Formation of Diastereomeric Benzo[a]pyrene Diol Epoxide-Guanine Adducts inp53Gene-Derived DNA Sequences

Abstract

G --> 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 (ligation-mediated 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 N(2)-BPDE-dG lesions within double-stranded oligodeoxynucleotides representing p53 lung cancer mutational hotspots and their surrounding DNA sequences. (15)N-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-N(2)-BPDE-dG, (+)-cis-N(2)-BPDE-dG, (-)-cis-N(2)-BPDE-dG, and (+)-trans-N(2)-BPDE-dG originating from the (15)N-labeled bases. We found that all four N(2)-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-N(2)-BPDE-dG, 5.6 and 16.7% for (-)-trans-N(2)-BPDE-dG, 2.1 and 8.5% for (-)-cis-N(2)-BPDE-dG, and 0.5 and 8.3% for (+)-cis-N(2)-BPDE-dG. The relative yields of the minor N(2)-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 N(2)-BPDE-dG diastereomers, probably a result of favorable hydrophobic interactions between BPDE and 5-methylcytosine. The targeted formation of N(2)-BPDE-dG at (Me)CG dinucleotides within the p53 gene is consistent with the high prevalence of G --> T transversions at these sites in smoking-induced lung cancer.

Ultra-performance liquid chromatography–tandem mass spectrometry for rapid and highly sensitive analysis of stereoisomers of benzo[a]pyrene diol epoxide–DNA adducts

“…This transition was used due to the observation that the major product ion of the adducted base, anti-BPDE-N 2 guanine, could be formed following ESI-MS/MS CID analysis. The observation is consistent with previous works [21,22]. Excellent linearity for each stereoisomer was achieved with a linear regression coefficient of R 2 ≥ 0.99 (Table 1).…”

“…Their structure and stereochemistry were identified and characterized by a combination of HPLC-Diode Array Detector (DAD)-MS/MS (Waters, MA, USA), UV spectroscopy (Hitachi, Tokyo, Japan), and circular dichroism (CD) spectroscopy (Jasco, Tokyo, Japan). The concentration of each purified anti-BPDE-N 2 dG stereoisomer was calculated by determining absorbance at 345 nm from triplicate measurements and reported maximum absorbance coefficient (ε 345 = 3.43 × 10 4 L/mol cm) [21].…”

“…A number of methods have been developed for detection of BPDE-DNA adducts, including capillary electrophoresis (CE)-laserinduced fluorescence (LIF) [13][14][15][16], accelerator mass spectrometry [17,18], liquid chromatography or capillary electrophoresis-mass spectrometry [19][20][21][22][23][24][25][26], and 32 P-postlabelling [27]. However, only few methods demonstrate the capability of resolving the stereoisomers of BPDE-DNA adducts.…”

“…The method development for stereoisomeric analysis of BPDE-DNA adducts takes multiple steps and labor-intensive. It often involves with the preparation of single stereoisomers of adducted nucleosides or nucleotides (which are not commercially available), and the structural and stereochemical characterization, enzymatic digestion of genomic DNA, and highly efficient and stereoselective separation and coupled sensitive detection [10,21,28]. Among these steps, the stereoselective separation of the adduct stereoisomers is most critical and tunable factor for achieving high sensitivity of detection.…”

“…m/z 570 → 454) can preclude the presence of BPDE tetrols and other interference substance, thus, only four stereoisomers of the adducted dG are needed to be resolved regardless of the other components. Because of the improved detection selectivity, it takes shorter time (20-50 min) for the stereoslective separation [21,22,30]. Due to large sample consumption (30-300 g DNA), the LC-MS based methods are often used for identification of new DNA damage and analysis of DNA adducts from cultured cells or animal tissues rather than human biomonitoring [19,31,32].…”