Guanosine labeled with 15N at N1, amino, and N7 and 13C at either C2 or C8 was oxidized by Rose Bengal photosensitization (singlet oxygen) in buffered aqueous solution. At pH > 7, spiroiminodihydantoin was the major product, while at pH < 7, guanidinohydantoin (Gh) was the principal product. 15N and 13C NMR studies confirmed that Gh was formed as a mixture of slowly equilibrating diastereomers. Experiments conducted in H218O indicated that Gh and Sp each contained one oxygen atom derived from O2 and one from H2O. Tandem mass spectrometry was used to identify the C4 carbonyl of Gh as the one labeled with 18O, supporting a mechanism involving attack of water at C5 of a dehydro-8-oxoguanosine intermediate.

Alkylating agents that react through highly electrophilic quinone methide intermediates often express a specificity for the weakly nucleophilic exocyclic amines of deoxyguanosine (dG N(2)) and deoxyadenosine (dA N(6)) in DNA. Investigations now indicate that the most nucleophilic site of dA (N1) preferentially, but reversibly, conjugates to a model ortho-quinone methide. Ultimately, the thermodynamically stable dA N(6) isomer accumulates by trapping the quinone methide that is transiently regenerated from collapse of the dA N1 adduct. Alternative conversions of the dA N1 to the dA N(6) derivative by a Dimroth rearrangement or other intramolecular processes are not competitive under neutral conditions, as demonstrated by studies with [6-(15)N]-dA. Both a model quinone methide precursor and its dA N1 adduct yield a similar profile of deoxynucleoside products when treated with an equimolar mixture of dC, dA, dG, and T. Consequently, the most readily observed products of DNA modification resulting from reversible reactions may reflect thermodynamic rather than kinetic selectivity.

The mutagenicity of a prominent tobacco carcinogen, benzo[a]pyrene (B[a]P), is believed to result from chemical reactions between its diol epoxide metabolite, (+)-anti-7r,8t-dihydroxy-c9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), and DNA, producing promutagenic lesions, e.g., (+)-trans-anti-7R,8S,9S-trihydroxy-10S-(N(2)-deoxyguanosyl)-7,8,9,10-tetrahydrobenzo[a]pyrene (N(2)-BPDE-dG). Previous studies used the DNA repair enzyme UvrABC endonuclease in combination with ligation-mediated PCR (LMPCR) to demonstrate an increased reactivity of BPDE toward guanine nucleobases within codons 157, 248, and 273 of the p53 tumor suppressor gene (Denissenko, M. F., Pao, A., Tang, M., and Pfeifer, G. P. Science 274, 430-432). These sites are also "hot spots" for mutations observed in lung tumors of smokers, suggesting an involvement of B[a]P in the initiation of lung cancer. However, the LMPCR approach relies on the ability of the repair enzyme to excise BPDE-induced lesions, and thus the slowly repaired lesions may escape detection. Furthermore, BPDE-DNA adduct structure and stereochemistry cannot be determined. In the present work, we performed a direct quantitative analysis of N(2)-BPDE-dG originating from specific guanine nucleobases within p53- and K-ras-derived DNA sequences by using a stable isotope labeling-mass spectrometry approach recently developed in our laboratory. (15)N-labeled dG was placed at defined positions within DNA sequences derived from the K-ras proto-oncogene and p53 tumor suppressor gene, the two genes most frequently mutated in smoking-induced lung cancer. (15)N-labeled DNA was annealed to the complementary strands, followed by BPDE treatment and liquid chromatography-electrospray ionization tandem mass spectrometry analysis (HPLC-ESI-MS/MS) of N(2)-BPDE-dG lesions. The extent of adduct formation at (15)N-labeled guanine was determined directly from the HPLC-ESI-MS/MS peak area ratios of (15)N-N(2)-BPDE-dG and N(2)-BPDE-dG. BPDE-induced guanine adducts were produced nonrandomly along K-ras and p53 gene-derived DNA sequences, with over 5-fold differences in adduct formation depending on sequence context. N(2)-BPDE-dG yield was enhanced by the presence of 5-Me substituent at the cytosine base-paired with the target guanine nucleobase, an endogenous DNA modification characteristic for CpG dinucleotides within the p53 gene. In the K-ras-derived DNA sequence, the majority of N(2)-BPDE-dG adducts originated from the first position of the codon 12 (GGT), consistent with the large number of G --> T transversions observed at this nucleotide in smoking-induced lung cancer. On the contrary, the pattern of N(2)-BPDE-dG formation within the p53 exon 5 sequences did not correlate with the mutational spectrum in lung cancer, suggesting that factors other than N(2)-BPDE-dG formation are responsible for these mutations. The stable isotope labeling HPLC-ESI-MS/MS approach described in this work is universally applicable to studies of modifications to isolated DNA by other carcinogens and alkylating drugs.