Formation of Diastereomeric Benzo [a] pyrene Diol Epoxide-Guanine

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Formation of Diastereomeric Benzo[a]pyrene Diol Epoxide-Guanine Adducts in p53 Gene-Derived DNA Sequences Brock Matter,† Gang Wang,‡ Roger Jones,‡ and Natalia Tretyakova*,† Department of Medicinal Chemistry, University of Minnesota Cancer Center, Minneapolis, Minnesota 55455, and Department of Chemistry, Rutgers University, Piscataway, New Jersey 08854 Received January 9, 2004

G f T transversion mutations in the p53 tumor suppressor gene are characteristic of smoking-related lung tumors, suggesting that these genetic changes may result from exposure to tobacco carcinogens. It has been previously demonstrated that the diol epoxide metabolites of bay region polycyclic aromatic hydrocarbons present in tobacco smoke, e.g., benzo[a]pyrene diol epoxide (BPDE), preferentially bind to the most frequently mutated guanine nucleotides within p53 codons 157, 158, 248, and 273 [Denissenko, M. F., Pao, A., Tang, M., and Pfeifer, G. P. (1996) Science 274, 430-432]. However, the methodology used in that work (ligationmediated polymerase chain reaction in combination with the UvrABC endonuclease incision assay) cannot establish the chemical structures and stereochemical identities of BPDE-guanine lesions. In the present study, we employ a stable isotope-labeling HPLC-MS/MS approach [Tretyakova, N., Matter, B., Jones, R., and Shallop, A. (2002) Biochemistry 41, 9535-9544] to analyze the formation of diastereomeric N2-BPDE-dG lesions within double-stranded oligodeoxynucleotides representing p53 lung cancer mutational hotspots and their surrounding DNA sequences. 15N-labeled dG was placed at defined positions within DNA duplexes containing 5-methylcytosine at all physiologically methylated sites, followed by (()-anti-BPDE treatment and enzymatic hydrolysis of the adducted DNA to 2′-deoxynucleosides. Capillary HPLC-ESI+MS/MS was used to establish the amounts of (-)-trans-N2-BPDE-dG, (+)-cis-N2-BPDE-dG, (-)-cis-N2-BPDE-dG, and (+)-trans-N2-BPDE-dG originating from the 15N-labeled bases. We found that all four N2-BPDE-dG diastereomers were formed preferentially at the methylated CG dinucleotides, including the frequently mutated p53 codons 157, 158, 245, 248, and 273. The contributions of individual diastereomers to the total adducts number at a given site varied between 70.8 and 92.9% for (+)-trans-N2-BPDE-dG, 5.6 and 16.7% for (-)-trans-N2-BPDE-dG, 2.1 and 8.5% for (-)-cis-N2-BPDE-dG, and 0.5 and 8.3% for (+)-cis-N2-BPDE-dG. The relative yields of the minor N2-BPDE-dG stereoisomers were elevated at the sites of inefficient adduction, while the major (+)-trans-BPDE lesion was even more dominant at the frequently adducted sites. The introduction of 5-methyl groups at adjacent cytosine bases increased the yields of N2-BPDE-dG diastereomers, probably a result of favorable hydrophobic interactions between BPDE and 5-methylcytosine. The targeted formation of N2-BPDE-dG at MeCG dinucleotides within the p53 gene is consistent with the high prevalence of G f T transversions at these sites in smoking-induced lung cancer.

Introduction Cigarette smoking is responsible for over 80% of all lung cancer cases and for over 30% of the total cancer mortality in industrialized nations (1). Smoking-induced carcinogenesis is thought to be triggered by the covalent binding of metabolically activated tobacco carcinogens to genomic DNA. If not repaired, the resulting chemically modified nucleobases (DNA adducts) are potentially misread by DNA polymerases, giving rise to heritable mutations and contributing to the initiation and progression of cancer. The p53 tumor suppressor gene plays an important role in cell cycle control, gene expression, DNA repair, and * To whom correspondence should be addressed. Tel: 612-626-3432. Fax: 612-626-5135. E-mail: [email protected]. † University of Minnesota Cancer Center. ‡ Rutgers University.

apoptosis (2). The p53 protein functions as a cell cycle checkpoint, allowing the cell to repair DNA damage before replication takes place, thus reducing the probability of polymerase errors. The p53 gene is a major target for the genetic damage observed in smokinginduced lung cancer (3), with approximately 56% of total lung tumors bearing p53 mutations (4). The majority of the p53 base changes observed in lung tumors of smokers are G f T transversions clustered in exons 5, 7, and 8 (Figure 1). Prominent mutational “hot spots” are observed at codons 157 (GTC f TTC), 158 (CGC f CTC), 245 (GGC f TGC), 248 (CGG f CTG), 249 (AGG f ATG), and 273 (CGT f CTT) (Figure 1) (5, 6). Because p53 exons 5-8 correspond to a sequence selective DNA binding domain of the p53 protein (4), mutations in this region inactivate the p53 protein by altering its ability to recognize promoter sequences (2). For example, p53

10.1021/tx049974l CCC: $27.50 © 2004 American Chemical Society Published on Web 05/14/2004

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Figure 1. Distribution of G f T transversions in human lung cancer mapped along exons 5-8 of the p53 gene. The frequency distribution of mutations was obtained from the AACR p53 mutation database (52). Cancers from nonsmokers and from occupational exposure were excluded.

gene products containing base substitutions in codons 248 and 273 can no longer act as transcription factors for certain downstream genes, e.g., p21 and BAX (7). The loss of functional p53 leads to genomic destabilization, clonal expansion of the affected cells, and an increased likelihood of further genetic damage (4). Among multiple constituents of cigarette smoke recognized as carcinogens by the International Agency for Cancer Research, polycyclic aromatic hydrocarbons (PAHs)1 are considered probable causative agents of smoking-induced lung cancer (8). PAHs are produced by the incomplete combustion of organic material during cigarette burning. The best studied PAH, B[a]P, is a potent systemic and local carcinogen known to induce skin, lung, and stomach tumors in animal models (9). B[a]P is a suspect human carcinogen based on its potent tumorigenic effects in multiple species, along with the similarity of B[a]P-induced DNA damage in laboratory animals and human cells and the accumulation of B[a]PDNA adducts in target tissues (10-14). B[a]P requires metabolic activation by P450 monooxygenases to yield DNA reactive diol epoxides, e.g., (+)anti-BPDE, (-)-anti-BPDE, (+)-syn-BPDE, and (-)-synBPDE (Scheme 1). The anti stereoisomers (Scheme 1) are produced in greater quantities and exhibit a greater mutagenic and carcinogenic activity and thus are considered the ultimate carcinogens of B[a]P (15, 16). Following intercalative binding of BPDE diol epoxides to the DNA duplex, the exocyclic amino group of guanine nucleophilically attacks the C-10 position of BPDE, giving rise to diastereomeric N2-guanine adducts, e.g., (+)-trans1 Abbreviations: B[a]P, benzo[a]pyrene; (+)-anti-BPDE, (+)-(7R, 8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; (-)-anti-BPDE, (-)-(7S,8R,9R,10S)-7,8-dihydroxy-9,10-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene; CD, circular dichroism; HPLCESI-MS/MS, high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry; LMPCR, ligation-mediated polymerase chain reaction; MeC, 5-methylcytosine; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; PAHs, polycyclic aromatic hydrocarbons; PDE, phosphodiesterase; SPE, solid phase extraction; SRM, selected reaction monitoring.

N2-BPDE-dG, (-)-trans-N2-BPDE-dG, (+)-cis-N2-BPDEdG, and (-)-cis-N2-BPDE-dG (Scheme 1) (17). Extensive structural studies of the DNA duplexes containing N2-BPDE-dG adducts by UV, NMR, fluorescence spectroscopy, and molecular dynamics simulations indicate that N2-BPDE-dG diastereomers have distinct spatial orientations in DNA. The BPDE moiety of the trans adducts is typically found in an external minor groove conformation within the DNA duplex (18). In contrast, the cis adducts assume an intercalative conformation by displacing the modified guanine residue and its partner cytosine into the major groove [(-)-cis isomer] or into the minor groove of DNA [(+)-cis isomer] (18). Interestingly, the (-)-trans adduct undergoes a shift to an intercalative conformation in the presence of neighboring 5-methylcytosine (MeC) (19). MeC is an endogenous DNA modification occurring at all CG dinucleotides within the p53 coding sequence (20). Although (+)-trans-N2-BPDE-dG (Scheme 1) is the predominant BPDE adduct in vivo, minor N2-BPDE-dG diastereoisomers may also play a role in mutagenesis. All four isomeric N2-BPDE-dG lesions represent strong blocks to DNA replication and, if bypassed, primarily induce G f T transversion mutations (21-23). The efficiency of translesion synthesis and the identity of the nucleotide incorporated opposite N2-BPDE-dG are dependent on the lesions’ stereochemistry, with the (-)cis-N2-BPDE-dG exhibiting the greatest mispairing efficiency in Escherichia coli and the (+)-trans adduct inducing a higher number of mutations in COS cells (24, 25). Importantly, nucleotide excision repair of the rare (+)-cis isomer of N2-BPDE-dG is inhibited in the presence of MeC at p53 codon 273, while the predominant adduct, (+)-trans-N2-BPDE-dG, is readily repaired (26). Local sequence context can further mediate N2-BPDE-dG mutagenicity and repair (23, 25, 27-29). Several groups have used an UvrABC nuclease incision assay in combination with LMPCR to demonstrate that lung cancer mutational hot spots within p53 codons 245, 248, and 273 are selectively modified by BPDE and other

Sequence Distribution of N2-BPDE-dG Diastereomers

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Scheme 1. Formation of Diastereomeric N2-BPDE-dG Adducts from (()-anti-BPDE

PAH diol epoxides (30, 31). Interestingly, the presence of neighboring MeC was essential for the observed sequence selectivity (32). These results suggest that targeted PAH binding to methylated CG sites may play a role in shaping the p53 mutational spectrum in smokinginduced lung cancer. However, the methodology used in that work (LMPCR in combination with the UvrABC endonuclease incision assay) cannot establish the chemical structures and stereochemical identities of BPDEguanine lesions. We now describe the use of a stable isotope-labeling HPLC-MS/MS approach (33) to analyze the distribution of diastereomeric N2-BPDE-dG lesions within physiologically methylated double-stranded DNA sequences derived from the frequently mutated regions of p53 exons 5, 7, and 8. This approach directly quantifies N2-BPDE-dG adducts at specific nucleobases, making it possible to investigate the effects of the local sequence environment and methylation status of neighboring cytosine bases on the formation of individual N2-BPDEdG diastereomers.

Materials and Methods Caution: BPDE is carcinogenic and should be handled with extreme caution. Materials. 15N-dG-labeled DNA oligodeoxynucleotides representing selected regions of the p53 tumor suppressor gene (Table 1) were prepared by standard phosphoramidite chemistry using a DNA synthesizer at the University of Minnesota Microchemical Facility. 1,7,NH2-15N-dG, 15N5-dG, or 1,7,NH2-15N-213C-dG were introduced at specified positions within oligodeoxynucleotide sequences by using the corresponding stable isotope-labeled dG phosphoramidites. 1,7,NH2-15N-dG and 1,7,NH2-15N-2-13C-dG phosphoramidites were prepared as described elsewhere (34, 35). 15N5-dG phosphoramidite was purchased from Martek Biosciences (Columbia, MD). (()-anti-BPDE was obtained from the NCI Chemical Carcinogen Repository (Midwest Research Institute). Ultraviolet spectra were recorded with a Hewlett-Packard P450A spectrophotometer. CD spectra were obtained with a Jasco Spectrapolarimeter (model J-710). Samples were scanned from 200 to 400 nm, with a resolution of 0.1 nm. N2-BPDE-dG diastereomers were prepared by treating calf thymus DNA with (()-anti-BPDE (36). Calf thymus DNA (10 mg) was dissolved in 6 mL of 10 mM Tris-HCl/15 mM MgCl2

buffer, pH 7, and (()-anti-BPDE (1 mg in 500 µL of THF) was added. After the mixture was incubated for 10 h at 37 °C, the unreacted BPDE was extracted with ethyl acetate, and the DNA was precipitated with NaCl and cold ethanol. The adducted DNA was dissolved in 3 mL of 10 mM Tris-HCl/15 mM MgCl2 buffer, pH 7, and hydrolyzed to 2′-deoxyribonucleosides with a mixture of deoxyribonuclease I (DNase I, 2800 U), PDE I (17 U), PDE II (PDE II, 7 U), and alkaline phosphatase (700 U). N2-BPDE-dG was isolated by SPE on C18 Sep-Pak cartridges (Waters Associates, Milford, MA), and individual diastereomers were purified by reversed phase HPLC as described below. HPLC Separation of BPDE-dG Diastereomers and Their CD Spectra. N2-BPDE-dG diastereomers were separated by HPLC with an Agilent Technologies HPLC system (model 1100) incorporating a diode array UV detector. An Extend-C18 column (4.6 mm × 150 mm, 5 µm, Agilent Technologies) was eluted at a flow rate of 1 mL/min. The buffer system was composed of 33% methanol in 150 mM ammonium acetate (A) and 25% buffer A in acetonitrile (B). The solvent composition was changed from 13 to 15% B in 24.5 min, and then further to 30% B at 30 min. N2-BPDE-dG adducts were detected by UV absorption at 345 nm. At these conditions, HPLC retention times for the individual diastereomers were as follows: (-)trans-N2-BPDE-dG (tR, 20.3 min), (+)-cis-N2-BPDE-dG (tR, 21.1 min), (-)-cis-N2-BPDE-dG (tR, 22.8 min), and (+)-trans-N2BPDE-dG (tR, 24.6 min). The HPLC peaks corresponding to individual N2-BPDE-dG diastereomers were collected and thoroughly dried under reduced pressure to remove most of the ammonium acetate and then dissolved in 33% methanol/water. CD spectra were obtained with a Jasco Spectrapolarimeter (model J-710) (see Appendix S-1 in the Supporting Information). Samples were scanned from 200 to 400 nm, with a resolution of 0.1 nm. N2-BPDE-dG stock solution concentrations were determined by UV spectrophotometry (345 ) 34 293; see Appendix S-2 in the Supporting Information). DNA Purification by HPLC and Purity Control. All DNA oligodeoxynucleotides were purified by HPLC with an Agilent Technologies HPLC system (model 1100). The DNA was separated on a Supelcosil LC-18-DB column (10 mm × 250 mm, 5 µm, Supelco, Bellefonte, PA) eluted at a temperature of 40 °C and a flow rate of 3 mL/min. HPLC buffers were 100 mM triethylammonium acetate, pH 7 (A), and 50% acetonitrile in 100 mM triethylammonium acetate, pH 7 (B). A linear gradient of 7.5-26% B in 42 min was employed. If necessary, DNA strands were further purified using the same Supelcosil LC18-DB column eluted with 150 mM ammonium acetate (buffer A) and acetonitrile (B) at a gradient of 5-12% B in 40 min.

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Matter et al. Table 1

id [15N3]-p53-exon 5-G1,Me2,9,11,14,16 [15N3]-p53-exon 5-G3,Me2,9,11,14,16 [15N3]-p53-exon 5-G4,Me2,9,11,14,16 [15N3]-p53-exon 5-G5,Me2,9,11,14,16 (-)-p53-exon 5,Me1,3,7,9,15 [15N3]-p53-exon 7-G4,Me6,16 [15N3]-p53-exon 7-G6,Me6,16 [15N3]-p53-exon 7-G7,Me6,16 [15N3]-p53-exon 7-G8,Me6,16 [15N3]-p53-exon 7-G9,Me6,16 (-)-p53-exon 7,Me9,19 [15N3]-p53-exon 8-G2,Me12 [15N3]-p53-exon 8-G3,Me12 [15N3]-p53-exon 8-G4,Me12 [15N3,13C]-p53-exon 8-G6,Me12 [15N5]-p53-exon 8-G6,Me12 [15N3]-p53-exon 8-G7,Me12 (-)-p53-exon 8,Me9 [15N3,13C]-p53-exon 7-codon245 [15N3,13C]-p53-exon 7-codon245,Me6 (-)-p53-exon 7-codon245 (-)-p53-exon 7-codon245,Me9 [15N3]-p53-exon 7-codon248 [15N3]-p53-exon 7-codon248,Me8 (-)-p53-exon 7-codon248 (-)-p53-exon 7-codon248,Me11 [15N3,13C]-p53-exon 8-G6 (-)-p53-exon 8 a

nucleotide no. 13136-13154 13136-13154 13136-13154 13136-13154 14054-14078 14054-14078 14054-14078 14054-14078 14054-14078 14475-14495 14475-14495 14475-14495 14475-14495 14475-14495 14475-14495 14054-14068 14054-14068 14062-14080 14062-14080 14475-14495

sequence

calcd MWa

CCMeC[15N3-G]GCACCMeCGMeCGTCMeCGMeCG CCMeCGGCACCMeC[15N3-G]MeCGTCMeCGMeCG CCMeCGGCACCMeCGMeC[15N3-G]TCMeCGMeCG CCMeCGGCACCMeCGMeCGTCMeC[15N3-G]MeCG MeCGMeCGGAMeCGMeCGGGTGCMeCGGG ATGGGMeC[15N3-G]GCATGAACMeCGGAGGCCCA ATGGGMeCGGCAT[15N3-G]AACMeCGGAGGCCCA ATGGGMeCGGCATGAACMeC[15N3-G]GAGGCCCA ATGGGMeCGGCATGAACMeCG[15N3-G]AGGCCCA ATGGGMeCGGCATGAACMeCGGA[15N3-G]GCCCA TGGGCCTCMeCGGTTCATGCMeCGCCCAT GCTTT[15N3-G]AGGTGMeCGTGTTTGTG GCTTTGA[15N3-G]GTGMeCGTGTTTGTG GCTTTGAG[15N3-G]TGMeCGTGTTTGTG GCTTTGAGGTGMeC[15N3,13C-G]TGTTTGTG GCTTTGAGGTGMeC[15N5-G]TGTTTGTG GCTTTGAGGTGMeCGT[15N3-G]TTTGTG CAC AAA CAMeC GCA CCT CAA AGC ATGGGC[15N3,13C-G]GCATGAAC ATGGGMeC[15N3,13C-G]GCATGAAC GTTCATGCCGCCCAT GTTCATGCMeCGCCCAT CATGAACC[15N3-G]GAGGCCCATC CATGAACMeC[15N3-G]GAGGCCCATC GATGGGCCTCCGGTTCATG GATGGGCCTCMeCGGTTCATG GCTTTGAGGTGC[15N3,13C-G]TGTTTGTG CAC AAA CAC GCA CCT CAA AGC

5784.7 5784.7 5784.7 5784.7 5981.9 7773.1 7773.1 7773.1 7773.1 7773.1 7614.0 6547.3 6547.3 6547.3 6548.3 6549.3 6547.3 6336.2 4646.1 4660.1 4504.0 4518.0 5785.8 5799.8 5835.8 5849.8 6534.3 6322.2

extinction observed coefficient (260 nm) MWa 5785.3 5785.2 5785.3 5785.2 5982.9 7774.2 7774.4 7774.1 7774.3 7774.1 7612.0 6546.8 5646.8 6546.8 6548.5 6549.4 6546.7 6336.4 4645.4 4659.5 4503.1 4517.4 5786.8 5800.1 5836.3 5850.3 6533.0 6321.6

174 970 174 970 174 970 174 970 199 770 278 169 278 169 278 169 278 169 278 169 236 049 213 343 213 343 213 343 213 343 213 343 213 343 233 016 170 823 171 173 142 328 142 678 202 184 202 534 191 718 192 068 212 993 232 666

Molecular weight.

The identities of HPLC-purified DNA strands were confirmed by HPLC-ESI-MS with an Agilent Technologies capillary HPLCMSD ion trap system (model 1100). The instrument was operated in the negative ion mode, with a mass range of m/z 600-2000 and a target ion abundance of 30 000. ESI was achieved at a spray voltage of +2.6 kV and a source temperature of 200 °C. The nebulizing gas (N2) pressure was set to 15 psi, and the drying gas (N2) flow rate was set at 5 L/min. The measured molecular masses of isotopically labeled DNA strands were within (2 Da of the theoretical values (Table 1). The concentration and purity of each DNA strand were established by HPLC analysis on a Supelcosil LC-18-DB column (2.1 mm × 250 mm, 5 µm, Supelco) eluted with 150 mM ammonium acetate (A) and acetonitrile (B) at a gradient of 5-12% B in 40 min. The DNA was detected by UV absorption at 260 nm. Standard curves constructed with the corresponding unlabeled DNA strands were used for quantification. The DNA was considered sufficiently pure if the impurities in the HPLC traces constituted less than 2% of the total HPLC peak area. To obtain double-stranded DNA, equimolar amounts of the two DNA strands were combined in 10 mM Tris buffer, pH 8.0, containing 50 mM NaCl, heated to 10°C above the melting temperature, and slowly cooled to room temperature. DNA Treatment with BPDE and Isolation of N2-BPDEdG. All experiments were performed in triplicate. Doublestranded DNA (2 nmol) was dissolved in 50 mM Tris-HCl buffer, pH 7.5, to yield a 40 µM concentration. (()-anti-BPDE was dissolved in dry DMSO, and the stock solution concentrations were established by UV spectrophotometry (345 ) 48 800). The BPDE solution was added to DNA at a 1:5 molar ratio [BPDE]: [DNA], and the reaction mixtures were incubated on ice for 18 h. The final concentration of (()-anti-BPDE was 8 µM. The DMSO percentage was 10% of the total volume for all treatments. The reaction mixtures were evaporated to dryness under reduced pressure. BPDE-treated DNA was dissolved in 10 mM Tris-HCl/15 mM MgCl2 buffer, pH 7. Enzymatic digestion of DNA to 2′-deoxyribonucleosides was achieved by incubating it with DNAse I (35 U), PDE I (105 mU), PDE II (120 mU), and alkaline phosphatase (22 U) for 18 h at 37 °C as previously described (33). To confirm the completeness of enzymatic

hydrolysis, small aliquots were removed and analyzed by HPLC with an Extend-C18 column (4.6 mm × 150 mm, 5 µm, Aglient Technologies) eluted at a flow rate of 1 mL/min. The mobile phase consisted of 150 mM ammonium acetate (A) and acetonitrile (B) eluted at the following gradient: 0-2 min, 0% B; 21.5 min, 4.5% B; 24.5-30 min, 30% B; 33 min, 0% B. The UV absorbance was monitored at 260 (UV maximum for DNA) and 345 nm (UV maximum for the BPDE chromophore) (33). Digestion was considered complete when the only UV absorbing peaks at 345 nm coeluted with N2-BPDE-dG isomers (29-30 min) and BPDE tetraol resulting from the hydrolytic decomposition of BPDE (30.7 min). N2-BPDE-dG was purified by SPE using 50 mg of C18 SPE cartridges (Waters Associates). N2BPDE-dG eluted in the 100% methanol fraction. SPE fractions containing N2-BPDE-dG were concentrated under vacuum and analyzed by HPLC-ESI-MS/MS as described below. HPLC-ESI-MS/MS. A 7000 Finnigan TSQ 7000 mass spectrometer (ThermoQuest, Palo Alto, CA) interfaced with an 1100 Agilent Technologies capillary HPLC system was used for these studies. Chromatographic separation of N2-BPDE-dG diastereomers was achieved with an Extend-C18 column (150 mm × 0.5 mm, 3.5 µm, Aglient Technologies) eluted at a flow rate of 10 µL/min and a temperature of 1 °C. The HPLC solvents were 33% methanol in 15 mM ammonium acetate (A) and 25% buffer A in acetonitrile (B), with a gradient of 13-15% B in 24.5 min, followed by a further increase to 30% B at 40 min. BPDEdG stereoisomers eluted as follows: (-)-trans-BPDE-dG (tR, 39.7 min), (+)-cis-BPDE-dG (tR, 40.4 min), (-)-cis-BPDE-dG (tR, 41.3 min), and (+)-trans-BPDE-dG (tR, 42.1 min). The mass spectrometer was operated in the positive ion mode. Nitrogen (40 psi) was used as a nebulizing and drying gas. Electrospray ionization was typically achieved at a spray voltage of 4.5 kV, and the temperature of the heated capillary was 220 °C. Quantitative analysis of N2-BPDE-dG was performed in SRM mode. The first quadrupole was set to isolate the protonated molecules ([M + H]+) of N2-BPDE-dG and 15N-N2-BPDE-dG (m/z ) 570.1 for N2-BPDE-dG, m/z ) 573.1 for 15N3-N2-BPDE-dG, m/z ) 574.1 for 15N3,13C1-N2-BPDE-dG, and m/z ) 575.1 for 15N5N2-BPDE-dG). Collision-induced dissociation was performed at an energy of 14 V and a collision gas pressure (Ar) of 2 mTorr.



Figure 2. HPLC-ESI-MS/MS analysis of N2-BPDE-dG adducts following the treatment of an [15N3]-labeled DNA duplex (5′-CCMeCGGCACCMeCGMeC[15N3]GTCMeCGMeCG + complementary strand) with (()-anti-BPDE (8 µM): N2-BPDE-dG standards (A), adducts originating from 15N3-labeled G (C), and lesions produced elsewhere in the sequence (B). Quantitative analyses were performed using the MS/MS transitions: N2-BPDE-dG, m/z 570.1 f m/z 454.1 (A,B); [15N3]-N2-BPDE-dG, m/z 573.1 f m/z 457.1 (C). An Aglient 1100 series capillary HPLC was interfaced to a Finnigan MAT TSQ 7000 triple quadrupole mass spectrometer (ThermoQuest, San Jose, CA) operated in the ESI+ mode. Chromatography was performed on an Extend-C18 column (150 mm × 0.5 mm, 3.5 µm) eluted at a flow rate of 10 µL/min with a gradient of acetonitrile/methanol/15 mM ammonium acetate and at a temperature of 1 °C. The mass spectrometer was operated at a spray voltage of 4.5 kV, a heated capillary temperature at 220 °C, and an electron multiplier setting at 2200 V. MS/MS data were generated with Ar as the collision gas (2 mTorr). The third quadrupole was set to the masses of [M-dR + 2H]+ (m/z ) 454.1 for N2-BPDE-dG, m/z ) 457.1 for 15N3-N2-BPDEdG, m/z ) 458.1 for 15N3,13C1-N2-BPDE-dG, and m/z ) 459.1 for 15N -N2-BPDE-dG). The scan time was 1 s, and the isolation 5 width was 0.7 amu. The instrument was tuned to maximum sensitivity by directly infusing the N2-BPDE-dG standard. The extent of N2-BPDE-dG formation at the 15N-labeled guanine (X) was calculated for each stereoisomer from the following equation:

to maintain an overall level of significance at 0.05, p values were adjusted using the Bonferroni method. A similar procedure was followed to compare the contributions of individual diastereomers N2-BPDE-dG to adduct formation at different sites within DNA sequences. The association between the number of adducts and the number of mutations was tested by calculating Pearson correlation coefficients.

% reaction at X ) ABPDE-dX/(ABPDE-dX + ABPDE-dG)

Targeted binding of PAH diol epoxides to the endogenously methylated CG dinucleotides within the coding sequence of the p53 tumor suppressor gene has been proposed to be responsible for the mutational hot spots at p53 codons 157, 245, 248, and 273 in smoking-induced lung cancer (30, 32). Studies based on UvrABC endonuclease incision assays suggest that PAH diol epoxides preferentially form guanine adducts at these sites (30, 32, 38, 39). However, an important limitation of gel electrophoresis-based techniques is that the structures and stereochemistry of N2-BPDE-dG adducts cannot be determined. In the present study, the formation of diastereomeric N2-BPDE-dG lesions within doublestranded oligodeoxynucleotides representing p53 lung cancer mutational hot spots is analyzed by a new mass spectrometry-based approach. Our ability to quantify individual N2-BPDE-dG diastereomers originating from specific guanine bases following (()-anti-BPDE treatment has enabled us to directly analyze the effects of the local sequence context and neighboring MeC on the stereochemical composition of N2-BPDE-dG adducts. Our experimental strategy was based on the recently described stable isotope-labeling HPLC-MS/MS approach

where ABPDE-dG and ABPDE-dX are the areas under the HPLCESI-MS/MS peaks corresponding to the unlabeled and [15N]labeled N2-BPDE-dG diastereomers, respectively. Statistical Analyses of the Data. All statistical analyses were performed at the Biostatistics Core of the University of Minnesota Cancer Center. ANOVA (37) was used to investigate the simultaneous effects of sequence on the total reactivity and on the contributions of individual stereoisomers to total adduct numbers at a given site. F-tests were conducted to examine the effects of sequence on the formation of each individual N2-BPDEdG stereoisomer and to compare the stereochemical compositions of N2-BPDE-dG originating from different sites within the same duplex. Subsequent pairwise differences were tested using posthoc t-tests. The reactivities of individual guanines were compared by t-test using the formula (µ1 - µ2)/[MSE(1/n1 + 1/n2)]1/2, where µ is the mean reactivity at positions 1 and 2, respectively, and MSE is the mean squared error from the ANOVA analysis. To compare the reactivity at a specified guanine nucleobase with the theoretical “random” reactivity value, the following formula was used (µi - c)/[MSE(1/ni)]1/2, where µi is the mean reactivity at position i, c is the theoretical reactivity value, and MSE is the mean squared error from ANOVA. To adjust for the number of multiple comparisons and

Results and Discussion

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Figure 3. Relative formation of N2-BPDE-dG at guanine nucleobases within a double-stranded DNA sequence derived from p53 exon 5: 5′-CCMeCGGCACCMeCGMeCGTCMeCGMeCG (blue bars) and the frequency of G f T transversions in human lung cancer obtained from the AACR p53 mutation database (black bars). The adduct formation data were compiled from two separate stable isotope-labeling HPLC-MS/MS experiments (N ) 3-6). The percent of reaction at each guanine was calculated from the area ratio of the 15N-labeled HPLC-ESI-MS/MS peak to the sum of unlabeled and 15N-labeled adduct peak areas (% reaction at X ) ABPDE-dX/ (ABPDE-dX + ABPDE-dG). The random percent reaction value was calculated from the total number of guanine nucleobases in both DNA strands. Insert: pie charts illustrating the relative contributions of (-)-trans-N2-BPDE-dG, (+)-cis-N2-BPDE-dG, (-)-cis-N2BPDE-dG, and (+)-trans-N2-BPDE-dG to the total N2-BPDE-dG adduct number at each guanine.

(33, 40). DNA duplexes were constructed representing the frequently mutated regions of p53 exons 5, 7, and 8 containing lung cancer hot spots at codons 157, 158, 245, 248, 249, and 273 (Figure 1). The formation of N2-BPDEdG diastereomers at a given guanine was investigated by inserting an 15N-labeled guanine at that site (Table 1). For each target sequence, a series of DNA oligomers was generated in which one of the guanine bases was replaced with 15N-Gua (Table 1). The presence of 15N does not affect the reactivity of the guanine nucleobase toward electrophiles but rather serves as an isotope “tag” to enable quantification of the lesions originating from that position (33). Following annealing to the complementary strand, treatment with (()-anti-BPDE, and enzymatic digestion to deoxynucleosides, HPLC-ESI-MS/MS was used to establish the extent of adduct formation at the 15 N-labeled guanine (Figure 2) (33). By preparing several oligomers of the same sequence but with different label positions (Table 1), the pattern of adduct formation within each sequence of interest was determined (33). Our previous experiments employing stable isotopelabeling methodology demonstrate that the oligodeoxynucleotides selected for our studies (Table 1) do not exhibit any significant “end effects” as a result of local duplex melting at the ends of the sequence (33, 40, 41). Our optimized HPLC-ESI-MS/MS method allows a baseline separation of (+)-trans, (-)-trans, (+)-cis, and (-)-cis diastereomers of N2-BPDE-dG to enable their

individual quantification (Figure 2A). The retention times of the authentic (-)-trans-, (+)-cis-, (-)-cis-, and (+)trans-N2-BPDE-dG under our HPLC conditions were tr ) 39.7, 40.4, 41.3, and 42.1 min, respectively (Figure 2A). Authentic standards of N2-BPDE-dG diastereomers were prepared independently as described in the Materials and Methods, and their chirality was established from the induced CD spectra (Appendix S-1 in the Supporting Information) (42). A representative HPLC-ESI+-MS/MS trace corresponding to 15N3-N2-BPDE-dG and N2-BPDE-dG originating from an 15N3-dG-containing DNA duplex following (()anti-BPDE treatment is shown in Figure 2B,C. HPLCESI-MS/MS quantification of N2-BPDE-dG is based on the loss of deoxyribose (M ) 116) from the protonated molecules of the adducted nucleoside (m/z ) 570.1, M + H) under collision-induced dissociation conditions, leading to the predominant fragment at m/z ) 454.1 (MS/ MS transition: m/z ) 570.1 [M + H]+ f 454.1 [M + H Gua]+) (Figure 2B) (33). Adducts originating from 15N3dG contain the 15N3 isotope tag and thus undergo a (+3) mass shift (m/z ) 573.1 [M + H]+ f 457.1 [M + H Gua]+) (see Figure 2C). The extent of adduct formation at 15N3-dG can be calculated directly from the ratios of the areas under the HPLC-ESI-MS/MS peaks corresponding to 15N3-labeled and unlabeled N2-BPDE-dG adducts, respectively: % reaction at X ) ABPDE-dX/ (ABPDE-dX + ABPDE-dG). A similar procedure is followed for



Figure 4. Relative formation of N2-BPDE-dG diastereomers at guanine nucleobases within a double-stranded DNA sequence derived from p53 exon 7: 5′-ATGGGMeCGGCATGAACMeCGGAGGCCCA (blue bars) and the corresponding frequency of G f T transversions in human lung cancer obtained from the AACR p53 mutation database (black bars). The adduct formation data were compiled from two separate stable isotope-labeling HPLC-MS/MS experiments (N ) 3-6). The percent (() of BPDE reaction at each guanine was calculated from the area ratio of the 15N-labeled HPLC-ESI-MS/MS peak to the sum of unlabeled and 15N-labeled adduct peak areas (% reaction at X ) ABPDE-dX/(ABPDE-dX + ABPDE-dG). The random reaction value was calculated from the total number of guanine nucleobases in both DNA strands. Insert: pie charts illustrating the relative contributions of (-)-trans-N2-BPDE-dG, (+)-cis-N2BPDE-dG, (-)-cis-N2-BPDE-dG, and (+)-trans-N2-BPDE-dG to the total adduct number at each guanine.

oligomers containing 15N3-dG at different sites, making it possible to map the patterns of adduct formation along each DNA duplex (33). Distribution of N2-BPDE-dG Diastereomers in p53 Exon 5-Derived DNA Sequence (Figure 3). Mutations at p53 codons 157 and 158 (GTC f TGC and CGC f CTC, respectively) are characteristic for smokinginduced lung cancer (43) but are rare in other cancer types, suggesting that these genetic changes may result from exposure to tobacco carcinogens (4) (Figure 1). To compare the relative reactivity of guanine bases in a region of the p53 gene containing codons 157 and 158 toward (()-BPDE, a series of double-stranded oligodeoxynucleotides [5′-CCMeCG1G2CACCMeCG3MeCG4TCMe CG5MeCG6 (+ strand)] was prepared containing MeC at all five physiologically methylated 5′-CG sites (20) (G1, G3, G4, G5, and G6, Table 1). 15N3-dG was introduced at one of the highlighted positions (G1, G3, G4, or G5, see Table 1), and the extent of N2-BPDE-dG formation at each G was established by HPLC-ESI+-MS/MS as described above (33). In the absence of any sequence effects, the extent of N2-BPDE-dG adducts formation at each guanine of the p53 exon 5-derived duplex should be 100%/17 ) 5.88% (17 is the total number of guanine bases in this duplex). However, the observed BPDE adduction patterns are not uniform, with the abundance of N2-BPDE-dG adducts

following the following order: G3 (MeCG3MeC) . G4 (MeCG4T) > G5 (MeCG5MeC) > G1 (MeCG1G) (Figure 3, blue bars). The number of adducts produced at G3 (codon 156) is 3.3× greater than the theoretical (random) value, and G4 (codon 157) is 1.7-fold more reactive than expected for a random distribution of N2-BPDE-dG adducts (p < 0.001). G5 (codon 158) exhibits an average reactivity (6.2%, p > 0.66), while the reactivity of G1 (codon 154) toward BPDE is less than the random value (3.9%, p < 0.001) (Figure 3). These variations in BPDE adduct yields cannot be explained solely by the nearest neighbor effects, as G3 and G5 are both flanked by methylated cytosines (MeC) but give rise to different amounts of N2-BPDE-dG adducts (Figure 3). These results (Figure 3) are consistent with our earlier study (33) in which the total numbers of N2-BPDE-dG lesions were analyzed. All four guanines investigated (G1, G3, G4, and G5) are preceded by MeC and are fairly reactive toward BPDE (Figure 3). However, the site of the greatest adduct formation (G3 in p53 codon 156) does not coincide with the lung cancer mutational hot spots in codons 157 and 158 (G4 and G5, black bars in Figure 3). This suggests that factors other than preferential N2BPDE-dG adduct formation are responsible for the high mutational frequency at p53 codons 157 and 158 (G4 and G5 in our numbering system). BPDE adducts formed at these sites may be slowly repaired or are more efficiently

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Figure 5. Relative formation of N2-BPDE-dG diastereomers at guanine nucleobases within a double-stranded DNA sequence derived from p53 exon 8: 5′-GCTTTGAGGTGMeCGTGTTTGTG (blue bars) and the frequency of G f T transversions in human lung cancer obtained from the AACR p53 mutation database (black bars). The adduct formation data were compiled from two separate stable isotope-labeling HPLC-MS/MS experiments (N ) 6). The percent of reaction at each guanine was calculated from the area ratio of the 15N-labeled HPLC-ESI-MS/MS peak to the sum of unlabeled and 15N-labeled adduct peak areas (% reaction at X ) ABPDE-dX/ (ABPDE-dX + ABPDE-dG). The random reaction value was calculated from the total number of guanine nucleobases in both DNA strands. Insert: pie charts illustrating the relative contributions of (-)-trans-N2-BPDE-dG, (+)-cis-N2-BPDE-dG, (-)-cis-N2-BPDE-dG, and (+)-trans-N2-BPDE-dG to the total adduct number at a given guanine.

bypassed by mammalian DNA polymerases than the lesions originating from codon 156 (G3). Alternatively, DNA modifications by other components of tobacco smoke or selection processes may be responsible for these mutations. Distribution of N2-BPDE-dG Diastereomers in p53 Exon 7-Derived DNA Sequence (Figure 4). The p53 exon 7 contains prominent lung cancer mutational hot spots at codons 245 (GGC f TGC), 248 (CGG f CTG), and 249 (AGG f ATG) (Figure 1). Unlike other cancer types, which exhibit primarily G f A transitions at these sites, G f T transversions are characteristic for smoking-associated lung tumors (3). A series of doublestranded oligodeoxynucleotides (5′-ATG1G2G3MeCG4G5CATG6AACMeCG7G8AG9G10CCCA) was used to examine the formation of N2-BPDE-dG diastereomers along a region of the p53 exon 7 including codons 243-250 (Table 1). MeC bases were inserted in both strands of the two endogenously methylated CG sites, MeCG4 and MeCG7. 15N -dG was placed at either G , G , G , G , or G of the 3 4 6 7 8 9 above sequence (Table 1), and the extent of N2-BPDEdG formation at each site was established by HPLC-MS/ MS as described above (33). The exon 7-derived DNA duplex contains a total of 17 guanines (Table 1); if each of them reacted with (()-antiBPDE to the same extent, an individual G would give rise to 5.88% of the total N2-BPDE-dG adducts. However, the actual N2-BPDE-dG yields vary ∼12-fold between the

sites of highest and lowest reactivity (Figure 4, blue bars). The majority of N2-BPDE-dG adducts originate from G7 (MeCG7G, codon 248), followed by G4 (MeCG4G, codon 245), G8 (GG8A, codon 248), G9 (AG9G, codon 249), and G6 (TG6A, codon 246). The number of adducts originating from G7 (the first G of p53 codon 248) is 2.7× times greater than the random value (p < 0.001). The second most reactive site is G4 in codon 245 (1.54-fold more adducts than expected from random distribution, p < 0.01). In contrast, the reactivities of G6 and G9 are below the statistical value (p < 0.002). Hence, the most frequently adducted positions in the p53 exon 7-derived sequence (G7 and G4) coincide with known p53 mutational hot spots (codons 248 and 245, respectively). Both of these sites are located within endogenously methylated CG dinucleotides (Table 1 and Figure 4). Distribution of N2-BPDE-dG Diastereomers in p53 Exon 8-Derived DNA Sequence (Figure 5). Another important lung cancer mutational hot spot is codon 273 located within p53 exon 8 (CGT f CTT) (Figure 1) (44). We examined the distribution of diastereomeric N2-BPDE-dG adducts within a DNA duplex containing p53 codon 273 and the surrounding sequence, 5′-G1CTTTG2AG3G4TG5MeCG6TG7TTTG8TG9 (Table 1 and Figure 5). Because this duplex contains a total of 11 guanines, sequence-independent alkylation would result in 9.09% N2-BPDE-dG formation at each G (100%/11 ) 9.09%). Stable isotope-labeling HPLC-MS/MS was em-



Figure 6. Effect of neighboring MeC on the formation of N2-BPDE-dG adducts (G) within p53 codon 245 in a double-stranded DNA sequence derived from p53 exon 7 (5′-ATGGGX[15N3,13C]GGCATGAAC (X ) C or MeC and X[15N3,13C]GG ) codon 245) as determined by stable isotope labeling. Samples were analyzed in triplicate. Insert: pie charts illustrating the relative contributions of (-)-transN2-BPDE-dG, (+)-cis-N2-BPDE-dG, (-)-cis-N2-BPDE-dG, and (+)-trans-N2-BPDE-dG to the total adduct number as a function of cytosine methylation.

ployed to quantify the formation of N2-BPDE-dG at G2, G3, G4, G6, and G7 (Table 1). We found that N2-BPDEdG adducts were overproduced at G6 within the endogenously methylated CG dinucleotide (MeCG6T, codon 273) (Figure 5, blue bars). Adduct yields at G6 (31.3%) were 15-30-fold greater than at the other positions examined (Figure 5). Therefore, the site of an increased N2-BPDEdG adduct formation (G6 in p53 codon 273) coincides with the known mutational hot spot for G f T transversions in smoking-related lung cancer (Figure 1). Statistical analyses indicate a significant association between the number of N2-BPDE-dG adducts and the G f T mutational frequency at a given G within p53 exon 8-derived DNA sequence (p < 0.002, Pearson correlation coefficient ) 0.987). Relative Contributions of N2-BPDE-dG Diastereomers to the Total Adduct Number. The stereochemical compositions of N2-BPDE-dG adducts originating from each guanine examined are shown as pie chart graphs in Figures 3-5. While (+)-trans-N2-BPDE-dG is the predominant isomer in all cases (70.8-92.9% of the total), the contributions of the (-)-trans isomer vary between 5.6 and 16.7%, the (-)-cis isomer accounts for 2.1-8.5% of total adducts, and the (+)-cis isomer is responsible for only 0.5-8.3% of the total number of lesions (pie charts in Figures 3-5). The molar ratios of N2-BPDE-dG diastereomers are dependent on the local sequence context. In general, the preferred sites for N2BPDE-dG adduct formation contain the highest share of (+)-trans adducts [e.g., G3 and G4 in exon 5 (Figure 3), G4 in exon 7 (Figure 4), and G6 in exon 8 sequence (Figure 5)]. In contrast, cis adduct formation appears to be more common at the least frequently modified guanines, e.g., G1 and G5 in the exon 5-derived sequence (Figure 3), G6 and G9 in the exon 7-derived sequence (Figure 4), and G2 and G7 in the exon 8-derived sequence (Figure 5). Statistical analyses with pairwise t-tests indicate that the contributions of (-)-trans, (+)-cis, and (-)-cis isomers

to the total adduct number at G6 within the p53 exon 246 are significally greater than at other p53 sequences (p < 0.001, Figure 4). The relative amounts of (+)-cisN2-BPDE-dG are elevated at G7 within the p53 exon 8-derived sequence (8.3% of the total adduct number vs 1-4% at other Gs, p < 0.001, Figure 5). While the exact mechanisms of sequence-mediated stereoselectivity for N2-BPDE-dG adduct formation are unknown, the local sequence environment may modulate the relative yields of N2-BPDE-dG diastereomers by favoring a particular geometry of the precovalent complex between BPDE and its DNA target. Effects of Neighboring MeC on the Formation of 2 N -BPDE-dG Diastereomers in p53 Context (Figure 6). MeC is an endogenous DNA modification present at all CG dinucleotides within p53 exons 5-8 (20). The presence of neighboring MeC has been shown to stimulate guanine reactions with carcinogens and drugs, e.g., mitomycin C (45), esperamicins (46), and BPDE (32, 33), while guanine modifications with N-methyl-N-nitrosourea, bleomycin, methylating agents, and NNK diazohydroxides are inhibited by cytosine methylation (47-49). Our results shown in Figures 3-5 indicate that the yields of N2-BPDE-dG adducts are increased at endogenously methylated CG sequences (e.g., p53 codons 156, 157, 245, 248, and 273). However, because each methylated CG dinucleotide contains two MeC nucleobases (one in each DNA strand), both of which may influence the reactivity of target guanines toward BPDE, questions remain about the mechanism of this effect. To directly analyze the effects of 5′-neighboring and base-paired MeC on the total N2-BPDE-dG adduct yields and their stereochemical composition, 15N3-labeled guanine bases were placed within hemimethylated and fully methylated CG dinucleotides representing p53 codons 245, 248, and 273 (Table 1 and Figure 6). The relative yields of N2-BPDE-dG at the 15N3-labeled position in the presence and in the absence of neighboring MeC were

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established by HPLC-ESI-MS/MS as described above. For the p53 codon 245-containing sequence, the formation of all four N2-BPDE-dG diastereomers was stimulated by the presence of a 5-methyl group at the 5′-flanking C and at the cytosine in the opposite strand from the target G (p < 0.001, Figure 6). A similar stimulating effect of the 5′-neighboring and base-paired MeC was observed for p53 codons 248 and 273 (25-40% increase in adduct yields; see Appendix S-3 in the Supporting Information). The enhancing effect of cytosine methylation on the formation of N2-BPDE-N2-BPDE-dG adducts is likely to involve favorable hydrophobic interactions between methylated CG dinucleotides and PAH diol epoxides (50). In general, BPDE reactivity patterns analyzed by stable isotope-labeling HPLC-MS/MS in the present work are consistent with those previously determined by the endonuclease incision assay (30). The use of short DNA sequences as models for nuclear DNA in our study is justified by the evidence that chromatin structure has little effect on the distribution of DNA damage induced by BPDE and methylating agents (32, 51). In comparison with gel electrophoresis-based techniques, our methodology affords additional details about the structural and stereochemical identities of the observed BPDE-guanine lesions. We demonstrate that while the molar ratios of N2-BPDE-dG diastereomers are moderately affected by the local sequence context, the formation of all four N2BPDE-dG adducts is stimulated by the presence of neighboring MeC. The preferential formation of N2-BPDEdG adducts at MeCG dinucleotides within the p53 gene (e.g., codons 245, 248, and 273) is consistent with the high prevalence of G f T transversions at these sites.

Acknowledgment. We thank Prof. Nicholas E. Geacintov (New York University) for helpful discussions, Dr. Peter Villalta (University of Minnesota) for assistance with the TSQ mass spectrometer, and Gregory Janis for editorial help. All statistical analyses were performed by Robin Bliss and Shelby Li at the University of Minnesota Cancer Center Biostatistics Core. Funding for this research was from the National Cancer Institute (CA095039).

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Supporting Information Available: Induced CD spectra of N2-BPDE-dG diastereomers recorded in 33% methanol/water. Ultraviolet spectra of N2-BPDE-dG diastereomers recorded in 33% methanol/water. Effect of neighboring MeC on the formation of N2-BPDE-dG adducts at the neighboring guanine (G) within p53 codon 248 as determined by stable isotope labeling. This material is available free of charge via the Internet at http:// pubs.acs.org.

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TX049974L

Formation of Diastereomeric Benzo [a] pyrene Diol Epoxide-Guanine

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