Tumor initiation by chemical carcinogens is thought to result from binding to DNA that leads to activation/deactivation of critical proto- oncogenes and suppressor genes. We have been studying the environmental pro-carcinogen benzo(a)pyrene (BaP), as a model for understanding molecular carcinogenesis. BaP is converted into a potent chemical carcinogen by cellular metabolism and this intermediate is 7R,8S- dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydroBP ((+)-anti-BPDE). A (-)- anti-BPDE enantiomer is also made but this metabolite has 50-fold less biological activity. Both of these stereoisomers bind to DNA to form major and minor adducts and conformational studies have been hampered by the availability of only microgram quantities of heterogeneously modified DNAs for study. Since the biological properties of the carcinogenic and non-carcinogenic stereoisomers may result from differences in conformation, one goal of this research is to synthesize sufficient quantities of both the major and minor BPDE adducts to carry out these studies. Another goal is to characterize important minor adducts, such as those formed by cis opening of the BPDE adducts to carry out these studies. Another goal is to characterize important minor adducts, such as those formed by cis opening of the BPDE epoxide and dCyd alkylation products, since they may be responsible for the mutagenic properties of the carcinogen.
Our specific aims are to (i) synthesize BPDE-modified deoxynucleotides and BPDE-modified oligodeoxynucleotides (ODNs), (ii) carry out conformational studies on the modified monomers and oligomers, (iii) study the mechanism of cis adduct formation, and (iv) analyze the structure and characterize the occurrence of dCyd adducts in DNA. The approach will be to synthesize BPDE-deoxynucleosides and incorporate them into site-specific, carcinogen-modified ODNs. The ODNs will be made with BPDE-modified dGuo and dAdo employing cis and trans deoxynucleoside adducts derived from both the (+)- and (-)-anti-stereoisomers. Synthetic methods for making BPDE-dCyd adducts will be developed, and this carcinogen-modified deoxynucleoside will also be used to construct ODNs containing a single, site-specific lesion. Conformations will be evaluated by UV, CD, fluorescence, and NMR spectroscopies and by X-ray crystallography. We will study the mechanism and properties of the halogen-catalyzed formation of cis adducts with DNA. We will also complete the characterization and occurrence of dCyd adducts in DNA, in order to assess their importance to the biological effects of BaP. The long term goal of this work is to determine the relative biological activity of each of the BPDE-DNA adducts that are formed, in order to assess which one(s) may be responsible for the tumor initiating properties of this important and ubiquitous environmental contaminant.
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