produced by a variety of oxidative treatments , 
 including riboflavin - mediated photooxidation , exposure to superoxide 
 radicals , and nitrosoperoxycarbonate treatment . while 
 og is readily oxidized to secondary products , z is resistant to further oxidation . mapping oxidative dna lesions along critical 
 regions of the p53 tumor suppressor gene and probing 
 their association with p53 mutational  hotspots may provide an insight into the origin of mutational hotspots observed 
 in smoking - induced lung cancer . the main advantage of the isotope 
 labeling methodology as compared to gel electrophoresis - based methods 
 is that it provides robust structural information for oxidized nucleobases , 
 in addition to accurate sequence distribution data . in order 
 to map the distribution of og and z adducts , synthetic 
 dna duplexes derived from a frequently mutated region of the p53 tumor suppressor gene were prepared in which guanine 
 bases of interest were sequentially replaced with c1,n3-guanine ( tables 1 and 2 ) . following photooxidation in 
 the presence of riboflavin or treatment with other reactive oxygen 
 species , dna was enzymatically digested to 2-deoxynucleosides , 
 and the formation of og and z adducts at each site was accurately 
 quantified by isotope ratio hplc  esi - ms / ms ( scheme 2 , figures 1 and 2 ) . we found that og and z formation within 
 duplexes derived from the p53 tumor suppressor gene 
 was nonrandom ( figure 5 ) . in general , og yields 
 followed the following 
 order : cgg > cgt  c gc  agg > tgt > tga 
  ggc  cgt  tgc  gga  ggt . while 
 z and og adducts exhibited similar sequence preferences in the central 
 regions of dna duplexes , the formation of z lesions was strongly favored 
 at solvent exposed regions at the ends of dna duplexes ( e.g. , g1 in 
 figure 5c ) . this can be explained by the effects 
 of sequence context and base pairing on the fate of their common intermediate , 
 the guanine radical cation produced upon one - electron oxidation of 
 g ( g , scheme 1 ) . deprotonation 
 of g ( pka  
 3.9 ) produces neutral guanine radicals ( g ) , which 
 react with oxygen and generate z as the main final product ( scheme 1 ) . in a stable dna duplex , 
 base pairing of g with c in the opposite strand prevents such deprotonation , 
 causing the radical cation intermediate to react with water and generate 
 og ( scheme 1 ) . as a result , z , but not og adducts , are specifically overproduced 
 at the ends of dna duplexes where watson  crick base pairing 
 of target gs to the complementary strand may be disrupted , e.g. , g1 in p53 exon 8-derived duplex ( figure 5c ) and g9 in exon 5-derived 
 sequence ( figure 5b ) . these results suggest 
 that the yield of highly mutagenic z adducts may be increased at the 
 regions of genome where base pairing is transiently or permanently 
 compromised ( e.g. , base mispairs or actively transcribed regions ) . in contrast , og and z formation in single stranded 
 dna exhibits little sequence specificity , consistent with a requirement 
 for charge transport along dna duplex ( figure s14 in the supporting information ) . in central regions 
 of dna with intact duplex structure , og and 
 z adduct yields are determined by two main factors : nucleobase sequence 
 and methylation status of neighboring cytosines . both lesions are 
 preferentially formed at endogenously methylated cg dinucleotides 
 and in runs of several gs , with the highest reactivity observed at 
 central gs of cgg 
 sequences ( figure 5 ) . the increased reactivity 
 of 5-gs of guanine runs toward one - electron oxidants is well 
 documented . it has been proposed that electron holes are preferentially trapped 
 at guanine residues located in the 5-position to another guanine 
 ( e.g. , 5-gg or 5-ggg ) . ab initio 
 calculations suggest that these are the most electron - rich sites in 
 the b - form of dna and therefore may act as thermodynamical  sinks  
 for oxidative damage following long - range charge transport from other 
 sites in the helix ( g - g stacking rule ) . perhaps 
 the most important result of this study is the enhanced 
 oxidation of guanines within endogenously methylated cg dinucleotides ( figures 35 ) . these methylated sequences are preferentially 
 oxidized in the presence of photosensitized riboflavin , nitrosoperoxycarbonate , 
 and co / benzoyl peroxide ( figure 4 ) . all cg dinucleotides within the coding region of the p53 gene contain 5-methylcytosine , and 
 many of these sites are known mutational hot spots  
 for smoking induced lung cancer ( figure s1 in the supporting information ) . furthermore , 
 targeted oxidation of cg sequence can induce epigenetic 
 changes , as og has been reported to inhibit human dna methyltransferases . previous experimental and computational 
 studies have revealed that 
 the nucleophilicity of guanine bases within c : g base 
 pairs is enhanced as a result of the inductive electronic effects 
 of the c-5 methyl group transmitted to the n - amino group of g through c : g hydrogen bonds . consistent with this explanation , the presence 
 of electron withdrawing fluoro group on the c-5 of cytosine reduces 
 the reactivity of g : c base pairs toward electrophiles . employed triplet - quenching experiments 
 to show that the one - electron oxidation rate of guanine was accelerated 
 upon hydrogen bonding with 5-methylcytosine . an opposite effect was observed when c-5 methyl group was replaced 
 with an electron withdrawing bromo substituent . these studies suggest that the transmitted electronic effect 
 of the methyl group may be an important factor responsible for the 
 accelerated oxidation of guanine bases within endogenously methylated cg dinucleotides . in addition to its electronic effects , 
 the presence of c5-methyl 
 group increases the molecular polarizability of cytosine , decreases 
 the major groove charge density , stabilizes dna helix , and enhances 
 base stacking . we have previously reported that 
 c-5 cytosine alkylation facilitates the formation of intercalative 
 complexes of diolepoxide metabolites of polycyclic aromatic hydrocarbons 
 with dna , facilitating the nucleophilic attack by the n position of guanine and increasing the guanine adduct 
 yields . our present results suggest that 
 the presence of c can similarly stabilize intercalative 
 complexes of riboflavin photosensitizer with dna via increased  
 stacking interactions , leading to increased yields of oxidative guanine 
 adducts at methylated cg dinucleotides . indeed , even greater z yields 
 were observed upon phootoxidation of phenylpyrroloc : g base pairs ( figure 3c ) . phenylpyrroloc is a highly aromatic analogue 
 of c previously shown to participate in intercalative 
 complex formation with flat polycondensed aromatic molecules . this finding suggests that riboflavin intercalation 
 adjacent to c : g base pairs enhances initial electron 
 transfer and facilitates subsequent oxidation steps . our results 
 differ from an earlier report that employed page methodology 
 to examine the influence of cytosine methylation on long - distance 
 radical cation transport following electron hole injection into duplexes 
 containing anthraquinone photosensitizer covalently linked to the 
 5-terminus . one possible explanation 
 is that the earlier study was limited 
 to two specific dna sequences ( tcgcgt and aggt ) and did not include 
 cgg trinucleotides as found in the p53 gene ( figure 5 ) . furthermore , our results may differ because the 
 approach employed in this work ( ild  ms ) quantifies specific dna adducts ( og and z ) rather than 
 determining total numbers of all alkali sensitive lesions by gel electrophoresis . finally , our photooxidation experiments intentionally employed riboflavin 
 in solution rather than covalently linked photosensitizer , allowing 
 for precovalent interactions to contribute to the observed sequence 
 specificity for photooxidation . typical riboflavin ( vitamin b2 ) concentrations 
 in human plasma are 300 nm , where it plays an important role 
 in oxidative folding and secretion of proteins including apolipoprotein 
 b-10 . it is also present in the cell 
 nucleus and has been reported to act as a photosensitizer , damaging 
 dna in vivo following uva irradiation . however , it should be noted that cytosine methylation also increased 
 oxidative adduct yields on neighboring guanines following treatment 
 with other ros such as nitrosoperoxycarbonate and co / benzoyl 
 peroxide ( figure 4 ) . these results are consistent 
 with the electronic effect of the methyl group transmitted through 
 hydrogen bonding within the c : g base pair . interestingly , c itself has been recently identified 
 as an oxidation  hotspot  when mispaired with a or t. in summary , our isotope labeling results 
 indicate that both og 
 and z are preferentially generated at endogenously methylated cg dinucleotides and at the 5 gs in runs of several 
 guanines . the central guanines in cgg sequences are highly susceptible to oxidation . in addition , 
 z but not og adducts are overproduced at solvent exposed regions of 
 dna such as the ends of dna duplexes . these results are important 
 because they provide the first sequence distribution data for structurally 
 defined oxidative guanine adducts , contributing to our understanding 
 of dna oxidation chemistry , and may help uncover the origins of mutational 
  hotspots  found at endogenously methylated cg dinucleotides 
 within the human genome . future studies are needed to establish whether 
 the same sequence specificity for oxidation is maintained in chromosomes 
 of human cells . however , it should be noted that cg sites 
 may also be targeted by other electrophiles due to the increased reactivity 
 of guanine bases adjacent to c. furthermore , the p53 mutational spectra found in 
 lung tumors are likely to be affected by sequence - dependent repair , mispairing efficiency , and biological selection 
 of mutants for growth .
dna 
 oxidation 
 by reactive oxygen species is nonrandom , potentially 
 leading to accumulation of nucleobase damage and mutations at specific 
 sites within the genome . 
 we now present the first quantitative data 
 for sequence - dependent formation of structurally defined oxidative 
 nucleobase adducts along p53 gene - derived dna duplexes 
 using a novel isotope labeling - based approach . 
 our results reveal 
 that local nucleobase sequence context differentially alters the yields 
 of 2,2,4-triamino-2h - oxal-5-one ( z ) and 8-oxo-7,8-dihydro-2-deoxyguanosine 
 ( og ) in double stranded dna . 
 while both lesions are overproduced within 
 endogenously methylated mecg dinucleotides and at 5 
 gs in runs of several guanines , the formation of z ( but not og ) is 
 strongly preferred at solvent - exposed guanine nucleobases at duplex 
 ends . 
 targeted oxidation of mecg sequences may be caused 
 by a lowered ionization potential of guanine bases paired with mec and the preferential intercalation of riboflavin photosensitizer 
 adjacent to mec : g base pairs . 
 importantly , some of the 
 most frequently oxidized positions coincide with the known p53 lung cancer mutational  hotspots  at codons 
 245 ( ggc ) , 248 ( cgg ) , and 158 ( cgc ) respectively , supporting a possible 
 role of oxidative degradation of dna in the initiation of lung cancer .