Preparation, identification and analysis of stereoisomeric anti-benzo[a]pyrene diol epoxide-deoxyguanosine adducts using phenyl liquid chromatography with diode array, fluorescence and tandem mass spectrometry detection.

Benzo[a]pyrene, a common environmental pollutant, can be metabolized into reactive anti-benzo[a]pyrene diol epoxide (anti-BPDE), which predominantly binds to deoxyguanine in DNA and forms four stereoisomeric adducts. To characterize the stereochemistry of these adduct isomers, preparation of single adducted deoxyguanosine (dG) is required for efficient enantiomeric analysis. Here, we demonstrate an improved method for preparation, identification, and analysis of four BPDE-adducted dGs, including (+)-trans-, (-)-trans-, (+)-cis-, and (-)-cis-anti-BPDE-N(2)-dG. These stereoisomerically adducted nucleosides were first synthesized by a direct reaction of (+/-)-anti-BPDE with dG, followed by optimized solid-phase extraction (SPE) and HPLC purification. The reaction of (+/-)-anti-BPDE and dG displayed a yield as high as 45%. The developed preparation method does not require any enzymatic digestion. Based on highly efficient separation achieved by optimization of stationary phase and mobile phase, LC-UV-MS/MS and LC-diode array detection (DAD)-fluorescence detection (FL) methods were established for characterization and analysis of the four stereoisomeric anti-BPDE-dGs. The established LC-DAD-FL method may provide characterization and analysis of four stereoisomeric anti-BPDE-dGs and two interfering anti-BPDE tetrols by taking advantage of their distinct fluorescence quenching.

Effects of base sequence context on translesion synthesis past a bulky (+)-trans-anti-B[a]P-N2-dG lesion catalyzed by the Y-family polymerase pol kappa.

The effects of bases flanking single bulky lesions derived from the binding of a benzo[a]pyrene 7,8-diol 9,10-epoxide derivative ((+)-7R,8S,9S,10R stereoisomer) to N(2)-guanine (G*) on translesion bypass catalyzed by the Y-family polymerase pol kappa (hDinB1) were examined in vitro. The lesions were positioned near the middle of six different 43-mer 5'-...XG*Y... sequences (X, Y = C, T, or G, with all other bases remaining fixed). The complementary dCTP is preferentially inserted opposite G* in all of the sequences; however, the proportions of other dNTPs inserted varies as a function of X and Y. The dCTP insertion efficiencies, f(ins) = (V(max)/K(m))(ins), are smaller in the XG*Y than in XGY sequences by factors of approximately 50-90 (GG*T and GG*C) or 5000-25000 (TG*G and CG*G). Remarkably, in XG*Y sequences, f(ins) varies by as much as 3 orders of magnitude, being smallest with G flanking the lesions on the 3'-side and highest with G flanking the adducts on the 5'-side. One-step primer extension efficiencies just beyond the lesions (f(ext)) are generally smaller than f(ins) and also depend on base sequence. However, reasonably efficient translesion bypass of the (+)-trans-[BP]-N(2)-dG adducts is observed in all sequences in running-start experiments with full, or nearly full, primer extension being observed under conditions of [dNTP] > K(m). The key features here are the relatively robust values of the kinetic parameters V(max) that are either diminished to a moderate extent or even enhanced in the presence of the (+)-trans-[BP]-N(2)-dG adducts. In contrast to the small effects of the lesions on V(max), the apparent K(m) values are orders of magnitude greater in XG*Y than in the unmodified XGY sequences. Thus the bypass of (+)-trans-[BP]-N(2)-dG adducts under conditions when [dNTP] < K(m) is quite inefficient. These considerations may be of importance in vivo where [dNTP]

Influence of bulky polynuclear carcinogen lesions in a TATA promoter sequence on TATA binding protein-DNA complex formation.

The TATA binding protein (TBP) is an essential component of the transcription initiation complex that recognizes and binds to the minor groove of the TATA DNA duplex consensus sequences. The objective of this study was to determine the effect of a carcinogen-modified adenine residue, positioned site-specifically within a regulatory TATA DNA sequence, on the binding of TBP. Two 25-mer oligonucleotides with stereoisomeric 10S (+)-trans-anti- or 10R (-)-trans-anti-BPDE-N(6)-dA residues at A(1) or A(2) within the TATA sequence element (5'-...TA(1)TAAA...-3')-(5'-...TTTA(2)TA...) were synthesized (anti-BPDE-N(6)-dA denotes an adduct formed from the reaction of r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydobenzo[a]pyrene). The formation of complexes with TBP of these two sequences in the double-stranded forms (1 nM) were studied employing electrophoretic mobility shift assays (EMSA) at different TBP concentrations (0-70 nM). The overall affinity of TBP for the BPDE-modified target DNA sequences was weakly enhanced in the case of the (+)-trans or (-)-trans lesions positioned at site A(1) with K(d) approximately 8 and 6 nM, respectively (K(d) approximately 9 nM for the unmodified TATA DNA). Higher-order TBP-DNA complexes were observed at TBP concentrations in excess of approximately 15 nM. However, the stabilities of the biologically significant monomeric TBP-DNA complexes was dramatically increased or decreased, depending on the position of the lesion (A(1) or A(2)), or on its stereochemical and conformational characteristics. A molecular docking modeling approach was employed to insert the stereoisomeric BPDE residues into the known TATA box-TBP structure [Nikolov, D. B., et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 4862-4867] to rationalize these observations. Native gel electrophoresis experiments with the same duplexes without TBP indicate that none of the modified sequences exhibit unusual bending induced by the lesions, nor that they differ from one another in this respect. These results suggest that the hydrophobic, bulky BPDE residues influence the binding of TBP by mechanisms other than prebending. The efficiency of RNA transcription of TBP-controlled promoters could be strongly influenced by the presence of such bulky lesions that could adversely affect the levels of gene expression.

Solvent-free synthesis of benzo[a]pyrene 7,8-diol 9,10-epoxide adducts at the N(2)-position of deoxyguanosine.

[reaction: see text] The first solid-state (or solvent-free) synthesis of protected deoxyguanosine (dG) adducts of benzo[a]pyrene diol epoxides at room temperature is reported. Whereas dG adducts derived from cis- and trans-opening of (+/-)-7beta,8alpha-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (DE-1 1) are formed as a 1:1 mixture, the direct opening of the diastereomeric (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (DE-2, 2) produced a 15:85 ratio favoring the trans-opened dG adduct 7.

Mass spectrometric sequencing of site-specific carcinogen-modified oligodeoxyribonucleotides containing bulky benzo[a]pyrene diol epoxide-deoxyguanosyl adducts.

Site-specific carcinogen-modified oligonucleotides are often used in site-directed mutagenesis and other biological and biochemical studies of structure-function relationships. Postsynthetic analysis and confirmation of the sites of carcinogen binding in such oligonucleotides is an important step in the characterization of these site-specific carcinogen-DNA adducts. It is shown here that negative ion mode electrospray tandem mass spectrometry methods and collision-induced dissociation offer a rapid and convenient approach for the sequencing of products derived from the reaction of the carcinogenic and mutagenic metabolite of benzo[a]pyrene, the diol epoxide r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE), with the 11-mer oligonucleotide d(CATGCGGCCTAC). The site of reaction of anti-BPDE with either one of the three dG residues in this oligonucleotide can be accurately established by comparing the mass/charge ratios of the observed collision-induced dissociation fragments with calculated values.

Highly mutagenic bypass synthesis by T7 RNA polymerase of site-specific benzo[a]pyrene diol epoxide-adducted template DNA.

We have previously developed an in vitro system that allows quantitative evaluation of the fidelity of transcription during synthesis on a natural template in the presence of all four nucleotides. Here, we have employed this system using a TAA ochre codon reversion assay to examine the fidelity of transcription by T7 RNA polymerase past an adenine residue adducted at the N6-position with (-)-anti-trans- or (+)-anti-trans-benzo[a]pyrene diol epoxide (BPDE). T7 RNAP was capable of transcribing past either BPDE isomer to generate full-length run-off transcripts. The extent of bypass was found to be 32% for the (-)-anti-trans-isomer and 18% for the (+)-anti-trans-isomer. Transcription past both adducts was highly mutagenic. The reversion frequency of bypass synthesis of the (-)-anti-trans-isomer was elevated 11,000-fold and that of the (+)-anti-trans-isomer 6000-fold, relative to the reversion frequency of transcription on unadducted template. Adenine was misinserted preferentially, followed by guanine, opposite the adenine adducted with either BPDE isomer. Although base substitution errors were by far the most frequent mutation on the adducted template, three- and six-base deletions were also observed. These results suggest that transcriptional errors, particularly with regard to damage bypass, may contribute to the mutational burden of the cell.

Peroxisome proliferators increase the formation of BPDE-DNA adducts in isolated rat hepatocytes.

Peroxisome proliferators are known to modulate the activity of xenobiotic-metabolising enzymes, including glutathione S-transferase (GST) and cytochrome P-450 (CYP). In this study the effect of peroxisome proliferators silvex and di(2-ethylhexyl)phthalate (DEHP) on the formation of (+)-anti-benzo(a)pyrene -7,8-dihydrodiol-9,10-epoxide (BPDE)-DNA adducts from a proximate mutagen and carcinogen (-)-transbenzo(a)pyrene-7,8-dihydrodiol (BPDD) has been investigated. Rat CYP1A1 metabolises BPDD to mutagenic BPDE, which may form DNA adducts or, alternatively, be detoxified by hydrolysis or glutathione conjugation. In this experiment the formation of BPDE-DNA adducts was significantly increased in hepatocytes isolated from all silvex treated rats and two out of four DEHP treated rats (14 day treatment). The activity of CYP1A1 was increased whereas GST was reduced by the peroxisome proliferator silvex. These changes were more significant than those induced by DEHP. We have hypothesised that the formation of BPDE-DNA adducts was primarily due to the increased BPDD activation to BPDE versus reduced detoxication of BPDE. Other hepatic changes induced by the peroxisome proliferators, e.g. peroxisome proliferation per se and increased mitotic activity of the liver could have an effect on the outcome of BPDD exposure.

Synthesis and characterization of adducts derived from the syn-diastereomer of benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide and the 5'-d(CCTATAGATATCC) oligonucleotide.

5'-d(CCTATAGATATCC) was reacted with each syn-enantiomer of trans-7,8-dihydroxy 9,10-epoxy 7,8,9,10-tetrahydrobenzo[a]pyrene (syn-BPDE). The (-)-enantiomer yielded one dominating adduct, whereas the (+)-enantiomer resulted in two major adducts. As indicated by optical spectroscopic methods, the major adduct derived from both (-)- and (+)-syn-BPDE involves cis addition of the C-10 position of the diol epoxide to the exocyclic amino group of deoxyguanosine [(-)-syn-BPDEc-N2-dG and (+)-syn-BPDEc-N2-dG, respectively], whereas the minor (+)-syn-BPDE adduct is identical to a trans adduct [(+)-syn-BPDEt-N2-dG]. The cis adducts as well as the (+)-syn-BPDEt-N2-dG adduct are chemically stable for several weeks when stored at < or = 4 degrees C in darkness. In duplexes composed of (-)-syn-BPDEc-N2-dG or (+)-syn-BPDEc-N2-dG modified 5'-d(CCTATAGATATCC) and the complement 5'-d(GGATATCTATAGG), the presence of an adduct, in particular the latter, substantially decreased the Tm value relative to the corresponding unmodified duplex. Addition of 5'-d(GGATATCTATAGG) or strands in which dC was replaced with dT, dG, or dA to (-)-syn-BPDEc-N2-dG modified 5'-d(CCTATAGATATCC) decreased the fluorescence intensity in all cases (25-45%). In similar experiments with the (+)-syn-BPDEc-N2-dG adduct, dC or dT opposite the adduct decreased the fluorescence intensity, whereas dA and dG caused an increase. With the (+)-syn-BPDEt-N2-dG adduct, duplex formation had no effect on the intensity with dC or dG opposite the adduct, while an increase could be noted with dT or dA. Acrylamide had no significant effect on the fluorescence intensity of duplexes with cis adducts in contrast to the marked quenching of the fluorescence of (+)-syn-BPDEt-N2-dG oligonucleotide duplexes. In single stranded form, both the cis adducts exhibited absorption and fluorescence excitation maxima at 352-353 nm while the (+)-syn-BPDEt-N2-dG adduct was around 350-351 nm. Addition of the complement or the sequence in which dA replaced dC to the (+)-syn-BPDEt-N2-dG adduct shifted the maxima to 347-349 nm, whereas addition of sequences containing dT or dG opposite the adduct affected the fluorescence maxima but had no effect on absorption maxima. Formation of duplexes with the cis adducts had no or very little effect on the absorption and fluorescence maxima. In conclusion, the results of this study imply an intercalative mode of interaction of the pyrenyl chromophores of the cis adducts and external localization of the (+)-syn-BPDEt-N2-dG adduct.

Synthesis of 3-nitrobenzo[a]pyrene bay-region trans-7,8-diol anti-9,10-epoxide and the corresponding N2-deoxyguanosine adduct.

3-Nitrobenzo[a]pyrene (3-nitro-BaP) is a potent mutagenic environmental contaminant, and its biological activities have been intensively studied. It is significant to prepare its reactive metabolites and the corresponding modified DNA adducts for biological studies. The synthesis of its oxidized proximate metabolite trans-7,8-dihydro-3-nitrobenzo[a]pyrene (3-nitro-BaP-trans-7,8-dihydrodiol, 1), its oxidized ultimate metabolite trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-3- nitrobenzo[a]pyrene (3-nitro-BaP-DE, 2), and the corresponding DNA adduct 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-3- nitrobenzo[a]pyrene is described.