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 havebeen 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. For the past two decades the initiation of tumors by BP has been viewed as a process in which epoxides directly alkylate DNA. However, we have recently discovered a new pathway in which chloride ions catalyze both adduct formation from BPDE and hydrolysis (passive detoxification) of the carcinogen. BPDE forms many adducts with DNA but the identity of the adduct that induces tumor initiation has not been determined. Conformational studies have focused on the major trans(+)-anti- and trans(-)-anti-BPDE-dGuo adducts but not on the minor dAdo and dCyd adducts have not been carried out, in part because of the poor synthetic reactions that are currently employed to make BPDE-DNA adducts. An important aspect of this proposal is the analysis of minor adducts since, in principle, even a single adduct could be biologically amplified. This research has three aims: (i) to study the mechanism of chloride-catalyzed adduct formation through in vitro model systems, (ii) to develop new synthetic schemes for making BPDE-deoxynucleoside adducts and use them to assemble oligodeoxynucleotides (ODNs) modified with the minor adducts for conformational studies, and (iii) to analyze the occurrence and properties of dCyd adducts in reaction profiles between BPDE and DNA.
Aim (i) will be pursued by synthesizing halohydrins of BPDE and comparing adducts formed with this intermediate and from BPDE. We will also study the role of bromide and iodide in adduct formation. An important question concerning carcinogenesis mechanisms is whether chloride participates in the tumor initiation properties of all electrophilic chemical carcinogens or whether the reaction is confined to epoxides.
Aim (ii) will be accomplished by substantially improving synthetic methods for making adducts. This will permit the synthesis and conformational study of ODNs modified with minor adducts.
Aim (iii) will be accomplished by development of separation systems capable of resolving dCyd adducts from other components in BPDE-DNA reactions. The long term goal of this work is to determine the mechanism by which chemical carcinogens attack DNA, and to assess the relative biological activity of each of the BPDE-DNA adducts that are formed from the carcinogen. These investigations will provide necessary information concerning which BPDE adduct is responsible for the biological activity of this important and ubiquitous environmental contaminant.
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