Preparation, Isolation, and Characterization of Dibenzo [a, l] pyrene

Eppley Institute for Research in Cancer and Allied Diseases, University of ... Center, 986805 Nebraska Medical Center, Omaha, Nebraska 68198-6805, and...
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Chem. Res. Toxicol. 1999, 12, 789-795

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Preparation, Isolation, and Characterization of Dibenzo[a,l]pyrene Diol Epoxide-Deoxyribonucleoside Monophosphate Adducts by HPLC and Fluorescence Line-Narrowing Spectroscopy Prabu Devanesan,† Freek Ariese,‡,§ Ryszard Jankowiak,‡ Gerald J. Small,‡ Eleanor G. Rogan,† and Ercole L. Cavalieri*,† Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, Nebraska 68198-6805, and Department of Chemistry and Ames Laboratory-U.S. Department of Energy, Iowa State University, Ames, Iowa 50011 Received August 31, 1998

Dibenzo[a,l]pyrene (DB[a,l]P) is the most potent carcinogenic polycyclic aromatic hydrocarbon that has been identified in the environment. Earlier studies in our laboratory indicated that more than 80% of the DB[a,l]P-DNA adducts formed in vitro were depurinating adducts and that most of the stable adducts were formed from diol epoxide intermediates. To complete the profile of both stable and depurinating adducts of DB[a,l]P, we have synthesized standard adducts by reacting 3′-dAMP or 3′-dGMP with either (()-anti- or (()-syn-dibenzo[a,l]pyrene 11,12-dihydrodiol 13,14-epoxide (DB[a,l]PDE). The adducts were separated by HPLC with an ion-pair column and were identified by fluorescence line-narrowing spectroscopy (FLNS). A total of six pairs of stereoisomers along with another stable DB[a,l]PDE-DNA adduct were successfully isolated and identified. Pairs of (()-trans and (()-cis isomers were expected to be formed from the reaction of anti-DB[a,l]PDE with either dAMP or dGMP. While we were able to identify two pairs of stereoisomeric (()-syn-DB[a,l]PDE-dAMP (cis and trans) and two pairs of stereoisomeric (()-anti-DB[a,l]PDE-dAMP (cis and trans) adducts, identification of all the stereoisomers of dGMP adducts proved to be impossible. A pair of (()-syn-trans-DB[a,l]PDEdGMP adducts, a pair of (()-anti-cis-DB[a,l]PDE-dGMP adducts, and one syn-cis-DB[a,l]PDEdGMP adduct were conclusively identified by FLNS. These standard adducts will be used to identify the stable DNA adducts formed by DB[a,l]P and DB[a,l]PDE in vitro and in vivo.

Introduction Dibenzo[a,l]pyrene (DB[a,l]P)1 is the most potent of the carcinogenic polycyclic aromatic hydrocarbons (PAHs) that have been studied thus far (1-4). DB[a,l]P has been tentatively identified in a biologically active fraction of cigarette smoke condensate (5). A definitive identification has been obtained in the particulates formed by combustion of smoky coal (6, 7). The identification and quantification of DNA adducts of DB[a,l]P formed in vitro reported earlier by our laboratory (8) indicated that 84% of the adducts were depurinating adducts. While the structures of the depurinating adducts of DB[a,l]P have been identified, the structures of the stable DNA adducts have not. Comparison of the stable adducts formed with DB[a,l]P and its diol epoxides (DB[a,l]PDE) showed that most of these adducts found after microsomal activation of DB[a,l]P were formed from diol epoxides (8). As with * To whom correspondence should be addressed. † University of Nebraska Medical Center. ‡ Iowa State University. § Present address: Institute for Environmental Studies, Free University, De Boelelaan 1115, 1081 HV Amsterdam, The Netherlands. 1 Abbreviations: BPDE, benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide; dA, deoxyadenosine; DB[a,l]P, dibenzo[a,l]pyrene; DB[a,l]PDE, dibenzo[a,l]pyrene 11,12-dihydrodiol 13,14-epoxide; dG, deoxyguanosine; FLNS, fluorescence line-narrowing spectroscopy; NLN, nonline-narrowed; PAH, polycyclic aromatic hydrocarbon.

other PAHs, the stable adducts of DB[a,l]P have only been quantitatively determined, i.e., the number and relative amounts of these adducts, as observed by the 32Ppostlabeling method. As a first step toward the goal of identifying the stable DNA adducts of DB[a,l]P, we have synthesized standard deoxyribonucleoside monophosphate adducts of (()-synand (()-anti-DB[a,l]]PDE (Figure 1) by reacting the respective epoxides with 3′-deoxyribonucleoside monophosphates. By combining HPLC separation and fluorescence line-narrowing spectroscopy (FLNS) techniques, we were able to successfully isolate and identify most of the expected adducts. These adducts will be used as standards in further studies for identifying the stable DNA adducts formed by DB[a,l]P and DB[a,l]PDE in vitro and in vivo (9).2

Materials and Methods Caution: DB[a,l]P and DB[a,l]PDE are hazardous chemicals and should be handled carefully in accordance with NIH guidelines (10). Preparation of Standard Adducts. Standard deoxyribonucleoside monophosphate adducts were prepared on the basis 2 R. Todorovic, P. Devanesan, E. G. Rogan, and E. L. Cavalieri, Identification and quantitation of stable DNA adducts of dibenzo[a,l]pyrene formed in vitro and in mouse skin and rat mammary gland, to be submitted for publication.

10.1021/tx980202x CCC: $18.00 © 1999 American Chemical Society Published on Web 08/05/1999

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Figure 1. Structures of DB[a,l]PDE adducts obtained by reaction of 3′-dAMP or 3′-dGMP with (A) (()-anti-DB[a,l]PDE and (B) (()-syn-DB[a,l]PDE. of the method employed by Canella et al. (11) for benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide (BPDE) adducts. A 0.1 mg (in 50 µL of Me2SO) solution of either (()-anti-DB[a,l]PDE or (()-synDB[a,l]PDE (National Cancer Institute Chemical Carcinogen Repository, Bethesda, MD) was mixed with 3 mg of 3′-dAMP or 3′-dGMP dissolved in 1 mL of 0.067 M sodium-potassium phosphate (pH 7.0). The mixture was incubated in the dark for 4 h at 37 °C. The reaction mixture was extracted six times with equal volumes of water-saturated ethyl acetate to eliminate the tetraols formed during the incubation. The aqueous phase was evaporated to dryness under argon and analyzed by HPLC. Analysis of Stable DNA Adducts by HPLC. The residue from the reaction mixture was dissolved in a minimal volume of 0.1 M ammonium acetate (pH 5.5)/acetonitrile (2:1). After sonication to enhance solubilization, the undissolved residue was removed by centrifugation and filtration. The adducts were analyzed on a Beckman 5 µm C18 Ultrasphere IP column (4.6 mm × 250 mm) on a Waters 600E multisolvent delivery system, together with a Waters 700 satellite WISP autoinjector (Millipore Corp., Wood Dale, IL). The unmodified nucleotides were eluted first by washing the column for 10 min with 10% acetonitrile (solvent B) in 0.1 M ammonium acetate (pH 5.5) (solvent A). The adducts were then separated by applying a linear gradient to 20% acetonitrile in 10 min, followed by an isocratic wash for 20 min with 20% acetonitrile, and then by applying successive linear gradients to 40% acetonitrile for 20 min and to 100% acetonitrile for 20 min at a flow rate of 1 mL/min. The effluent was monitored for UV absorbance at 254 nm with a Waters 990 photodiode array detector and for fluorescence (λex ) 332 nm, λem ) 388 nm) with a Jasco FP-920 fluorescence detector. Peak fractions were collected for analysis by FLNS. The non-line-narrowed (NLN) fluorescence spectra were obtained with an excitation wavelength of 308 nm at 77 K. Analysis and Identification of Stable DNA Adducts by FLNS. For FLNS characterization, the collected HPLC fractions were dried in a Speedvac instrument and redissolved in 100 µL of a glass-forming mixture (50:50 glycerol/water) with sonication. Aliquots (30 µL) were transferred to 2 mm i.d. quartz tubes, sealed with a rubber septum, and cooled to 4.2 K in a doublenested glass cryostat with quartz optical windows. For FLNS fingerprint characterization, adduct samples were excited using a Lambda Physik Lextra 100 excimer laser/FL-2002 dye laser system. Fluorescence was dispersed by a McPherson 2061 1 m

monochromator (0.08 nm resolution) and detected with a Princeton Instruments IRY-1024/GRB intensified photodiode array detector. For time-resolved detection (to eliminate scattered laser light), a Princeton Instruments FG-100 high-voltage pulse generator was used. The detector delay and gate width were typically 30 and 200 ns, respectively. For FLNS experiments, vibronic excitation into the first excited state (S1 r S0) was employed; the resulting linenarrowed spectra exhibit a multiplet of (0,0) origin transitions, with the energy difference between each FLN peak and the laser excitation energy corresponding to a vibrational level of the excited state of the molecule. Detailed discussions about FLNS and its application to the fingerprint identification of carcinogenDNA adducts have been reported elsewhere (12, 13). Adduct characterization was accomplished by comparison with synthesized DB[a,l]PDE-deoxyadenosine (dA) and DB[a,l]PDE-deoxyguanosine (dG) adduct standards, for which the stereochemistry had been established (14-19).

Results and Discussion Extraction of the reaction mixtures with watersaturated ethyl acetate resulted in elimination of most of the tetraols, the major products from the reaction of DB[a,l]PDE and nucleoside monophosphates. Small amounts of tetraols, however, were still detected on the HPLC system. Washing the column with 90% ammonium acetate (pH 5.5) in acetonitrile after application of the sample resulted in nearly complete elution of the unmodified nucleotides at the beginning of the HPLC run. Lowering the concentration of the buffer at this step, however, reduced the efficiency of this process, leading to incomplete separation of the adducts. Acetonitrile was found to be a better solvent than methanol for the separation of these adducts. The structures of the DB[a,l]PDE adducts obtained by reaction with 3′-dAMP and 3′-dGMP are shown in panels A and B of Figure 1. Adduct Characterization by FLNS. FLNS is a lowtemperature fluorescence technique that yields highresolution, vibrationally resolved fluorescence spectra that can serve as fingerprints for unambiguous identification of PAH and derivatives (12, 13). In recent years,

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Table 1. Comparison of the Fluorescence Origin Bands of Conformation I [(0,0) ∼ 382-385 nm] and Conformation II [(0,0) ∼ 388-390 nm] Observed for syn- and anti-DB[a,l]PDE-dA, -dAMP, -dG, and -dGMP Adductsa adduct

(0,0) (nm) trans

(0,0) (nm) cis

382.0 (major) 388.0 (minor) syn-DB[a,l]PDE-14-N6dAMP 382.5 (major) 388.0 (minor) anti-DB[a,l]PDE-14-N6dA 382.0 (major) ∼389 (minor) 6 anti-DB[a,l]PDE-14-N dAMP 382.5 (major) 388.3 (minor) syn-DB[a,l]PDE-14-N2dGb ∼382.5 (minor) 390.5 (major) 2 syn-DB[a,l]PDE-14-N dGMP 382.5 (major) ∼390 (minor) anti-DB[a,l]PDE-14-N2dG not observedc 389.1 anti-DB[a,l]PDE-14-N2dGMP not observedd

384.0 (major) 389.0 (minor) 384.0 (major) 389.0 (minor) 385.0 (major) 389.5 (minor) ∼385 (major) 389.5 (minor) ∼384 (minor) 390.5 (major) 384.0 (major) ∼390 (minor) not observedc 388.3 not observedc ∼388

syn-DB[a,l]PDE-14-N6dA

T ) 77 K. λex ) 308 nm. The matrix was a 50:50 glycerol/ water glass. Details about structural differences of various adduct conformations can be found elsewhere (18, 19). See also ref 16 in which various conformations of DB[a,l]P tetraols are discussed. b The relative distribution of adduct conformations depends on matrix composition (ref 22 and unpublished results). c Conformation I was not formed. d None of the isolated fractions exhibited spectra identical to those of anti-trans-DB[a,l]PDE-14-N2dG standards. a

we have used FLNS extensively for the identification of DNA adducts from various PAHs, both in vitro and in vivo (20). The usual approach is to synthesize the appropriate standards, record FLN spectra, and then use these standard spectra for the identification of trace amounts of adducts produced in biological systems. In this article, however, we demonstrate that characterization of dGMP and dAMP adducts derived from DB[a,l]PDE can also be accomplished by comparison with FLN spectra of related dG and dA adducts having the same stereochemistry. Since the structure and stereochemistry of dG and dA adducts derived from DB[a,l]PDE have been unambiguously determined (17, 18), these adducts can be used for structure identification of DB[a,l]PDEderived dAMP and dGMP adducts. This has already been demonstrated in the case of BPDE-derived mononucleotide adducts (21). Since some DB[a,l]PDE derivatives show the existence of multiple conformations, indicated by multiple fluorescence origin bands (15, 16, 19), the wavelengths of the fluorescence origin bands of DB[a,l]PDE-dG, -dGMP, -dA, and -dAMP adducts are summarized in Table 1. syn-DB[a,l]PDE-dAMP Adducts. As expected, the reaction of racemic syn-DB[a,l]PDE with 3′-dAMP yielded a mixture consisting of several distinct peaks in the HPLC chromatogram (Figure 2); however, only four fractions corresponded to two (()-syn-trans and (()-syncis-DB[a,l]PDE-derived adducts. The cis adducts were formed in larger amounts than the trans adducts, in contrast to the reaction with anti-DB[a,l]PDE (see below). Three-fold more cis adducts were obtained than trans adducts, with approximately equal amounts of the (+)and (-)-isomers in the cis and trans pairs (Figure 2). The UV spectra of cis and trans adducts are only marginally different from each other. The definitive structures of these adducts were established with the aid of FLNS. Identification of these fractions is based on the similarity of FLN spectra with the spectra obtained for the cis-

Figure 2. HPLC separation profile of syn-DB[a,l]PDE-dAMP adducts.

Figure 3. FLNS characterization of dAMP adducts formed from syn-DB[a,l]PDE (refer to the chromatogram of Figure 2): (a) FLN spectrum of syn-trans-DB[a,l]PDE-dA, (b) FLN spectrum of fraction 1, (c) FLN spectrum of fraction 2, (d) FLN spectrum of syn-cis-DB[a,l]PDE-dA, (e) FLN spectrum of fraction 3, and (f) FLN spectrum of fraction 4. Peaks are labeled with their excited state vibrational frequencies in cm-1. λex ) 374.0 nm. T ) 4.2 K.

and trans-syn-DB[a,l]PDE-dA standards (Figure 3). Spectra a and d were obtained for syn-trans- and syncis-DB[a,l]PDE-dA standards, and are compared with FLN spectra acquired for fractions 1 and 2 (spectra b and c) and fractions 3 and 4 of Figure 2 (spectra e and f), respectively. All spectra shown in Figure 3 were obtained for an excitation wavelength of 374.0 nm. Comparison of the data shown in Figure 3 reveals that the frequencies and relative intensities of the vibrational modes of fractions 1 and 2 (spectra b and c) and fractions 3 and 4 (spectra e and f) are practically indistinguishable from those of the corresponding FLN spectra obtained for the syn-trans- and syn-cis-DB[a,l]PDE-dA standards. This indicates that spectra b and c (fractions 1 and 2 of Figure 2) correspond to a pair of (()-trans stereoisomers of synDB[a,l]PDE-dAMP, whereas spectra e and f (fractions 3 and 4) correspond to the pair of (()-cis stereoisomers

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Figure 4. HPLC separation profile of anti-DB[a,l]PDE-dAMP adducts.

of syn-DB[a,l]PDE-dAMP adducts. As expected, the presence of the phosphate group, which is many bonds away from the chromophore, has no dramatic effect on the FLN spectra. This assignment was confirmed for several other excitation wavelengths (data not shown). The fact that cis- and trans-opened adducts yield very different spectra is in accordance with our previous experience with BPDE adducts (21) and DB[a,l]P tetraols (16). The assignment of the absolute stereochemistry of these adducts cannot be determined by FLNS because the (+)- and (-)-isomers are indistinguishable. anti-DB[a,l]PDE-dAMP Adducts. The reaction of racemic anti-DB[a,l]PDE with 3′-dAMP produced several peaks in the HPLC separation profile corresponding to either adducts or metabolites (Figure 4). Four of the fractions corresponded to anti-DB[a,l]PDE-dAMP-derived adducts, one pair of trans isomers and one pair of cis isomers. More trans adducts were obtained than cis adducts, whereas each of the cis or trans isomers was formed in equal amounts. The UV spectra of the trans adducts were distinguishable from those of the cis isomers, but the structures of these adducts were definitively established by FLNS. Detailed high-resolution FLNS studies of all fractions yielded two pairs of identical spectra for peaks 1 and 2, and fractions 3 and 4. The FLN spectrum of one of each pair is shown in Figure 5 (spectra b and d, respectively). These spectra are compared with the FLN spectra obtained for anti-trans-DB[a,l]PDE-dA (spectrum a) and anti-cis-DB[a,l]PDE-dA (curve c) adduct standards. The FLN spectra of fraction 1 (curve b) and of anti-trans-DB[a,l]PDE-dA (curve a) are very similar, indicating that these compounds must have the same stereochemistry of substitution (i.e., trans). Similarly, the FLN spectrum d obtained for fraction 3 is very similar to that of anticis-DB[a,l]PDE-dA (curve c). FLN spectra for fractions 2 and 4 were identical to the spectra of fractions 1 and 3 (data not shown). Therefore, peaks 1 and 2 of Figure 4 correspond to the trans-opened (+)- and (-)-stereoisomers of anti-DB[a,l]PDE-dAMP, whereas peaks 3 and 4 contain the corresponding (+)- and (-)-cis adducts. The (()-stereochemistry of the adduct pairs has not been assigned. The results are very similar to those obtained by reaction of DB[a,l]PDE with dA in dimethylformamide (17); namely, (()-anti-DB[a,l]PDE produced more ad-

Figure 5. FLNS characterization of dAMP adducts formed from anti-DB[a,l]PDE (refer to the chromatogram of Figure 4): (a) FLN spectrum of anti-trans-DB[a,l]PDE-dA, (b) FLN spectrum of fraction 1, (c) FLN spectrum of anti-cis-DB[a,l]PDEdA, and (d) FLN spectrum of fraction 3. Peaks are labeled with their excited state vibrational frequencies in cm-1. λex ) 378.0 nm. T ) 4.2 K.

Figure 6. HPLC separation profile of syn-DB[a,l]PDE-dGMP adducts. The insets are HPLC separation profiles of reinjected peaks 1 and 2.

ducts trans-opened at C-14, whereas (()-syn-DB[a,l]PDE yielded more cis-opened adducts (see above). syn-DB[a,l]PDE-dGMPAdducts.Theadductsformed by the reaction of syn-DB[a,l]PDE with 3′-dGMP do not separate as well as the dAMP adducts under any of the various chromatographic conditions that were tried. synDB[a,l]PDE is about half as reactive with dGMP as with dAMP. The individual isomers were obtained by reinjecting the collected fractions and separating them under isocratic conditions [25% acetonitrile/75% 0.1 M ammonium acetate (pH 5.5)]. The separation profile of synDB[a,l]PDE-dGMP adducts is shown in Figure 6. The first major peak (1) separated into two peaks which were identified as trans adducts, as described below. Reinjection of the second peak (2) also yielded two fractions, one of which was identified as a cis isomer. The identity of the adducts was established by using FLNS.

DB[a,l]PDE-Deoxynucleoside Monophosphate Adducts

Figure 7. NLN fluorescence spectra obtained for syn-transDB[a,l]PDE-dG (spectrum a) and syn-cis-DB[a,l]PDE-dG (spectrum c) adducts. Spectra b and d were obtained for fractions 1a and 2a of the chromatogram shown in Figure 6, respectively. λex ) 308 nm. T ) 77 K. Spectral differences are due to differences in conformational equilibria; see the text.

Identification of dGMP adducts and assignment of the cis and trans stereochemistry was carried out by comparison with corresponding dG adduct standards characterized in detail elsewhere (18). In this case, however, the comparison was not as straightforward as in the case of dAMP adducts, since syn-dGMP and syn-dG adducts do not separate very well, and these dGMP and dG adducts also appear to exist in different distributions of adduct conformations (see Table 1). More specifically, the minor conformation of the well-characterized syn-transdG and syn-cis-dG adducts becomes the major conformation of the corresponding dGMP adducts. The presence of multiple conformations of DB[a,l]P tetraols and of N7Ade and N7Gua adducts derived from DB[a,l]PDE has already been demonstrated (15, 16). Specifically, it has been shown that DB[a,l]PDE-derived species may adopt two conformations (I and II) with their fluorescence origin bands shifted by several nanometers (15, 16). Adduct conformations differ in the structure of the cyclohexenyl ring and the position of Ade (or Gua) relative to the PAH-type fluorescent chromophore of these adducts. This aspect, however, is beyond the scope of this paper and is discussed in detail elsewhere (15, 16, 19). Fractions 1a and 2a (Figure 6) were expected to correspond to one of the stereoisomers of the syn-transdGMP and syn-cis-dGMP adducts, respectively. Both yielded, however, NLN fluorescence spectra that were different from those of the corresponding dG adduct standards. Although two similar fluorescence origin bands have been observed for both the dG adduct standards and dGMP adduct fractions, their relative contribution is reversed (Table 1). Namely, fractions 1a and 2a exhibit much stronger emission in the region of conformation I, as shown in Figure 7 (spectra b and d, respectively). Spectra a and c were obtained from standard syn-trans-dG and syn-cis-dG adducts (18). These data indicate that minor conformations with their (0,0) bands observed at 382.5 nm for syn-trans- and ∼384 nm for syn-cis-dG adducts may have become major conforma-

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Figure 8. Identification of syn-DB[a,l]PDE-dGMP adducts based on similarity with syn-DB[a,l]PDE-dG adducts. Spectra a and c were obtained for syn-trans-dG and syn-cis-dG adduct standards, respectively, and spectra b and d were obtained for fractions 1a and 2a of the HPLC chromatogram shown in Figure 6, respectively. λex ) 376.0 nm. T ) 4.2 K.

tions in the case of syn-dGMP adducts, since strong (0,0) bands are observed at ∼383 nm for these dGMP adduct fractions. We emphasize that the relative distribution of conformations I and II of syn-dG adducts depends on matrix composition (data not shown). This provides additional support that these adducts exist in different conformations. The hypothesis of whether the minor conformation I of syn-trans-dG and syn-cis-dG adducts really corresponds to the respective (in this case, major) conformation I of syn-trans-dGMP and syn-cis-dGMP can be confirmed via FLN spectroscopy. This is demonstrated in Figure 8. Spectra a and c were obtained for minor conformation I of syn-trans-dG and syn-cis-dG standards (18), whereas spectra b and d correspond to peaks 1a and 2a of Figure 6. Comparison reveals that spectrum a is very similar to spectrum b, and spectrum c is similar to d; therefore, peak 1a of Figure 6 corresponds to one of the stereoisomers of the trans-opened adducts of (()-syn-DB[a,l]PDE with dGMP, whereas fraction 2a corresponds to either the (+)- or (-)-stereoisomer of syn-cis-DB[a,l]PDEdGMP. The FLN spectra for fraction 1b of Figure 6 were identical to those of fraction 1a (data not shown); thus, 1a and 1b must correspond to a pair of (+)- and (-)stereoisomers of the syn-trans-dGMP adducts. Spectra obtained for fraction 2b, however, were different from those obtained for fraction 2a; thus, the main contribution in fraction 2b must correspond to an unidentified product. Therefore, only one stereoisomer of the syn-cis-dGMP adducts has been identified. Fractions 1a and 1b, identified as syn-trans-dGMP adducts, also seem to contain a small contribution from syn-cis-dGMP (deconvolution spectra not shown), which could indicate that the second minor stereoisomer of syn-cis-dGMP does not separate well from the syn-trans-dGMP adducts, and elutes in the vicinity of fractions 1a and 1b. This would indicate that even with two successive HPLC runs, the separation of adducts and/or metabolites may not be complete. Reinjection of peak 3 (Figure 6) revealed the presence of several compounds (chromatogram not shown); none

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Figure 9. HPLC separation profile of anti-DB[a,l]PDE-dGMP adducts.

of those fractions, however, was identified as a syn-DB[a,l]PDE-dGMP adduct. Finally, assignment of fraction 2a as syn-cis-DB[a,l]PDE-dGMP is supported by the fact that FLN spectra for fraction 2a and for syn-cis-DB[a,l]PDE-dAMP exhibit similarities in terms of vibrational frequencies and intensities of modes. While there are some differences in the vibrational modes, the general similarity is not surprising, since FLN spectra of dAMP and dGMP adducts can be expected to feature many similarities, provided the stereochemistry and conformation of the saturated ring are identical. This is especially true if the bases (Gua and Ade) are attached via the same type of bond, i.e., the exocyclic amino group. anti-DB[a,l]PDE-dGMP Adducts. Characterization of dGMP adducts formed with anti-DB[a,l]PDE proved to be the most difficult. Although, as shown in Figure 9, the standard HPLC gradient conditions resolved several peaks that could correspond to anti-dGMP adducts, highresolution FLN spectra indicated that only two of the fractions are anti-dGMP adducts. FLNS identification, based on comparison of the FLN spectra of anti-dGMP adduct fractions with those of authentic standards of anti-cis-dG (18), is shown in Figure 10. Several excitation wavelengths were chosen to reveal mode structure in the 450-950 cm-1 range. Figure 10 shows the FLN spectra obtained for a λex of 379.0 nm (spectra a and b) and a λex of 376.0 nm (spectra c and d). Spectra a and c were obtained for the anti-cis-DB[a,l]PDE-dG standard, whereas spectra b and d correspond to fraction 1 of the chromatogram shown in Figure 9. Small differences in the relative intensity distribution of the zero-phonon lines (Figure 10) for the dGMP-type adducts is caused by relatively small “blue” shifts (∼0.5 nm) of their (0,0) origin bands. Since spectrum a is very similar to spectrum b, and spectrum c is very similar to spectrum d, we concluded that fraction 1 corresponds to one of the stereoisomers of anti-cis-DB[a,l]PDE-dGMP. Fractions 2-4, however, did not resemble any of our available standard adducts or DB[a,l]P tetraols (ref 16 and unpublished results); therefore, these fractions were reinjected to carry out a second isocratic HPLC separation, as described earlier, to further separate the analytes. The reseparated fraction 1 exhibited only one peak and was not analyzed again. Reseparated fraction 2 exhibited a contribution from anti-cis-DB[a,l]PDE-

Figure 10. Characterization of anti-cis-dGMP adducts formed from anti-DB[a,l]PDE. Spectra a and b, and c and d, were obtained at a λex of 379.0 nm, and a λex of 376.0 nm, respectively. Curves a and c correspond to the anti-cis-dG standard, and spectra b and d are FLN spectra obtained for fraction 1 of Figure 9. Peaks are labeled with their excited state vibrational frequencies in cm-1. T ) 4.2 K. Table 2. Summary of Identified Adducts (()-syn-cis-DB[a,l]PDE-14-N6dAMP (()-syn-trans-DB[a,l]PDE-14-N6dAMP (()-anti-cis-DB[a,l]PDE-14-N6dAMP (()-anti-trans-DB[a,l]PDE-14-N6dAMP (()-syn-trans-DB[a,l]PDE-14-N2dGMP (()-anti-cis-DB[a,l]PDE-14-N2dGMP (()-syn-cis-DB[a,l]PDE-14-N2dGMP

dGMP and an unknown product. None of the HPLCreseparated fractions 3 and 4, however, exhibited spectra identical to those of anti-trans-DB[a,l]PDE-dG standards. Therefore, only the two stereoisomers of anti-cisDB[a,l]PDE-dGMP were identified, whereas the antitrans adducts were not. The latter is consistent with our recent, unpublished data showing that in general syndGMP adducts are more stable than anti-dGMP adducts, with the anti-trans-DB[a,l]PDE-dGMP being the most unstable. In conclusion, six of the eight possible pairs of adducts and one additional adduct were separated by HPLC and identified by FLNS (Table 2).

Conclusions A relatively simple methodology has been developed for separation and identification of standard nucleoside monophosphate adducts of syn- and anti-DB[a,l]PDE. A total of six pairs of stereoisomeric adducts along with another adduct have been successfully identified from the reaction of (()-anti-DB[a,l]PDE and (()-syn-DB[a,l]PDE with 3′-dAMP and 3′-dGMP and isolated by means of HPLC. The structures of these adducts were confirmed by comparing their FLN spectra with those of syn- and anti-dA and dG standard adducts. With this technique, adducts from anti-DB[a,l]PDE were easily distinguished from syn-DB[a,l]PDE adducts, and the spectra of cisopened adducts were shown to be very different from those of trans-opened adducts. Although six pairs along with another single adduct of the expected eight pairs of syn- and anti-DB[a,l]PDE-dAMP and -dGMP adducts

DB[a,l]PDE-Deoxynucleoside Monophosphate Adducts

have been identified, the (()-stereochemistry of all of these mononucleotide adducts was not established. In fact, although the FLN spectra obtained for the (+)- and (-)-fractions for a given adduct are identical, the adducts separate into distinct peaks on HPLC. Thus, FLNS can distinguish a (+)- and (-)-pair from other pairs of adducts. As shown in Table 1, seven of the eight antiand syn-dAMP and dGMP adducts have been identified. Although the anti-trans-DB[a,l]PDE-dGMP adduct has not been positively identified, this particular adduct could be very unstable (see above) or may be trapped at low temperatures in a conformation that is significantly different from that of the corresponding dG adduct standard. If this is the case, a direct comparison cannot be made. This issue may require detailed theoretical modeling. Nevertheless, these adducts serve as standards for the FLNS identification of stable DB[a,l]PDE-DNA adducts formed both in vitro and in vivo (9).2

Acknowledgment. We thank Dr. Kai-Ming Li for the synthesis of DB[a,l]PDE-dA and DB[a,l]PDE-dG adducts and Mr. Dan Zamzow for his contributions to the FLNS analysis. This research was supported by U.S. Public Health Service grants from the National Cancer Institute (R01 CA49917 and P01 CA49210). Core support at the Eppley Institute was provided by NCI Laboratory Cancer Research Center Support Grant CA36727.

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