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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 Ingrid Ponte´n,† Albrecht Seidel,‡ Astrid Gra¨slund,§ and Bengt Jernstro¨m*,† Institute of Environmental Medicine, Division of Biochemical Toxicology, Karolinska Institutet, Box 210, S-17177 Stockholm, Sweden, Department of Toxicology, University of Mainz, D-55131 Mainz, Germany, and Department of Biophysics, Stockholm University, S-10691 Stockholm, Sweden Received March 6, 1995X

5′-d(CCTATAGATATCC) was reacted with each syn-enantiomer of trans-7,8-dihydroxy 9,10epoxy 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 e4 °C in darkness. In duplexes composed of (-)-syn-BPDEc-N2-dG or (+)-synBPDEc-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-BPDEtN2-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.

Introduction Polycyclic aromatic hydrocarbons (PAH)1 are common environmental pollutants, and several are carcinogenic following their metabolism to electrophilic intermediates and their subsequent covalent binding to critical targets in DNA (1-3). Furthermore, formation of specific transversion mutations (GC f TA or AT f TA) in certain proto-oncogenes and suppressor genes seems to be necessary events for tumor induction by PAH (4-6). Benzo[a]pyrene (BP),1 the most studied PAH, may be metabolized by one-electron oxidation or monooxygenation to yield radical cations or bay-region diol epoxides, * To whom correspondence should be addressed. † Karolinska Institutet. ‡ University of Mainz. § Stockholm University. X Abstract published in Advance ACS Abstracts, December 1, 1995. 1 Abbreviations: PAH, polycyclic aromatic hydrocarbons; BP, benzo[a]pyrene; BPDE, benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide; LD, linear dichroism; CD, circular dichroism.

0893-228x/96/2709-0188$12.00/0

respectively (1, 2, 7, 8). Metabolism of BP by the monooxygenation pathway (CYP1A1) yields the diastereomers anti- and syn-benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide (BPDE,1 Figure 1), in particular, the (+)anti- and (-)-syn-enantiomers (reviewed in ref 9). Systematic studies of both racemic anti- and syn-BPDE or the resolved enantiomers have clearly shown that (+)anti-BPDE is the most mutagenic and carcinogenic form in mammalian systems, although all isomers covalently bind to DNA in target cells (10-13). Interestingly, the BPDE isomers exhibiting low mutagenic activity in mammalian cells have been found to be highly active in bacterial systems (13-16). The covalent binding of the BPDE diastereomers to DNA takes place with high preference at the exocyclic amino group of deoxyguanosine (N2-dG) via trans or cis addition to the benzylic C-10 position in the diol epoxide (17-20). Previous results on binding of BPDE to DNA, polynucleotides, or oligonucleotides by optical spectroscopy have demonstrated a heterogeneous adduct distribution and distinguished two different types of com© 1996 American Chemical Society

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Figure 1. Partial structures of anti- and syn-BPDE-N2-dG adducts including absolute configurations and verified or proposed adduct type and binding geometries. References are given in parenthesis.

plexes, denoted type I and type II (21-24; Figure 1). Type I adducts are characterized by a substantial shift (5-10 nm) to longer wavelengths of the pyrene light absorption spectrum compared to benzo[a]pyrene tetraols in water and a negative linear dichroism (LD)1 spectrum. Type II adducts exhibit a small shift (2-3 nm) to longer wavelengths and a positive LD spectrum. Binding of (+)anti-BPDE to DNA results predominantly in type II adducts whereas binding of (-)-anti-BPDE results in a mixture of type I and type II adducts (22-24). Very little is known about the binding mode of syn-BPDE. However, available information indicates that this diastereomer binds mainly in the type I manner (24, 25). On the basis of spectral data, type I adducts were suggested to have intercalative binding characteristics, whereas type II adducts were suggested to exhibit external binding (21; Figure 1). Recent studies by two-dimensional NMR spectroscopy on oligonucleotide duplexes containing single trans adducts derived from (+)- or (-)anti-BPDE have shown that the pyrenyl chromophores are positioned in the minor groove and directed toward the 5′- or 3′-end of the binding strand, respectively (26, 27; Figure 1). A similar study on the same oligonucleotide duplex containing a single cis adduct derived from (+)-anti-BPDE demonstrated that the pyrenyl chromophore in this case is intercalated between base pairs (28). Thus, the geometries suggested from the optical studies of type I or II adducts as being located in the minor groove or intercalated (29) are verified by the NMR solution structure studies. The mixtures of adducts observed with certain enantiomers, e.g., (-)-anti-BPDE, are due to about equal yields of trans as well as cis adducts, which have different characteristics.

The lower mutagenic and carcinogenic potency generally observed with (-)-anti- and the syn-BPDE enantiomers may well be explained by the difference in binding mode and structural features of the adducts, which in turn may affect how these lesions are recognized and treated by DNA repair, DNA transcription, and DNA replication systems. Alternatively, the lower biological effect of the syn-BPDE diastereomer may be related to the lower chemical stability of the corresponding DNA adducts (25, 30). 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 the adduct structure and base preference and recognition during replication seems to be of great relevance. In a former study, we investigated trans adducts derived from the reaction of (+)- or (-)anti-BPDE with 5′-d(CCTATAGATATCC) by employing optical spectroscopic techniques (31). Using similar methods, we have now studied adducts derived from the reaction of (+)- or (-)-syn-BPDE with the same oligonucleotide.

Materials and Methods Caution: Benzo[a]pyrene diol epoxides are mutagenic and/ or carcinogenic agents and should be handled with care, as outlined in National Cancer Institute guidelines. Chemicals. Oligonucleotides were purchased from Symbicom AB (Umeå, Sweden), from Innovagen AB (Lund, Sweden), or from Kebo (Stockholm, Sweden). Prior to use, the oligonucleotides were tested for purity by HPLC (see below for chromatographic conditions). The (-)- and (+)-enantiomer of syn-BPDE were synthesized from optically active benzo[a]pyrene 7,8-

190 Chem. Res. Toxicol., Vol. 9, No. 1, 1996 dihydrodiol as described below. Racemic benzo[a]pyrene 7,8dihydrodiol was synthesized as previously reported (32, 33) and for resolution of enantiomers it was converted into the diastereomeric bis-(-)-menthoxyacetate derivatives (34, 35). Separation of the diastereomeric mixture was performed by preparative HPLC using a Labomatic HD 200 chromatograph equipped with a Knauer 32 × 250 mm silica gel (LiChrosorb Si 60, 5 µm) column. Elution with 5% diethyl ether in cyclohexane, a solvent system earlier used by Yagi et al. (36) for similar separations, allowed the resolution of 50 mg samples of the bis-(-)-menthoxyacetates injected in the same solvent. The pure diastereomeric esters obtained after one rechromatography were saponified in methanol/tetrahydrofuran with sodium methoxide to give the (+)- and (-)-enantiomer of the benzo[a]pyrene 7,8dihydrodiol. The conversion of (-)- and (+)-benzo[a]pyrene 7,8dihydrodiol into (-)- and (+)-syn-BPDE, respectively, was accomplished via the intermediate bromohydrin derivatives according to procedures published in detail for the racemic material (37). The specific rotation values of each optically active benzo[a]pyrene 7,8-dihydrodiol and syn-BPDE were in good agreement to those reported by Yagi et al. (38). Phosphodiesterase from Crotalus durissus and alkaline phosphatase from calf intestine were obtained from Boehringer-Mannheim Scandinavia AB (Bromma, Sweden). NAP-10 columns were purchased from Pharmacia (Stockholm, Sweden). Other chemicals were obtained from local suppliers. Site-Specific Binding of BPDE to 5′-d(CCTATAGATATCC). The samples were prepared and purified by HPLC according to the method described for anti-BPDE-modified oligonucleotides in Ponte´n et al. (31). Hydrolysis of oligonucleotides to deoxyribonucleosides used for CD measurements was performed as described for anti-BPDE modified oligonucleotides (31) except that the incubation time was 2 h. Isolation of the deoxyribonucleoside adducts was performed in a modified HPLC system as compared to the system used previously (31). A Microsorb MV reversed phase column (C18, 5 µm, 300 A, 4.6 i.d. × 250 mm, Rainin Instrument Co., Woburn, MA) was used in conjunction with a solvent system composed of 0.1 M triethylammonium acetate and acetonitrile. The following elution system was used: 90% A in B for 7 min, a gradient from 90% A to 50% A for 13 min, 50% A to 40% A for 9 min, and 40% A in B for 6 min (solvent A ) 0.1 M triethylammonium acetate, solvent B ) 50% A in acetonitrile). Prior to CD measurements, the samples were freed of acetonitrile. Chemical Stability of syn-BPDE Adducts. The chemical stability of the oligonucleotide adducts was measured by HPLC using the same system as for the purification (see above). Aliquots of unpurified or pure adducts (stored at 4 °C in darkness) were injected at various time points up to 8 weeks after termination of the reaction. Thermal Stability of Oligonucleotide Duplexes. 5′d(CCTATAGATATCC) or the analog containing the (-)- or (+)syn-BPDE-N2-dG adduct A (see Results and Discussion) was annealed to the complementary sequence 5′-d(GGATATCTATAGG) or the partially complementary sequences 5′-d(GGATATATATAGG), 5′-d(GGATATTTATAGG), and 5′-d(GGATATGTATAGG) (2 µM final duplex concentration in 0.5 M NaCl and 0.5 mM EDTA) by mixing at 60 °C for 10 min followed by slow cooling to room temperature. The samples were kept at 4 °C in darkness for at least 12 h before measurements. The stability of the oligonucleotide duplexes was studied by monitoring the change in absorbance at 260 nm as a function of time as described (31). Fluorescence of Oligonucleotide Duplexes. The complement 5′-d(GGATATCTATAGG) or 5′-d(GGATATTTATAGG), 5′d(GGATATGTATAGG) or 5′-d(GGATATATATAGG) was gradually added to 0.17 µM 5′-d(CCTATAGATATCC) containing (-)syn-BPDE-N2-dG adduct A or (+)-syn-BPDE-N2-dG adduct A or B (see Results and Discussion), final ratio unmodified:modified oligonucleotide 3:1. The fluorescence excitation (λemission ) 400 nm) and emission (λexitation ) 355 nm) spectra were recorded after allowing the sample to equilibrate for 15 min. Acrylamide was used as a fluorescence quencher. All measurements were

Ponte´ n et al.

Figure 2. HPLC elution profiles of reaction products of (-)and (+)-syn-BPDE and 5′-d(CCTATAGATATCC) monitored by absorbance at 260 nm (A and B), and by fluorescence emission at 400 nm (λexcitation ) 350 nm) (C and D). carried out at 11 °C and in the presence of 0.5 M NaCl and 0.5 mM EDTA. Circular Dichroism Measurements. Circular dichroism (CD)1 measurements were carried out with a JASCO J-720 spectropolarimeter. The oligonucleotide samples were dissolved in water and measured in 1 cm cuvettes at 20 °C. The unmodified and modified single stranded oligonucleotide containing (+)-anti-BPDEt-N2-dG adduct, (-)-anti-BPDEt-N2-dG adduct, (-)-syn-BPDE-N2-dG adduct A, or (+)-syn-BPDE-N2dG adduct A were measured at 6-8 µM concentration whereas the (+)-syn-BPDE-N2-dG adduct B was measured at 4.3 µM. The deoxyribonucleoside adducts [(+)-anti-BPDEt-N2-dG (2.5 µM), (-)-anti-BPDEt-N2-dG (3.2 µM), and the (-)- and (+)-syn-BPDEN2-dG (1.9 and 0.6 µM, respectively)] obtained by hydrolysis of adducted oligonucleotides were measured at 4 °C in ≈0.1 M triethylammonium acetate (see above).

Results and Discussion Isolation of Modified Oligonucleotides. As shown in Figure 2, the reaction of (-)- or (+)-syn-BPDE with 5′-d(CCTATAGATATCC) gives rise to several products with both UV-absorbing (Figure 2A,B) and fluorescent properties (Figure 2C,D). The UV-absorbing but nonfluorescent peak eluting at ≈12 min corresponds to the unmodified oligonucleotide and the peaks eluting at 3940 min corresponds to hydrolysis products of BPDE (tetraols) not removed by ethyl acetate extraction (cf. ref 31). The reaction with (-)-syn-BPDE gives rise to one major adduct, the cis adduct as will be shown below (Rt ≈16 min), with both UV-absorbing and fluorescent properties. Under our experimental conditions a reaction yield of about 5% with respect to oligonucleotides was generally obtained. The reaction with (+)-syn-BPDE results in two major adducts, the cis and trans adducts as will be shown below (Rt ≈16 min and ≈18 min, respectively). With this enantiomer the reaction yields were about 2% and 0.6%, respectively. The major adduct containing peaks were collected, freed of solvent by lyophilization, and rechromatographed on HPLC. Part of the apparently homogeneous products were enzymatically hydrolyzed to deoxyribonucleosides and subse-

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Table 1. Light Absorption and Fluorescence Excitation Maxima of (-)-syn-BPDE Adduct A, (+)-syn-BPDE Adduct A, and (+)-syn-BPDE Adduct B Modified Oligonucleotides as Single Strands or Annealed with Complementary or Partially Complementary Strands system (-)-syn-BPDE adduct A (+)-syn-BPDE adduct A (+)-syn-BPDE adduct B a

single strand 352a 352 350

353b 353 351

adduct/dC 352 353 349

353 351 347

adduct/dT 352 353 351

353 353 348

adduct/dA 352 353 349

353 353 347

adduct/dG 352 353 351

353 353 348

Light absorption maxima, uncertainty (0.3 nm. b Fluorescence excitation maxima (λemission ) 400 nm), uncertainty (1 nm.

quently analyzed by HPLC (ref 31 and Materials and Methods; cf. ref 39). The results demonstrated an equal distribution of dC, dT, and dA and the absence of nonadducted dG in all cases (results not shown). Thus we conclude that the major adducts derived from the synenantiomers involve dG. The deoxyribonucleoside adducts were purified by HPLC and analyzed by CD. The CD results verified the dG adduct formation (see below). Although the ratio of dA and dG in the oligonucleotide used is 4:1, the major adduct formed is with deoxyguanosine, a result that is in agreement with the observation on syn-BPDE modified calf thymus DNA (20). In this case, the amount of dG adducts was 5-7-fold larger than dA adducts. The preferential formation of cis adducts of syn-BPDE in a well-defined oligonucleotide is shown here for the first time. With (-)-syn-BPDE no detectable amounts of trans adduct was obtained. The results are consistant with those of Sayer et al. (20) on calf thymus DNA modified by (-)- or (+)-syn-BPDE. Wolfe et al. (40) reported recently that chloride ions promoted the formation of cis adducts of racemic anti-BPDE. If, and to what extent, the presence of chloride ions affects the cis/trans ratio of syn-BPDE in our system is not known. Chemical Stability of syn-BPDE Modified Oligonucleotides. The major oligonucleotide adducts obtained from (-)- and (+)-syn-BPDE were stored at 4 °C in the darkness and examined with respect to chemical stability by HPLC analysis of aliquots as a function of time. With all three adducts, no significant decomposition could be observed up to 8 weeks after preparation. This observation is in contrast to previous findings with native double stranded DNA modified by racemic syn-BPDE (25, 30). A substantial decomposition of adducts to products fully consistent with tetraols was observed. The reason for the adduct lability in previous studies is not known, although it may be related to the double stranded conformation of DNA in conjunction with adduct structure. Geacintov et al. (41) have shown that the cis adducts in double stranded DNA are considerably more labile than trans adducts derived from antiBPDE. More recently, Drouin et al. (42) demonstrated the destabilizing effect of duplex formation on the stability of the trans adducts of (+)-anti-BPDE. Light Absorbance and Fluorescence Studies on syn-BPDE-Modified Oligonucleotides. The absorption and fluorescence excitation maxima related to the pyrenyl residue of the syn-BPDE derived adducts are compiled in Table 1. The differences observed for some adducts between absorption and excitation maxima are outside the error limits and indicate a certain heterogeneity in the adduct distribution (cf. refs 25 and 43). The first eluting peak in the HPLC trace (adduct A), derived from either (-)- or (+)-syn-BPDE (Figure 2, panels A and B, respectively) show absorption and excitation maxima at 352 and 353 nm, respectively, when

Figure 3. Light absorption spectra of 5′-d(CCTATAGATATCC) containing (-)-syn-BPDE-N2-dG-adduct A in single stranded form (A) and as a duplex with 5′-d(GGATATTTATAGG) (B).

present in single stranded form. No changes in maxima are observed upon duplex formation with the complementary strand or strands where the dC opposite the adduct has been replaced by dT, dA, or dG. Figure 3 shows an example of the results obtained. In contrast, the maximal absorption and excitation of adduct B derived from (+)-syn-BPDE and present in single stranded form is at 350 and 351 nm, respectively. Duplex formation with the complementary strand or the strand where the dC opposite the adduct has been replaced by dA cause a shift to 349 and 347 nm, respectively. When dT or dG is situated opposite the adduct the absorption maxima are at 351 nm and the excitation maxima at 348 nm. Previous studies have shown that anti-BPDE derived adducts present in single stranded DNA or oligonucleotides exhibit a maximal absorption at about 350-352 nm, a shift of 7-9 nm relative to the absorption at 343 nm of pyrenyl chromophores free in solution (22-24). The difference in absorption maxima has been attributed to base stacking interactions with the BPDE chromophore (39, 43, 44). In fully complementary duplexes, type II complexes (trans adducts of anti-BPDE) show absorption maxima at 346-348 nm, whereas type I complexes (cis adducts of anti-BPDE) typically exhibit maxima at 350352 nm (21, 29). Since the spectroscopic properties obtained with anti-BPDE are likely to be representative also for the syn-diastereomer, we propose the following: (a) oligonucleotide adduct A derived from the (-)- or (+)syn-BPDE (with type I spectroscopic properties) is identical to a cis adduct [(-)-syn-BPDEc-N2-dG and (+)-synBPDEc-N2-dG, respectively]; and (b) adduct B derived from (+)-syn-BPDE (with type II spectral properties) is identical to a trans adduct [(+)-syn-BPDEt-N2-dG].

192 Chem. Res. Toxicol., Vol. 9, No. 1, 1996

Ponte´ n et al.

Figure 4. Change in fluorescence intensity of 5′-d(CCTATAGATATCC) containing (-)-syn-BPDE-N2-dG-adduct A upon gradual addition of 5′-d(GGATATATATAGG) (b). In the presence of acrylamide (2).

It has been demonstrated that fluorescence intensities vary substantially in oligonucleotides modified by the anti-BPDE enantiomers and that the effect depends on the adduct environment. For instance, fully complementary duplexes containing trans adducts demonstrate a decreased fluorescence intensity (29, 31) relative to single stranded modified sequences, whereas oligonucleotide duplexes containing cis adducts exhibit increased intensity (29). The effect has been attributed to the nature of the adduct and to changes in polarity of the environment to which the adducts are exposed. For a cis adduct it seems that decreased polarity in the environment results in increased fluorescence yields (29, 45, 46; cf. ref 47). For trans adducts the fluorescence intensity change between single and double stranded environment depends on the nature of the complementary base (31). In order to gain further information on the properties of the syn-BPDE derived adducts, 5′-d(GGATATCTATAGG) or partially complementary sequences were gradually added to 5′-d(CCTATAGATATCC) containing (-)syn-BPDEc-N2-dG, (+)-syn-BPDEc-N2-dG, or (+)-synBPDEt-N2-dG. The process was followed by measuring the fluorescence emission intensity. Furthermore, acrylamide was used as a fluorescence quencher to probe adduct environment since intercalating adducts are less accessible to acrylamide as compared to adducts located at external sites (48). An example of the result is shown in Figure 4. The fluorescence intensity of (-)-syn-BPDEc-N2-dG changes markedly when titrated with the complementary or partially complementary strands, the intensity is reduced linearly up to 1:1 concentration of both strands (30-45% reduction) and does not change any further at higher concentrations. Data from a number of experiments are compiled in Table 2. With (+)-syn-BPDEc-N2-dG there is also a reduction in intensity with dC or dT opposite the adduct, while dA or dG opposite gives a 30-35% intensity increase. The quenching effect of acrylamide

is moderate on the duplexes containing either cis adduct (in general, 10-30%). Addition of acrylamide to single stranded oligonucleotides containing these adducts had no significant effect. Experiments with the oligonucleotide containing (+)syn-BPDEt-N2-dG show a different pattern. Addition of the complementary or the partially complementary strand with dG was not associated with any significant changes in fluorescence yield relative to the single stranded oligonucleotide, but dT or dA gives an increased intensity of approximately 20-40%. In contrast to the results obtained with the cis adducts, addition of acrylamide to the modified duplex caused a significant quenching of the fluorescence. The lack of effect on fluorescence yield observed here on the assigned cis adducts are in contrast to previous results on the corresponding adducts from anti-BPDE (29). The difference may be due to the difference in sequence context of the oligonucleotide 5′-d(CACATGTACAC) used by Geacintov et al. (29) and 5′-d(CCTATAGATATCC) used in this study. Measurements by Circular Dichroism on synBPDE-Modified Oligonucleotides. Circular dichroism (CD) measurements were carried out on single stranded oligonucleotides containing trans and cis adducts derived from (-)- and (+)-syn-BPDE and, for comparison, the trans adducts of (+)- and (-)-anti-BPDE, respectively. In addition, CD measurements have been performed on deoxyribonucleoside adducts obtained by enzymatic hydrolysis of these oligonucleotides (with the exception of the trans adduct of (+)-syn-BPDE, where the yield was too low, Figure 5). The CD spectra of nucleoside adducts of the various enantiomers of BPDE arise from exciton interaction between the purine ring and the BPDE chromophore in the wavelength region where both absorb light. The sign of the different bands depends on the average orientation of the purine relative to the BPDE chromophore. This means that the CD spectrum should be highly sensitive to e.g., cis or trans configuration corresponding to absolute R or S configuration at C-10 of the BPDE added to the purine, but should be relatively insensitive to other variables such as substitutions on the purine (e.g., base vs nucleoside). Studies by NMR and CD on various BPDE enantiomers and model compounds such as dG and G (20, 49) clearly show that the CD spectra and particularly the sign of the major band around 250 nm, can be considered as signatures for the absolute configuration at C-10: a positive major band is evidence for absolute S configuration and a negative band for absolute R configuration. These results are in complete agreement with those of Cheng et al. (50) who used NMR and CD to study and assign the absolute configuration of the anti-BPDE adducts of dG. We have used a strategy based on these results to assign the absolute configuration of the syn and anti

Table 2. Relative Fluorescence Emission Intensity of Single Stranded (-)-syn-BPDE Adduct A, (+)-syn-BPDE Adduct A, or (+)-syn-BPDE Adduct B Modified Oligonucleotide (Concentration 0.17 µM) or Annealed to the Complementary Strand or Partially Complementary Strands and the Effect of 0.5 M Acrylamide (AA)a single strand system (-)-syn-BPDE adduct A (+)-syn-BPDE adduct A (+)-syn-BPDE adduct B

adduct/dC

+AA 1.0 1.0 1.0

0.88 0.93 0.73

adduct/dT

+AA 0.71 0.63 0.96

0.51 (0.72) 0.38 (0.60) 0.46 (0.48)

adduct/dA

+AA 0.74 0.76 1.39

0.50 (0.68) 0.59 (0.76) 0.42 (0.30)

adduct/dG

+AA 0.55 1.34 1.23

0.49 (0.89) 1.11 (0.82) 0.53 (0.43)

+AA 0.58 1.31 1.04

0.47 (0.81) 1.12 (0.85) 0.59 (0.57)

a The numbers are mean values from all results with unmodified/modified oligonucleotide ratio g1:1. Figures in parentheses show relative intensity after quenching as compared to duplexes.

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Figure 5. Circular dichroism spectra of unmodified, (+)- and (-)-anti-BPDEt-N2-dG, and (-)- and (+)-syn-BPDE-N2-dG modified 5′-d(CCTATAGATATCC) and their corresponding deoxyribonucleosides. (A) (+)-anti-BPDEt-N2-dG modified oligonucleotide (s), (+)-anti-BPDEt-N2-dG (- - -). (B) Oligonucleotide containing (-)-syn-BPDE-N2-dG adduct A (s), (-)-syn-BPDEN2-dG (- - -). (C) (-)-anti-BPDEt-N2-dG modified oligonucleotide (s), (-)-anti-BPDEt-N2-dG (- - -). (D) Oligonucleotide containing (+)-syn-BPDE-N2-dG adduct A (s), (+)-syn-BPDE-N2-dG (- - -). (E) Oligonucleotide containing (+)-syn-BPDE-N2-dG adduct B. (F) Unmodified oligonucleotide.

adducts of BPDE to oligonucleotides and the corresponding nucleosides after hydrolysis. It is obvious that modified oligonucleotides and the corresponding nucleosides give spectra with quite similar characteristics, and that there are in principle two kinds of spectra: those with a major positive band around 250 nm and those with a major negative band around 250 nm (Figure 5). Table 3 summarizes the band maxima and minima and their corresponding ∆ or θ for the spectra of the modified nucleosides. The table also includes data from previous studies by Moore et al. (49) and Cheng et al. (50) including their assigned absolute adduct configurations, as well as the ones deduced from the present results.

Thus, we conclude that (-)-syn-BPDEc-N2-dG and (+)-syn-BPDEc-N2-dG have absolute configuration 7R,8S,9S,10S and 7S,8R,9R,10R, respectively, and (+)syn-BPDEt-N2-dG 7S,8R,9R,10S (Figure 1). The cis configuration of the two first adducts (9S,10S and 9R,10R) and the trans configuration of the last (9R,10S) are therefore demonstrated in a manner completely independent on the initial assignment based on light absorption maxima.The characteristics of the induced CD are an independent method to define the cis and trans adducts of a particular diol epoxide enantiomer. Previous studies by 2D-NMR on the oligonucleotide 5′d(CCATCGCTACC) have shown that the pyrenyl chromophore of the trans adduct derived from (+)-anti-BPDE is located in the minor groove of the duplex and oriented toward the 5′-end of the binding strand. The chromophore of the corresponding adduct of the (-)-enantiomer is oriented in the opposite direction (26, 27; Figure 1). Recent studies have shown that the bases adjacent to the anti-BPDE adduct strongly affect the conformation (51, 52). Thus, it seems premature to conclude that trans adduct of anti-BPDE in general are externally located. Other studies have indicated the importance of the relative conformation of the hydroxyl groups at the C-7 and C-8 position of the diol epoxide in determining both reactivity and to which extent the reaction yields trans or cis adducts (53). The results obtained by optical spectroscopy on the (+)-syn-BPDEt-N2-dG adduct are consistent with minor groove localization of the pyrenyl chromophore. However, the results do not allow any conclusion on the orientation of the chromophore. Assuming that the conformation of the hydroxyl groups is the major determinant in orienting the chromophore in type II complexes toward the 5′- or 3′-end of the binding strand, the recent results would indicate that the chromophore of (+)-syn-BPDEt-N2-dG adduct is oriented toward the 3′-end. If the orientation of the oxirane ring is the major determinant, the chromophore is, like the trans adduct of (+)-anti-BPDE, oriented toward the 5′end of the binding strand. Thermal Stability of Oligonucleotide Duplexes. 5′-d(CCTATAGATATCC) or the oligonucleotide containing (-)-syn-BPDEc-N2-dG or (+)-syn-BPDEc-N2-dG adduct was allowed to form duplexes with the complementary sequence or partially complementary sequences. The thermal stability of the duplexes were studied by monitoring the double stranded-single stranded transition as a function of temperature. The results are compiled in Table 4. As can be seen, the presence of (-)-syn-BPDEc-

Table 3. Band Maxima and Minima and Their Corresponding ∆Ea of the anti- and syn-BPDE Modified dG or G Nucleosides adduct

absolute configuration

(+)-syn-BPDEc-dG (+)-syn-BPDEc-Gb

(9R,10R) (9R,10R)

(-)-syn-BPDEc-dG (-)-syn-BPDEc-Gb

λ (nm)d

∆e

λ (nm)d

∆e

231 239

44 56

250.5 249

-44 -115

274

-14

280

-42

(9S,10S) (9S,10S)

241 240

-28.5 -59

251 247

55 117

275 274

6 7

282 280

25 40

(+)-anti-BPDEt-dG (+)-anti-BPDEt-Gb (+)-anti-BPDEt-dGc

(9R,10S) (9R,10S) (9R,10S)

241.5 237 240

-32 -58 -27f

252 249 248

77 121 54f

276 274 273

-11 -19 -7f

284 280 279

20 27 15f

(-)-anti-BPDEt-dG (-)-anti-BPDEt-Gb (-)-anti-BPDEt-dGc

(9S,10R) (9S,10R) (9S,10R)

241.5 240 240

34 49 27.5f

251.5 249 249

-62 -118 -57f

λ (nm)d

275.5 273 273

∆e

12 14 9f

λ (nm)d

283.5 280 281

∆e

-14 -27 -12f

a The differences in ∆ could be high due to varying accuracy in concentration determination. b Data from Figure 8 in Moore et al. (49), with correct assignment from Sayer et al. (20). c Data from Figure 3 in Cheng et al. (50). d Wavelengths at maxima and minima (nm). e,f CD at maxima and minima (∆ ) M-1 cm-1) or f(θ obs ) m‚deg/AU).

194 Chem. Res. Toxicol., Vol. 9, No. 1, 1996

Ponte´ n et al.

Table 4. Melting Points of Unmodified and (-)- and (+)-syn-BPDE-Modified Matched and Mismatched Duplexes duplex

Tm (°C)(0.5 M NaCl)a

duplex

Tm (°C)(0.5 M NaCl)a

dG/dC (-)-syn-BPDEc-N2-dG/dC (+)-syn-BPDEc-N2-dG/dC

47.0 31.5 28.0

dG/dA (-)-syn-BPDEc-N2-dG/dA (+)-syn-BPDEc-N2-dG/dA

35.0 30.0 27.0

dG/dT (-)-syn-BPDEc-N2-dG/dT (+)-syn-BPDEc-N2-dG/dT

33.0 30.5 29.0

dG/dG (-)-syn-BPDEc-N2-dG/dG (+)-syn-BPDEc-N2-dG/dG

35.0 30.0 26.0

a The results are the means of the T values obtained from the melting profiles (low to high temperature and the reversed order) and m from at least two independent experiments. The variation was in general better than (1.0 °C (SD). Concentration ) 1 µM/strand.

N2-dG or (+)-syn-BPDEc-N2-dG adduct lowered the Tm value by 16.5 and 20 °C, respectively, relative to the unmodified duplex. The destabilizing effect of a cis adduct on the duplex seems to be larger than when trans adducts of anti-BPDE are present in the oligonucleotide (31). This was unexpected since previous results have indicated an increased orientation in flow LD measurements consistent with increased stability of poly-d[(GC)‚(G-C)] modified by racemic syn-BPDE and thus the likely formation of type I complexes (54). Recent results by Ya et al. (55; cf. ref 56) using the oligonucleotide 5′d(CCATCGCTACC) have shown that cis adducts from (+)- or (-)-anti-BPDE had less effect on the Tm value than the corresponding trans adducts; results consistent with a stabilizing effect of molecules which intercalate between base pairs. The reason why the intercalating cis adducts of syn-BPDE appear to be more destabilizing than the corresponding minor groove located trans adducts cannot be explained in simple terms. However, the sequence context and special properties of the cis adducts of syn-BPDE may be important. Regarding the thermal stability of modified mismatched duplexes, replacing dC opposite the adduct with dT, dA, or dG had no further effect on the Tm values relative to the matched duplexes. These results are in contrast to previous observations with the trans adducts of anti-BPDE (31), which showed that dT, dA, or dG inserted opposite the adduct further reduced the thermal stability of the mismatched duplexes compared to the fully complementary duplex (se also refs 55 and 56).

Concluding Remarks The cis adducts of (-)- and (+)-syn-BPDE as well as the trans adduct of (+)-syn-BPDE in 5′-d(CCTATAGATATCC) have been separated and characterized by optical spectroscopy. The results show that each adduct population is homogeneous and can be described with type I and II characteristics, as they were previously defined for anti-BPDE adducts. As illustrated in Figure 1, the spectral types of anti-BPDE adducts are correlated with binding modes, determined independently by NMR. In analogy, we suggest probable binding modes for the observed syn-BPDE adducts; see Figure 1. With the exception of the trans adduct of (-)-syn-BPDE, all possible enantiomeric forms of BPDE-dG adducts in DNA (or model systems) have now been characterized by optical spectroscopy. The previously puzzling heterogeneities observed in optical spectra of BPDE adducts (2124, 29) can now be understood in terms of mixtures of cis and trans adducts in varying proportions.

Acknowledgment. This study has been supported by grants from the Swedish Cancer Society and the Swedish Work Environmental Fund. Kerstin Stro¨m is acknowleded for most valuable help with isolating the

deoxyribonucleoside adducts. Professor Nicholas Geacintov is acknowledged for his very helpful views on the manuscript.

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