Formation of Diastereomeric Benzo[a]pyrene Diol Epoxide-Guanine Adducts in p53 Gene-Derived DNA Sequences
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- 作者：Brock Matter ; Gang Wang ; Roger Jones ; and Natalia Tretyakova
- 刊名：Chemical Research in Toxicology
- 出版年：2004
- 出版时间：June 2004
- 年：2004
- 卷：17
- 期：6
- 页码：731 - 741
- 全文大小：323K
- 年卷期：v.17,no.6(June 2004)
- ISSN：1520-5010
文摘
G 
T transversion mutations in the p53 tumor suppressor gene are characteristic ofsmoking-related lung tumors, suggesting that these genetic changes may result from exposureto tobacco carcinogens. It has been previously demonstrated that the diol epoxide metabolitesof bay region polycyclic aromatic hydrocarbons present in tobacco smoke, e.g., benzo[a]pyrenediol epoxide (BPDE), preferentially bind to the most frequently mutated guanine nucleotideswithin 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 incisionassay) cannot establish the chemical structures and stereochemical identities of BPDE-guaninelesions. 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] toanalyze the formation of diastereomeric N2-BPDE-dG lesions within double-stranded oligodeoxynucleotides representing p53 lung cancer mutational hotspots and their surrounding DNAsequences. 15N-labeled dG was placed at defined positions within DNA duplexes containing5-methylcytosine at all physiologically methylated sites, followed by (±)-anti-BPDE treatmentand 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. Wefound that all four N2-BPDE-dG diastereomers were formed preferentially at the methylatedCG 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 variedbetween 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 relativeyields of the minor N2-BPDE-dG stereoisomers were elevated at the sites of inefficientadduction, while the major (+)-trans-BPDE lesion was even more dominant at the frequentlyadducted sites. The introduction of 5-methyl groups at adjacent cytosine bases increased theyields of N2-BPDE-dG diastereomers, probably a result of favorable hydrophobic interactionsbetween BPDE and 5-methylcytosine. The targeted formation of N2-BPDE-dG at MeCGdinucleotides within the p53 gene is consistent with the high prevalence of G T transversionsat these sites in smoking-induced lung cancer.
T transversion mutations in the p53 tumor suppressor gene are characteristic ofsmoking-related lung tumors, suggesting that these genetic changes may result from exposureto tobacco carcinogens. It has been previously demonstrated that the diol epoxide metabolitesof bay region polycyclic aromatic hydrocarbons present in tobacco smoke, e.g., benzo[a]pyrenediol epoxide (BPDE), preferentially bind to the most frequently mutated guanine nucleotideswithin 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 incisionassay) cannot establish the chemical structures and stereochemical identities of BPDE-guaninelesions. 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] toanalyze the formation of diastereomeric N2-BPDE-dG lesions within double-stranded oligodeoxynucleotides representing p53 lung cancer mutational hotspots and their surrounding DNAsequences. 15N-labeled dG was placed at defined positions within DNA duplexes containing5-methylcytosine at all physiologically methylated sites, followed by (±)-anti-BPDE treatmentand 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. Wefound that all four N2-BPDE-dG diastereomers were formed preferentially at the methylatedCG 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 variedbetween 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 relativeyields of the minor N2-BPDE-dG stereoisomers were elevated at the sites of inefficientadduction, while the major (+)-trans-BPDE lesion was even more dominant at the frequentlyadducted sites. The introduction of 5-methyl groups at adjacent cytosine bases increased theyields of N2-BPDE-dG diastereomers, probably a result of favorable hydrophobic interactionsbetween BPDE and 5-methylcytosine. The targeted formation of N2-BPDE-dG at MeCGdinucleotides within the p53 gene is consistent with the high prevalence of G T transversionsat these sites in smoking-induced lung cancer.