pyrene and Dibenzo[a,l] - ACS Publications - American Chemical

Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical. Center, 986805 Nebraska Medical Center, Omaha, Nebraska ...
0 downloads 0 Views 90KB Size
Download PDF
796

Chem. Res. Toxicol. 1999, 12, 796-801

A Novel Method for the Isolation and Identification of Stable DNA Adducts Formed by Dibenzo[a,l]pyrene and Dibenzo[a,l]pyrene 11,12-Dihydrodiol 13,14-Epoxides in Vitro 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

Our laboratory previously reported the identification and quantification of depurinating DNA adducts of dibenzo[a,l]pyrene (DB[a,l]P) in vitro, which comprise about 84% of all the DNA adducts that are formed [Li, K.-M., et al. (1995) Biochemistry 34, 8043-8049]. To determine a complete adduct profile and identify both stable and depurinating DNA adducts, we have developed a relatively simple, nonradioactive method for the identification of stable DNA adducts by combining enzymatic digestion, HPLC, and fluorescence line-narrowing spectroscopy (FLNS) techniques. Calf thymus DNA, bound to either (()-anti- or (()-syn-DB[a,l]PDE or rat liver microsome-activated DB[a,l]P, was first digested to 3′-mononucleotides with micrococcal nuclease and spleen phosphodiesterase. The adducts were then separated by HPLC with an ion-pair column and identified by FLNS by using the spectra of standards for comparison. In reactions with (()-anti-DB[a,l]PDE, three adducts, an anti-cis-DB[a,l]PDE-dGMP, an antitrans-DB[a,l]PDE-dAMP, and an anti-cis-DB[a,l]PDE-dAMP, were identified by HPLC and FLNS. In reactions with (()-syn-DB[a,l]PDE, a pair of syn-trans-DB[a,l]PDE-dGMP adducts as well as a syn-cis-DB[a,l]PDE-dGMP, a syn-cis-DB[a,l]PDE-dAMP, and a pair of syn-transDB[a,l]PDE-dAMP adducts were identified. From the digest of microsome-activated DB[a,l]Pbound DNA, a syn-trans-DB[a,l]PDE-dGMP, an anti-cis-DB[a,l]PDE-dGMP, a syn-transDB[a,l]PDE-dAMP, and a syn-cis-DB[a,l]PDE-dAMP adduct were identified. An anti-cisDB[a,l]PDE-dAMP adduct was identified only by 32P-postlabeling. A total of five of the stable adducts formed by DB[a,l]P and nine of the stable adducts formed by DB[a,l]PDE in vitro have been identified. These adducts were also correlated to adduct spots in the 32P-postlabeling method by cochromatography with standards. Approximately 93% of the stable adducts formed in reactions with (()-anti-DB[a,l]PDE, 90% of adducts with (()-syn-DB[a,l]PDE, and 85% of adducts formed with microsome-activated DB[a,l]P have been identified as Gua or Ade adducts. Equal amounts of stable Gua and Ade adducts were observed in the microsome-catalyzed binding of DB[a,l]P to calf thymus DNA, while 1.4 times more Gua adducts than Ade adducts were obtained in reactions with (()-anti- or (()-syn-DB[a,l]PDE.

Introduction Formation of DNA adducts is thought to be a pivotal event in the tumor-initiating activity of polycyclic aromatic hydrocarbons (PAHs)1 and other carcinogens. PAHs, such as dibenzo[a,l]pyrene (DB[a,l]P) and benzo[a]pyrene (BP), are metabolically activated primarily by two mechanisms, one-electron oxidation and the diol epoxide pathway (1, 2). Activation by either mechanism and subsequent binding to DNA give rise to both stable * To whom correspondence should be addressed. † University of Nebraska Medical Center. ‡ Iowa State University. § Present address: Institute for Environmental Studies, Free University, De Boelalaan, 1115, 1081 HV Amsterdam, The Netherlands. 1 Abbreviations: BP, benzo[a]pyrene; DB[a,l]P, dibenzo[a,l]pyrene; DB[a,l]PDE, dibenzo[a,l]pyrene 11,12-dihydrodiol 13,14-epoxide; FLNS, fluorescence line-narrowing spectroscopy; PAH, polycyclic aromatic hydrocarbon.

and depurinating DNA adducts. Depurinating adducts are lost from DNA by cleavage of the glycosidic bond of the Gua and Ade nucleotides, whereas stable adducts result from covalent binding of a carcinogen at the exocyclic amino group of either Gua or Ade. Formation of DNA adducts is thought to be an essential step in tumor initiation. Stable DNA adducts are normally identified by the 32Ppostlabeling technique. This method, however, suffers from a major flaw. Although it provides information about the number and the relative amounts of the adducts formed, the method does not provide much information about the actual identity of the adducts. The few researchers who have tried to identify stable DNA adducts have relied on performing HPLC with 32Pradiolabeled nucleotide fractions. Such methods have been used to identify BP diol epoxide adducts (3, 4), other

10.1021/tx980203p CCC: $18.00 © 1999 American Chemical Society Published on Web 08/05/1999

Identification of Stable DB[a,l]P-DNA Adducts

PAH adducts (5, 6), and alkylguanine adducts (7-9). Lau and Baird have also used 35S-labeling and HPLC to identify BP adducts (10). Identification and quantitation of the depurinating DNA adducts and quantitation of the stable adducts formed in vitro by reaction of DB[a,l]P 11,12-dihydrodiol 13,14-epoxide (DB[a,l]PDE) or microsome-activated DB[a,l]P was reported by our laboratory (11). When DB[a,l]PDE was activated by microsomes, 84% of all the adducts were depurinating adducts formed either by one-electron oxidation (50%) or via the diol epoxide pathway (34%). The stable adducts, representing 16% of the total adducts, were predominantly formed by the DB[a,l]PDE pathway. While the depurinating adducts were identified, the structures of the stable adducts were not determined. To complete the identification of the stable adducts, we developed a nonradioactive technique capable of identifying these adducts, as well as those formed by other PAH. Ralston et al. (12) identified some of the stable adducts of DB[a,l]PDE formed in the human mammary carcinoma cell line MCF-7 by using 33P-postlabeling and HPLC. Jankowiak et al. (13) recently reported identification of stable DB[a,l]P-DNA adducts formed in vitro by microsomal activation and in vivo in mouse skin. The identification utilized the 32P-postlabeling technique. However, until now no one has succeeded in isolating and identifying individual stable DB[a,l]P-DNA adducts without using a radiolabel. Our aim was to develop a sensitive, nonradioactive technique for purifying and identifying stable adducts of DB[a,l]P. As a first step, we synthesized standard nucleoside monophosphate adducts of DB[a,l]PDE (14). With the aid of these standard adducts, we have developed a relatively simple nonradioactive method for elucidating the identities of the stable DNA adducts by combining DNA digestion, HPLC, and fluorescence linenarrowing spectroscopy (FLNS). The identified adducts were further correlated to the adduct spots observed in the 32P-postlabeling method by using cochromatography with standards. In this paper, we report the successful identification of five of the stable adducts (85% of the stable adducts) obtained by reaction of microsomeactivated DB[a,l]P with DNA and nine of the stable adducts (at least 90% of the stable adducts) formed by DB[a,l]PDE and DNA in vitro.

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 (15). Binding of PAH to Calf Thymus DNA. Reactions of DB[a,l]PDE with calf thymus DNA were carried out in 15 mL mixtures by incubating (()-syn- or (()-anti-DB[a,l]PDE (80 µM) with 3 mM calf thymus DNA in 150 mM Tris-HCl (pH 7.5), 150 mM KCl, and 5 mM MgCl2 at 37 °C for 2 h in the dark. DB[a,l]P was bound to calf thymus DNA in reactions catalyzed by 3-methylcholanthrene-induced rat liver microsomes (1 mg/mL). Each 15 mL reaction mixture containing 3 mM DNA in 150 mM Tris-HCl (pH 7.5), 150 mM KCl, 5 mM MgCl2, 0.6 mM NADPH, 80 µM DB[a,l]P, and microsomal protein was incubated for 1 h at 37 °C (16). Both reaction mixtures were extracted twice with equal volumes of chloroform. The DNA was precipitated with absolute ethanol and redissolved in 10 mL of DNA digestion buffer [50 mM Tris-HCl and 10 mM CaCl2 (pH 8.5)]. Digestion of DNA to 3′-Monophosphates. DNA digestion to 3′-monophosphates was carried by using a modified procedure employed by Gorelick and Wogan (6). PAH-bound DNA (1.5 mM)

Chem. Res. Toxicol., Vol. 12, No. 9, 1999 797 in 10 mL of digestion buffer was incubated with 10 000 units of micrococcal nuclease for 6 h at 37 °C. Incubation was continued for another 16 h after addition of 5 mL of 250 mM HEPES buffer (pH 6.8) and 20 units of spleen phosphodiesterase. The mixture was evaporated to dryness, and the stable adducts of DNA were analyzed by HPLC. Separation and Analysis of Stable 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, followed by linear gradients to 40% acetonitrile over the course of 20 min and to 100% acetonitrile over the course of 20 min at a flow rate of 1 mL/min. The effluent was monitored for UV absorbance with a Waters 990 photodiode array detector and for fluorescence (λex ) 332 nm, λem ) 388 nm) with a Jasco FP920 fluorescence detector. Peak fractions were collected for analysis by FLNS and by 32P-postlabeling. Before further structural analysis, each of the collected fractions was purified for a second time by reinjection and separation with a second HPLC gradient. This gradient consisted of an isocratic wash with 20% acetonitrile in 0.1 M ammonium acetate (pH 5.5) for 10 min, followed by linear gradients to 40% acetonitrile over the course of 30 min and then to 100% acetonitrile over the course of 30 min at a flow rate of 1 mL/min. Analysis of Stable Adducts by FLNS. For FLNS characterization, the collected HPLC fractions were dried in a Speedvac apparatus and redissolved in 100 µL of a glass-forming mixture (50:50 glycerol/water) with the help of sonication. Aliquots (30 µL) were transferred to 2 mm i.d. quartz tubes, and the tubes were sealed with a rubber septum and cooled to 4.2 K in a double-nested glass cryostat with quartz optical windows. For FLNS fingerprint characterization, adduct samples were probed with 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, a Princeton Instruments FG-100 high-voltage pulse generator was used with delay and gate widths of 30 and 200 ns, respectively. For the 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; the energy difference between each FLN peak and the laser excitation energy corresponds to a vibrational level of the excited state of the molecule. FLNS and its application to the fingerprint identification of carcinogen-DNA adducts have been described in detail previously (17, 18). Adduct characterization was accomplished by comparison with previously synthesized DB[a,l]PDE-dAMP and DB[a,l]PDE-dGMP adduct standards (14). Analysis of Stable Adducts by 32P-Postlabeling. A 1 µL aliquot of the collected fraction(s), diluted 50-100-fold, containing PAH-adducted nucleoside monophosphates was labeled with 32P by using T4 polynucleotide kinase. The reaction mixture was applied to a PEI-cellulose TLC plate, and the adduct spots were separated by two-dimensional TLC on 10 cm × 13 cm PEIcellulose plates as previously described (16). The mobilities of the standard adducts on the TLC plate were compared with the adduct profile obtained for calf thymus DNA reacted with DB[a,l]PDE or microsome-activated DB[a,l]P. These adduct profiles were obtained by using the regular 32P-postlabeling procedure

798 Chem. Res. Toxicol., Vol. 12, No. 9, 1999

Devanesan et al.

Figure 1. HPLC separation profile of DNA reacted with antiDB[a,l]PDE and digested to 3′-monophosphates. Peaks are labeled with adducts identified by FLNS. described elsewhere (16). The standards were also cochromatographed with the in vitro DNA samples to further confirm their assigned identity.

Results and Discussion Elution of the adduct peaks from the HPLC column was monitored by their characteristic fluorescence. Despite several attempts to preclean the reaction mixture with Sep-Pak cartridges prior to HPLC, the best results were obtained when the samples were not subjected to such a procedure. Unmodified nucleotides were removed first by washing the column with 90% ammonium acetate in acetonitrile. Dipple and Pigott (19) previously reported that prolonging the time of enzymatic digestion increased the rate of release of Ade adducts of 7,12-dimethylbenz[a]anthracene. Neither the HPLC profiles nor the amount of recovered stable Ade or Gua adducts from DB[a,l]PDE or DB[a,l]P was significantly different with prolonged incubation times, even up to 24 h. Approximately the same amount of Ade adducts was obtained after incubations with micrococcal nuclease and spleen phosphodiesterase for g2 hours each. Adducts Formed by Reaction of (()-anti-DB[a,l]PDE with DNA. Reaction of anti-DB[a,l]PDE with calf thymus DNA and subsequent digestion to 3′-monophosphates yielded five major HPLC peaks and a number of smaller peaks in the adduct region (Figure 1). Since the peaks are crowded together in the chromatogram, identification could not be based solely on retention times. The peaks were collected and analyzed independently for identification. As an illustration, FLNS identification of two fractions is shown in Figure 2. The fraction collected at 54.3 min yielded an FLN spectrum identical to that of anti-cis-DB[a,l]PDE-dGMP. The similarity of the vibronic transitions using excitation at 378.00 nm is shown in Figure 2A. Matching spectra were also obtained at other excitation wavelengths probing different vibronic regions (not shown). The spectra in Figure 2B illustrate the identification of one of the minor peaks; the fraction collected at 56.6 min yielded an FLN spectrum identical with that of the anti-trans-DB[a,l]PDE-dAMP standard. A second small peak (58.9 min) had FLN spectra corresponding to one of the anti-cis-DB[a,l]PDE-dAMP adducts (data not shown). The identified adducts were correlated to adduct spots observed with the 32P-postlabeling technique, based on comigration with their respective standards (Figure 3). The three TLC spots corresponding to the adducts

Figure 2. Illustration of FLNS identification of stable DNA adducts formed from anti-DB[a,l]PDE (refer to the chromatogram of Figure 1). (A) FLN spectrum of the anti-cis-DB[a,l]PDE-dGMP standard (spectrum a) in comparison with that of the 54.3 min fraction (spectrum b); λex ) 378.00 nm. (B) FLN spectrum of anti-trans-DB[a,l]PDE-dAMP (spectrum c) in comparison with that of the 56.6 min fraction (spectrum d); λex ) 379.00 nm. Peaks are labeled with their excited state vibrational frequencies in cm-1. T ) 4.2 K.

Figure 3. Autoradiogram of 32P-postlabeled DNA containing stable adducts formed by reaction of (()-anti-DB[a,l]PDE with DNA. The film was exposed at room temperature for 2 min.

confirmed by FLNS, anti-cis-DB[a,l]PDE-dGMP, antitrans-DB[a,l]PDE-dAMP, and anti-cis-DB[a,l]PDEdAMP, were identified. In addition, two of the adduct spots, corresponding to the second and third major peaks eluting after 50 min in the HPLC chromatogram (Figure 1), comigrated with Gua-derived adduct standards (Figure 3). Although they appear to be Gua adducts, their structure could not be assigned unequivocally by FLNS due to the complicated behavior of Gua adducts (14). The unidentified HPLC peaks did not have FLN spectra corresponding to the known standards. The spots not designated as adducts did not comigrate with any of the standards. On the basis of the quantitation of radioactive adduct spots from the 32P-postlabeling technique, 93% of all the adducts were identified as either Ade or Gua adducts. It was estimated that approximately 58% of these were Gua adducts, while 42% were Ade adducts. The smaller size of the Ade adduct peaks in the HPLC chromatogram can be attributed to weaker fluorescence of these Ade adducts that was observed under the HPLC conditions. Differences in the fluorescence of Ade and Gua adducts were expected since such differences have also been observed with other PAHs (unpublished results).

Identification of Stable DB[a,l]P-DNA Adducts

Chem. Res. Toxicol., Vol. 12, No. 9, 1999 799

Figure 6. Autoradiogram of 32P-postlabeled DNA containing stable adducts formed by reaction of (()-syn-DB[a,l]PDE with DNA. The film was exposed at room temperature for 2 min.

Figure 4. HPLC separation profile of DNA reacted with synDB[a,l]PDE and digested to 3′-monophosphates. Peaks are labeled with adducts identified by FLNS.

Figure 7. HPLC separation profile of DNA reacted with DB[a,l]P in the presence of rat liver microsomes and digested to 3′-monophosphates. Peaks are labeled with adducts identified by FLNS.

Figure 5. Illustration of FLNS identification of stable DNA adducts formed from syn-DB[a,l]PDE (refer to the chromatogram of Figure 4). (A) FLN spectrum of the syn-cis-DB[a,l]PDEdGMP standard (spectrum a) in comparison with that of fraction 56A (spectrum b). (B) FLN spectrum of syn-cis-DB[a,l]PDEdAMP (spectrum c) in comparison with that of the 60.5 min fraction (spectrum d). Peaks are labeled with their excited state vibrational frequencies in cm-1. λex ) 376.00 nm. T ) 4.2 K.

Adducts Formed by Reaction of (()-syn-DB[a,l]PDE with DNA. Four major peaks and several smaller peaks can be observed in the adduct region of the chromatogram for the separation of the syn-DB[a,l]PDEDNA digestion products (Figure 4). Several peaks could be identified on the basis of a combination of HPLC retention times and FLN spectra of the collected fractions. Two of the major adduct peaks were identified as corresponding to the isomeric pair of syn-trans-DB[a,l]PDE-dGMP adducts, while a third was identified as a syn-cis-DB[a,l]PDE-dGMP adduct. A pair of syn-cis-DB[a,l]PDE-dAMP adducts and a syn-trans-DB[a,l]PDEdAMP adduct were identified among the minor peaks. The FLN spectrum of fraction 56A (reinjected) was nearly identical to that of the syn-cis-DB[a,l]PDE-dGMP standard (Figure 5A). One of the minor adducts was identified as syn-cis-DB[a,l]PDE-dAMP (Figure 5B). It was observed that there are only small differences between the spectra of the dAMP and dGMP adducts. The spectra, recorded using identical excitation wavelengths, exhibit only minor differences, for instance in the 540-560 cm-1 region. As the two adducts have

identical stereochemistries (syn-cis) and the Gua and Ade moieties are bound to the chromophore via the same exocyclic amino group, this similarity could be expected and agrees with previous findings (20, 21). The unidentified HPLC peaks did not exhibit FLN spectra matching any of the standards described in an accompanying article (14). In the 32P-postlabeling method, syn-cis-DB[a,l]PDEdAMP, syn-trans-DB[a,l]PDE-dGMP, and syn-trans-DB[a,l]PDE-dAMP were identified as corresponding to unique adduct spots (Figure 6) on the basis of their mobilities on the TLC plate. It should be noted that the standards for syn-cis-DB[a,l]PDE-dGMP and syn-transDB[a,l]PDE-dGMP comigrate as one spot on the TLC plate. The rest of the adduct spots could not be identified since they did not comigrate with any of the standard adducts. Quantitation of the radioactive adduct spots indicates that 90% of all stable adducts were either Ade or Gua adducts, and of these, 58% were Gua adducts and 42% were Ade adducts formed with (()-syn-DB[a,l]PDE; the same relative percentage of Gua and Ade adducts was found in the reaction of (()-anti-DB[a,l]PDE and DNA (see above). Adducts Formed by Reaction of MicrosomeActivated DB[a,l]P with DNA. Adduct peaks of microsome-activated DB[a,l]P bound to calf thymus DNA could not be detected by means of UV because of the small amounts that were formed, but were easily observed with the fluorescence detector. The unmodified nucleotides elute from the column with the initial wash and do not interfere with the separation of the adducts. Five major peaks and a number of smaller ones were detected (Figure 7). Upon reinjection and separation with an isocratic HPLC run, some of the peaks were found to contain more than one adduct. Sometimes even reinjection did not lead

800 Chem. Res. Toxicol., Vol. 12, No. 9, 1999

Devanesan et al.

Figure 9. Autoradiogram of 32P-postlabeled DNA containing stable adducts formed by reaction of microsome-activated DB[a,l]P with DNA. The film was exposed at room temperature for 30 min. Table 1. Stable Adducts Identified after Reaction of (()-syn- or (()-anti-DB[a,l]PDE or Microsome-Activated DB[a,l]P with DNA in Vitro

Figure 8. Illustration of FLNS identification of stable DNA adducts formed from microsome-activated DB[a,l]P (refer to the chromatogram of Figure 7). FLN spectrum of the 54.8 min fraction (spectrum a) in comparison with those of two Gua adduct standards: syn-trans-DB[a,l]PDE-dGMP (spectrum b) and anti-cis-DB[a,l]PDE-dGMP (spectrum c). Peaks are labeled with their excited state vibrational frequencies in cm-1. λex ) 376.00 nm. T ) 4.2 K.

to full separation. This is illustrated in Figure 8; two Gua adducts coeluting at 54.8 min were still not fully separated after a second HPLC run. The FLN spectrum of the HPLC peak (Figure 8, spectrum a), however, is composed of two spectra sufficiently different to allow both adducts to be individually identified (Figure 8, spectra b and c). The high-energy component of the FLN spectrum near 383 nm matches the FLN spectrum of syntrans-DB[a,l]PDE-dGMP (spectrum b), whereas the linenarrowed peaks at the low-energy side of the spectrum near 388 nm match that of anti-cis-DB[a,l]PDE-dGMP (spectrum c). As the adduct yield was much lower than in the case of DNA reacted with DB[a,l]PDE, FLNS was not always sufficiently sensitive for the identification of minor Ade adducts. Low-resolution fluorescence spectra (77 K, excitation at 308 nm), however, could be recorded for fractions at 59 and 61.5 min, which were in agreement with the corresponding spectra from syn-trans-DB[a,l]PDE-dAMP and syn-cis-DB[a,l]PDE-dAMP, respectively (not shown). Peaks that are not labeled did not have FLN spectra corresponding to any of the standards. Three separate and distinct adducts, syn-cis-DB[a,l]PDE-dAMP, syn-trans-DB[a,l]PDE-dGMP, and anti-cisDB[a,l]]PDE-dGMP, were identified by the 32P-postlabeling technique (Figure 9). The major identified adduct spot comigrated on TLC with the standards for both syntrans-DB[a,l]PDE-dAMP and anti-cis-DB[a,l]PDEdAMP adducts, although the latter was not identified as one of the HPLC adduct fractions and may not be present. The adduct spot labeled as anti-DB[a,l]PDEdGMP comigrated with one of the anti-DB[a,l]PDEdGMP-derived standard adduct fractions which could not be identified conclusively by FLNS, as indicated above in the discussion of the reaction of (()-anti-DB[a,l]PDE with DNA. Thus, 85% of the adducts in the microsomecatalyzed binding of DB[a,l]P to DNA were determined to be either Ade or Gua adducts by radiocounting of excised adduct spots; Gua and Ade adducts were observed in approximately equal amounts.

anti-DB- syn-DB[a,l]PDE [a,l]PDE DB[a,l]P (()-anti-cis-DB[a,l]PDE-dGMP (()-anti-trans-DB[a,l]PDE-dGMP (()-anti-cis-DB[a,l]PDE-dAMP (()-anti-trans-DB[a,l]PDE-dAMP (()-syn-cis-DB[a,l]PDE-dGMP (()-syn-trans-DB[a,l]PDE-dGMP (()-syn-cis-DB[a,l]PDE-dAMP (()-syn-trans-DB[a,l]PDE-dAMP a

+ or NDa + or + or -

Not detected. b Detected only by

+ or ND + or -b ND + or ND + and - + or + or + or + and - + or -

32P-postlabeling.

In summary, reaction of (()-anti-DB[a,l]PDE with DNA produced three identified adducts (Table 1), anticis-DB[a,l]PDE-dGMP, anti-cis-DB[a,l]PDE-dAMP, and anti-trans-DB[a,l]PDE-dAMP, that constitute 93% of the total stable adducts that are observed. The reaction of (()-syn-DB[a,l]PDE with DNA yielded four identified adducts (Table 1), syn-cis-DB[a,l]PDE-dGMP, a pair of syn-trans-DB[a,l]PDE-dGMP, syn-cis-DB[a,l]PDE-dAMP, and a pair of syn-trans-DB[a,l]PDE-dAMP, comprising 90% of the total stable adducts. The DNA adducts formed after microsomal activation of DB[a,l]P included five identified adducts (Table 1), anti-cis-DB[a,l]PDE-dGMP, anti-cis-DB[a,l]PDE-dAMP, syn-trans-DB[a,l]PDE-dGMP, syn-cis-DB[a,l]PDE-dAMP, and syn-trans-DB[a,l]PDE-dAMP, that constitute 85% of the total stable adducts that were observed.

Conclusions A relatively simple nonradioactive method combining HPLC and FLNS has been developed for determining the structure of the stable DNA adducts of DB[a,l]P. One major advantage of this method is that no radioactivity of any kind is required at any stage of the procedure. The FLNS method does require the availability of adduct standards for fingerprint identification. The reaction of microsome-activated DB[a,l]P with DNA yields a very large number of stereoisomeric adducts, leading to very crowded chromatograms. Therefore, selective detection is needed as an extra independent identification method. FLNS is very suitable, as it provides complementary information; cis and trans adducts from syn- or anti-DB[a,l]PDE are easily distinguished on the basis of their high-resolution FLN spectra. On the other hand, the spectral differences between Gua and Ade adducts of the same stereochemistry are often rather small (Figure 5), but these compounds usually have very different HPLC retention times.

Identification of Stable DB[a,l]P-DNA Adducts

Approximately 85-90% of all stable adducts observed by the 32P-postlabeling method in the microsome-activated reaction of DB[a,l]P with DNA have been identified by this technique. HPLC peaks that were not identified did not have FLN spectra corresponding to any of the standards. Equal amounts of Ade and Gua adducts were identified by the 32P-postlabeling method after reaction of microsome-activated DB[a,l]P with DNA, whereas more of the identified stable Gua than Ade adducts were found after (()-syn- or (()-anti-DB[a,l]PDE reacted with DNA. This significant finding is in contrast to reactions of DB[a,l]PDE with single nucleotides, in which Ade adducts were formed much more readily and in higher yields (14). Jankowiak et al. (13) reported the presence of more Ade than Gua adducts in the microsomal reaction, but equal amounts of Ade and Gua adducts are reported here. Since the 32P-postlabeling procedure used was similar in both cases, perhaps the difference can be attributed to the rat liver microsomal preparations that were used. The method developed here could be extended to identifying stable DNA adducts of other fluorescent carcinogenic compounds.

Acknowledgment. We thank 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.

References (1) Cavalieri, E., and Rogan, E. (1992) The approach to understanding aromatic hydrocarbon carcinogenesis. The central role of radical cations in metabolic activation. Pharmacol. Ther. 55, 183-199. (2) Cavalieri, E. L., and Rogan, E. G. (1998) Mechanisms of tumor initiation by polycyclic aromatic hydrocarbons in mammals. In The Handbook of Environmental Chemistry: PAHs and Related Compounds (Neilson, A. H., Ed.) Vol. 3J, pp 81-117, Springer, Heidelberg, Germany. (3) Amin, S., Misra, B., Desai, D., Huie, K., and Hecht, S. (1989) Chromatographic conditions for separation of 32P-labeled phosphates of major polynuclear aromatic hydrocarbon-deoxyribonucleoside adducts. Carcinogenesis 10, 1971-1974. (4) Canella, K., Peltonen, K., and Dipple, A. (1991) Identification of (+) and (-) anti-benzo[a]pyrene dihydrodiol epoxide-nucleic acid adducts by the 32P-postlabeling assay. Carcinogenesis 12, 11091114. (5) Zeisig, M., and Moller, L. (1995) 32P-HPLC suitable for characterization of DNA adducts formed in vitro by polycyclic aromatic hydrocarbons and derivatives. Carcinogenesis 16, 1-9. (6) Gorelick, N., and Wogan, G. (1989) Fluoranthene-DNA adducts: identification and quantification by an HPLC-32P-postlabeling method. Carcinogenesis 10, 1567-1577. (7) Kato, S., Petruzzelli, S., Bowman, E. D., Turteltaub, K. W., Blomeke, B., Weston, A., and Shields, P. G. (1993) 7-Alkyldeox-

Chem. Res. Toxicol., Vol. 12, No. 9, 1999 801

(8)

(9)

(10)

(11)

(12)

(13)

(14)

(15)

(16)

(17)

(18)

(19)

(20)

(21)

yguanosine adduct detection by two-step HPLC and the 32Ppostlabeling assay. Carcinogenesis 14, 545-550. Kang, H., Konishi, C., Eberle, G., Rajewsky, M. F., Kuroki, T., and Huh, N. (1992) Highly sensitive, specific detection of O6methylguanine, O4-methylthymine and O4-ethylthymine by the combination of high performance liquid chromatography prefractionation, 32P-postlabeling and immunoprecipitation. Cancer Res. 52, 5307-5312. Shields, P. G., Povey, A. C., Wilson, V. L., Weston, A., and Harris, C. C. (1990) Combined high-performance liquid chromatography/ 32P-postlabeling assay of N7-methyldeoxyguanosine. Cancer Res. 50, 6580-6584. Lau, H. H. S., and Baird, W. M. (1991) Detection and identification of benzo[a]pyrene-DNA adducts by [35S]phosphorothioate labeling and HPLC. Carcinogenesis 12, 885-893. Li, K.-M., Todorovic, R., Rogan, E. G., Cavalieri, E. L., Ariese, F., Suh, M., Jankowiak, R., and Small, G. J. (1995) Identification and quantitation of dibenzo[a,l]pyrene-DNA adducts formed by rat liver microsomes in vitro: Preponderance of depurinating adducts. Biochemistry 34, 8043-8049. Ralston, S. L., Seidel, A., Luch, A., Platt, K. L., and Baird, W. M. (1995) Stereoselective activation of dibenzo[a,l]pyrene to (-)-anti(11R,12S,13S,14R)- and (+)-syn-(11S,12R,13S,14R)-11,12-diol13,14-epoxides which bind extensively to deoxyadenosine residues of DNA in the human mammary carcinoma cell line MCF-7. Carcinogenesis 16, 2899-2907. Jankowiak, R., Ariese, F., Hewer, A., Luch, A., Zamzow, D., Hughes, N. C., Philips, D., Seidel, A., Platt, K. L., Oesch, F., and Small, G. J. (1998) Structure, conformations and repair of DNA adducts from dibenzo[a,l]pyrene: 32P-postlabeling and fluorescence studies. Chem. Res. Toxicol. 11, 674-685. Devanesan, P., Ariese, F., Jankowiak, R., Small, G. J., Rogan, E. G., and Cavalieri, E. L. (1999) Preparation, isolation and characterization of dibenzo[a,l]pyrene diol epoxide-deoxyribonucleoside monophosphate adducts by HPLC and fluorescence linenarrowing spectroscopy. Chem. Res. Toxicol. 12, 789-795. NIH Guidelines for the Laboratory Use of Chemical Carcinogens (1981) NIH Publication 81-2385, U.S. Government Printing Office, Washington, DC. Bodell, W. J., Devanesan, P. D., Rogan, E. G., and Cavalieri, E. L. (1989) 32P-Postlabeling analysis of benzo[a]pyrene-DNA adducts formed in vitro and in vivo. Chem. Res. Toxicol. 2, 312315. Jankowiak, R., and Small, G. J. (1989) Fluorescence linenarrowing spectroscopy in the study of chemical carcinogenesis. Anal. Chem. 61, 1023A-1032A. Jankowiak, R., and Small, G. J. (1991) Fluorescence linenarrowing: a high-resolution window on DNA and protein damage from chemical carcinogens. Chem. Res. Toxicol. 4, 256269. Dipple, A., and Pigott, M. A. (1987) Resistance of 7,12-dimethylbenz[a]anthracene-deoxyadenosine adducts in DNA to hydrolysis by snake venom phosphodiesterase. Carcinogenesis 8, 491-493. Suh, M., Ariese, F., Small, G. J., Jankowiak, R., Liu, T.-M., and Geacintov, N. E. (1995) Confirmational studies of the (+)-trans, (-)-trans, (+)-cis, and (-)-cis adducts of anti-benzo[a]pyrene diolepoxide to N2-dG in duplex oligonucleotides using polyacrylamide electrophoresis and low-temperature fluorescence spectroscopy. Biophys. Chem. 56, 281-296. Ariese, F., Small, G. J., and Jankowiak, R. (1996) Conformational studies of depurinating DNA adducts from syn-dibenzo[a,l]pyrene diolepoxide. Carcinogenesis 17, 829-837.

TX980203P