Studies on oligonucleotide adducts formed from 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene stereoisomers

Abstract: The widespread environmental pollutant benzo[a]pyrene (BP), is formed from incomplete combustion of fossil fuels and is mutagenic and carcinogenic after metabolic activation to bay-region diol epoxides and subsequent covalent binding to DNA. The metabolic activation pathway to diol epoxides is via BP-7,8-oxide and BP-7,8-dihydrodiol to yield preferentially 7R,8S-dihydroxy-9S,lOR-epoxy-7,8,9,10-tetrahydrobenzo[a]py- rene [(+)-anti-BPDE] and to lesser extents, 7R,8S-dihydroxy-9R,lOS-epoxy-BP [(-)-syn-BPDE], 7S,8R- dihydroxy-9S,lOR-epoxy-BP [(+)-syn-BPDE] and 7S,8R-dihydroxy-9R,lOS-epoxy-BP [(-)-anti-BPDE]. Although all stereoisomeric diol epoxides bind to DNA (via C-10), (+)-anti-BPDE is the most active form. In order to better understand the mechanisms underlying the carcinogenic and mutagenic properties of DNA adducts derived from BPDE, studies of the physical relationship between adduct structure and base preference and recognition during replication, repair and transcription seems to be of great relevance. We have studied the spectroscopic properties and thermal stabilities of a 13-mer oligonucleotide site-specifically modified with anti- or syn-BPDE enantiomers. The oligonucleotide chosen. 5'-d(CCTATAGATATCC), contains only one target dG, the preferred binding site for covalent binding of BPDE diastereomers. An HPLC system for purifying the dG adducts was developed and the modified oligonucleotides were characterized by optical spectroscopy. One major N2-dG adduct was detected with the (+)-anti-BPDE, whereas (-)-anri-BPDE resulted in two N2-dG adducts, resulting from cis and trans additon of BPDE to'N2-dG. From syn-BPDE one major adduct was obtained from the (-)-enantiomer whereas the (+)-enantiomer resulted in two N2-dG adducts. It was concluded that the major adducts from (-)- and (+)-syn-BPDE were cis adducts. The minor adduct from (+)-syn-BPDE was identified as a dG adduct with trans configuration. The adduct environment is strongly influenced by duplex formation and interactions between the adduct and the base localized opposite the adduct as shown by the fluorescence behaviour. In view of thermal stability, the presence of a cis syn-BPDE adduct destabilizes the helix more than a (+)-anti-BPDE adduct. In order to improve adduct yields various parameters relevant for the formation of dG adducts of BPDE stereoisomers with oligonucleotides were studied. The optimal experimental conditions found was different depending on the base sequence context employed, but in general low temperature to achieve long reaction time. near neutral Tris-HCI buffer or alkaline phosphate buffer, low concentration of organic solvent and high molar ratio of BPDE and oligonucleotide favour adduct formation. Several studies have confirmed that the extent of binding of anti-BPDE to DNA is highly dependent on the sequence context, but very little is known about the selectivity of syn-BPDE. Therefore, using the same oligonucleotide as above and in addition, by exchanging the bases flanking the target dG, sequences containing all possible base combinations surrounding the central dG were reacted with (-)-syn-BPDE and, for comparison (+)-anti-BPDE. The adduct amounts were in the range 3-30% and 0-4% trans and cis (+)-anti- BPDE adducts respectively, whereas the range of cis and trans (-)-syn-BPDE adducts were 1-10% and 0-0.5% , respectively. The binding to dG of the stereoisomers studied differs in base selectivity. However, it is clear that the base sequence has a large impact on the amount of adducts formed from both diastereomers. Extensive knowledge is available concerning the regulation, structure and function of the transcription factor. Activator Protein-l (AP-I, consisting of of homo- and heterodimers of Fos/Jun proteins), as well as its response to different stimuli. However, the effect of an alteration of the transcription binding site in DNA, for instance by the presence of covalently bound carcinogenic PAH intermediates has so far received little attention. We have specifically modified a 28-mer oligonucleotide containing the AP-I binding site, with (+)-anti- or (-)-syn-BPDE (yielding trans and cis adducts, respectively) and studied the effect on AP-I binding with HeLa cell nuclear extracts or truncated Fos/Jun proteins. The presence of a BPDE adduct extensively reduced the affinity for binding of proteins from HeLa cell nuclear extracts as compared to controls. No apparent differences in the response between the different adducts were obtained, suggesting that the binding geometry of the adduct is of little importance. From experiments with antibodies directed against c-Fos and c-Jun proteins it was clear that the binding to BPDE modified oligonucleotide seems to involve AP-I related proteins other than c-Fos and c-Jun. The data presented here clearly suggest that chemical modification of the AP- I transcription factor binding site with BPDE may seriously alter the extent and/or duration of expression of genes containing AP-l sites in their promoter regions. Doctoral Thesis ~) 1996 Ingrid Ponten ISBN 91-628-2153-9