Chem. Res. Toxicol. 1996, 9, 1113-1116
1113
Depurinating and Stable Benzo[a]pyrene-DNA Adducts Formed in Isolated Rat Liver Nuclei Prabhakar D. Devanesan,† Sheila Higginbotham,† Freek Ariese,‡ Ryszard Jankowiak,‡ Myungkoo Suh,‡ Gerald J. Small,‡,§ Ercole L. Cavalieri,† and Eleanor G. Rogan*,† Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, Nebraska 68198-6805, and Department of Chemistry and Ames Laboratory-USDOE, Iowa State University, Ames, Iowa 50011 Received March 25, 1996X
Polycyclic aromatic hydrocarbons are bound to DNA by two major pathways, one-electron oxidation and monooxygenation, to form adducts that are stable in DNA under normal conditions of isolation and depurinating adducts that are released from DNA by cleavage of the bond between the purine base and deoxyribose. Isolated rat liver nuclei have been used as an in vitro model for studying covalent binding of aromatic hydrocarbons to DNA, but the depurinating adducts formed by nuclei have not been identified or compared to those formed by the more commonly used rat liver microsomes. To examine the profiles of stable and depurinating adducts, nuclei from the livers of 3-methylcholanthrene-induced male MRC Wistar rats were incubated with [3H]benzo[a]pyrene (BP) and NADPH. Three depurinating adducts, 8-(BP-6-yl)Gua, 7-(BP-6-yl)Gua, and 7-(BP-6-yl)Ade, were obtained from the nuclei, as seen previously with rat liver microsomes or in mouse skin. The profile of stable adducts analyzed by the 32P-postlabeling method was qualitatively similar to that found in the microsomal activation of BP or in mouse skin treated with BP. Low-temperature fluorescence studies of the nuclear DNA revealed the presence of stable BP adducts originating from syn- and antiBP diol epoxide.
Introduction Polycyclic aromatic hydrocarbons (PAH)1 are bound to DNA by two major pathways: one-electron oxidation to form radical cations and monooxygenation to form diol epoxides (1-9). Two types of adducts are formed, stable adducts that remain covalently bound to DNA under normal conditions of isolation and depurinating adducts that are removed from DNA by cleavage of the glycosidic bond. Binding of PAH to DNA by one-electron oxidation requires that the highly reactive radical cation intermediate be formed in close proximity to the DNA (3), whereas the less reactive diol epoxide intermediate generally does not necessarily need to be formed close to DNA. In cells, cytochrome P450 present in the nuclear membrane is mostly responsible for binding of PAH to nuclear DNA (10, 11). Isolated rat liver nuclei have served as an in vitro model for studying covalent binding of PAH to DNA (1013). To use this model for further studies of PAH-DNA adducts, it is necessary to determine whether the profile of stable and depurinating adducts formed in isolated rat liver nuclei resembles that seen with the more commonly used rat liver microsomal activating system (4, 5, 7, 9). Following activation of benzo[a]pyrene (BP) by microsomes, the three major depurinating adducts are * To whom correspondence should be addressed. † Eppley Institute, University of Nebraska Medical Center. ‡ Department of Chemistry, Iowa State University. § Ames Laboratory-USDOE, Iowa State University. X Abstract published in Advance ACS Abstracts, August 15, 1996. 1 Abbreviations: BP, benzo[a]pyrene; BP-6-C8Gua, 8-(benzo[a]pyren-6-yl)Gua; BP-6-N7Gua, 7-(benzo[a]pyren-6-yl)Gua; BP-6-N7Ade, 7-(benzo[a]pyren-6-yl)Ade; BPDE, benzo[a]pyrene 7,8-diol-9,10-epoxide; BPDE-10-N2dG, 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10tetrahydrobenzo[a]pyrene; FLNS, fluorescence line-narrowing spectroscopy; PAH, polycyclic aromatic hydrocarbon(s).
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7-(BP-6-yl)Gua (BP-6-N7Gua), 8-(BP-6-yl)Gua (BP-6C8Gua), and 7-(BP-6-yl)Ade (BP-6-N7Ade), and the major stable adduct is BP diol epoxide (BPDE) bound at C-10 to the 2-amino of deoxyguanosine (BPDE-10-N2dG) (1, 2, 4, 6, 7). To determine whether the profile of adducts formed in vitro by nuclei is qualitatively and quantitatively similar to that obtained by using microsomes with exogenous DNA, the profile of stable and depurinating BP-DNA adducts formed in nuclei isolated from the livers of 3-methylcholanthrene-induced male rats was determined by analyzing the stable adducts by the 32Ppostlabeling method (14-16) and the depurinating adducts by HPLC (4-9). In addition, low-temperature fluorescence studies of the DNA reveal the presence of stable BP adducts originating from syn- and anti-BPDE.
Experimental Section Caution: BP is hazardous and should be handled carefully in accordance with NIH guidelines (17). Ten to fifteen 3-methylcholanthrene-induced (10) 3-week-old male MRC Wistar rats (Eppley Colony) were used to prepare liver nuclei (10). Freshly prepared nuclei (3 mg of protein/mL) were incubated with 50 µM [3H]BP (Amersham, Arlington Heights, IL, sp act. 400 Ci/mol) and 0.6 mM NADPH in 50 mM Tris-HCl (pH 7.5) for 20 min at 37 °C. Analysis of Total DNA Adducts. At the end of the incubation, nuclei were lysed in 1% sodium dodecyl sulfate and DNA was precipitated in 70% ethanol. The DNA was purified by extraction with phenol and chloroform and by precipitation in 70% ethanol (10), and analyzed for bound [3H]BP and stable DNA adducts. The water-ethanol supernatant was analyzed for depurinating DNA adducts by HPLC/fluorescence linenarrowing spectroscopy (FLNS). Procedure for 32P-Postlabeling Analysis of Stable Adducts. Purified DNA (4 µg) was enzymically digested to 3′dNp’s
© 1996 American Chemical Society
1114 Chem. Res. Toxicol., Vol. 9, No. 7, 1996 Chart 1. Structures of BP Depurinating Adducts
Devanesan et al. 20), transferred to quartz tubes (3.2 o.d. × 2.0 i.d. × 10 mm), and sealed with a rubber septum. The same sample tubes were used for FLNS analysis of stable adducts in ethanol-precipitated DNA pellets. Synthetic standards were used for comparison.
Results and Discussion
at 37 °C for 2 h with micrococcal nuclease and spleen phosphodiesterase (16). P1-Nuclease was added to the digest to remove the phosphate from unmodified nucleotides, and the incubation was continued at 37 °C for 1 h. The modified nucleotides were converted into [32P]-labeled 3′,5′dpNp’s by incubation with 150 µCi of [γ-32P]ATP and T4 polynucleotide kinase for 60 min at 37 °C. Potato apyrase was added, and the mixture was incubated for 30 min at 37 °C. The postlabeled mixtures were applied to 10 cm × 10 cm PEI-cellulose thin-layer chromatography plates and developed in 1 M NaH2PO4 (pH 6), followed by 3.0 M lithium formate and 7.0 M urea (pH 3.5). The plates were then developed at a right angle to the first direction in 0.72 M NaH2PO4, 0.45 M TrisHCl, and 7.6 M urea (pH 8.7), followed by 1.7 M NaH2PO4 (pH 6.0). The adduct spots were located by autoradiography and counted by liquid scintillation. The amount of stable adducts was calculated by the 32P-postlabeling method as previously described (16). Procedures for Analysis of Depurinating Adducts by HPLC. The water-ethanol supernatant containing adducts formed in the nuclei was evaporated under vacuum with gentle heating. The residue was resuspended in 0.5 mL of methanoldimethyl sulfoxide (1:1), and undissolved material was removed by centrifugation. Adducts were analyzed by HPLC on a YMC 5-µm ODS-AQ reverse-phase analytical column (6.0 × 250 mm) eluted with methanol-water and acetonitrile-water gradients as previously described (4). The electrochemically-synthesized (18) adducts BP-6-C8Gua, BP-6-N7Gua, and BP-6-N7Ade (Chart 1) were used as chromatographic standards. The HPLC effluent was analyzed by a photodiode array detector and a Radiomatic Flow-1β radiation monitor (Packard, Meridian, CT). Adduct structures were confirmed by FLNS (19). Fluorescence Line-Narrowing Spectroscopy. Stable BP adducts in intact DNA and depurinating BP adducts separated by HPLC were analyzed by means of FLNS (19). The excitation source was a Lambda Physik FL-2002 dye laser pumped by a Lambda Physik EMG 102 MSC XeCl excimer laser. Samples were cooled in a double-nested glass cryostat fitted with quartz optical windows (H. S. Martin Inc., Vineland, NJ). A 1 m focal length McPherson 2016 monochromator with a 2400 grooves/ mm grating was used to disperse the fluorescence. The detector was a Princeton Instruments IRY 1024/G/B intensified blueenhanced photodiode array (spectral window 8 nm; resolution ca. 0.08 nm). FLN spectra were obtained using several different excitation wavelengths, each revealing a portion of the S1 excited-state vibrational frequencies. For time-resolved detection, an FG-100 high voltage gate pulse generator was used; typical detector delay times and gate widths were 30/100 ns for BP chromophores (one-electron oxidation adducts) and 70/400 ns for pyrene chromophores (BPDE adducts). HPLC-separated fractions were stored dry, then dissolved in a glass-forming solvent mixture, water/glycerol/ethanol (40:40:
Profile of Depurinating BP-DNA Adducts Formed in Nuclei. Isolated rat liver nuclei incubated in the presence of NADPH were previously shown to form the depurinating adduct BP-6-N7Gua (12, 13). In both rat liver microsomes in vitro (4, 6, 7) and mouse skin in vivo (6, 7), three depurinating adducts arising from BP radical cation are formed and constitute 70-80% of the total adducts, whereas the N7Ade and N7Gua depurinating adducts formed by BPDE are formed in much smaller amounts (e5%). The depurinating adducts isolated from rat liver nuclei, BP-6-C8Gua, BP-6-N7Gua, and BP-6N7Ade, were identified by their retention times in two different solvent systems and quantitated radiometrically. Further confirmation of their identity was obtained by means of FLNS (19). HPLC fractions were collected, dried, dissolved in a glass-forming solvent mixture, and cooled to 4.2 K in liquid helium. Selective laser excitation, a few nanometers higher in energy than the (0,0) origin band at 405 nm, yields highly resolved fluorescence spectra that allow distinction between closely related BP adducts (4, 6, 7, 19). The narrow lines in the FLN spectra (Figure 1) represent a multiplet of (0,0) transitions, each excited through overlapping vibrational modes of the S1 excited state. As shown in Figure 1, the three depurinating one-electron oxidation adducts BP-6-C8Gua (frame A), BP-6-N7Gua (frame B), and BP-6-N7Ade (frame C) can be identified on the basis of their “fingerprint” FLN spectra when excited into the 500-600 cm-1 vibronic region (λexc ) 395.59 nm). All three adduct fractions yielded FLN spectra matching those of the synthetic standards; matching spectra were also obtained at other excitation wavelengths (not shown). The fluorescence data showed no sign of contamination of the HPLC fractions with possible coeluting BP metabolites. The profile of depurinating adducts found in this study (Table 1) is similar to that found with rat liver microsomes (4, 6, 7). BP-6-N7Ade comprises more than half of the adducts isolated from the nuclei. No depurinating adducts formed by BPDE were detected in the HPLC profiles at the times such adducts would elute. If any are formed in the nuclei, their levels are too low to be detected. Analysis of Stable BP-DNA Adducts Formed in Nuclei. Analysis of the 18% stable adducts in nuclear DNA by the 32P-postlabeling method also yielded an adduct distribution profile (Table 1) qualitatively and quantitatively similar to that found with rat liver microsomes (4, 6, 7, 13). The stable adducts were identified by means of 32P-postlabeling (Figure 2) and FLNS. BP adduct #1 (Table 1) comigrated in 32P-postlabeling with the (+)-trans-anti-BPDE-N2dG adduct, and adduct #3 comigrated with one of the minor (()-syn-BPDE adducts (data not shown), but the identity of adduct #2 could not be determined. Stable BP adducts were also analyzed by means of FLNS analysis of the DNA. The FLN spectrum of the intact nuclear DNA sample excited at 369.48 nm is shown in Figure 3a. The major peak at 579 cm-1 corresponds to a strong S1 vibrational frequency of trans-anti-BPDE adducts, as seen by comparison with the FLN spectrum (curve 2b) of the previously studied
BP-DNA Adducts Formed in Isolated Nuclei
Chem. Res. Toxicol., Vol. 9, No. 7, 1996 1115
Figure 1. FLNS identification of depurinating BP one-electron oxidation adducts from rat liver nuclei. Solid lines: synthetic standards; dashed lines: corresponding HPLC fractions. Frame A: BP-6-C8Gua; frame B: BP-6-N7Gua; frame C: BP-6-N7Ade. Peaks are labeled with their excited-state vibrational frequencies in cm-1; intensities are not on scale. Solvent matrix: water/glycerol/ ethanol 40:40:20; T ) 4.2 K; λexc ) 395.59 nm. Table 1. Stable and Depurinating BP-DNA Adducts Formed by Isolated Rat Liver Nucleia % of total adducts total adducts µmol/mol of DNA-P 7.7
depurinating adducts
stable adducts
BP-6BP-6BP-6#1 C8Gua N7Gua N7Ade (anti-BPDE) #2 #3 5
20
57
9
3
6
a
Values are the average of determinations on three preparations. Stable adducts were quantitated by the 32P-postlabeling method, and depurinating adducts by two consecutive HPLC separations. The percentage of each adduct varied between 5% and 15% in the three preparations.
Figure 2. Autoradiogram of 32P-postlabeled DNA containing BP adducts formed after nuclear activation. The film was exposed at room temperature for 3 h.
synthetic standard (+)-trans-anti-BPDE-N2dG in duplex oligonucleotides (20). This is in full agreement with the TLC assignment of adduct #1 as a trans-anti-BPDE adduct. Other strong vibronic lines in spectrum 2a at 553 and 613 cm-1 match the frequencies observed for DNA treated with (()-syn-BPDE (spectrum 2c; modified base and stereochemistry unknown). A similar spectrum of DNA modified with (-)-syn-BPDE was reported by Jankowiak et al. (21). It is, therefore, concluded that the stable DNA adducts consist of a mixture of anti- and synBPDE adducts.
Figure 3. FLNS identification of stable BPDE adducts in DNA from rat liver nuclei. Curve a: undigested total DNA sample (ethanol-precipitated pellet). Curve b: standard FLN spectrum of the synthetic (+)-trans-anti-BPDE-N2dG adduct in double stranded oligonucleotide obtained from the laboratory of Dr. N. E. Geacintov; solvent matrix: aqueous buffer (ref 20 and references therein). Curve c: sample of DNA treated with (()syn-BPDE. Peaks are labeled with their excited-state vibrational frequencies in cm-1; intensities are not on scale. T ) 4.2 K; λexc ) 369.48 nm. 32P-Postlabeling
experiments showed, however, that under the separation conditions applied the major synBPDE adduct also comigrated with the (+)-trans-antiBPDE-N2dG adduct. In addition, a minor contribution of a cis-anti-BPDE adduct in the DNA sample is indicated by the presence of a 742 cm-1 mode (see spectrum 2a). Thus, adduct #1 (Table 1) contains a mixture of transand cis-anti-BPDE adducts, whereas adduct #3 originates from (()-syn-BPDE. The (+)-trans- and (+)-cis-antiBPDE-N2dG adducts were also found in mouse skin epidermal DNA by 32P-postlabeling and FLNS (22). When using low resolution fluorescence spectroscopy at 77 K for the DNA samples, only BPDE-type emission was
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observed. Stable one-electron oxidation adducts (containing a full BP chromophore, expected to show emission in the 405 nm region and a fluorescence lifetime of ca. 35 ns) were not observed in the nuclear DNA sample. It is not known whether no stable BP one-electron oxidation adducts are present or their fluorescence is completely masked by the DNA.
Devanesan et al.
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Conclusions Isolated rat liver nuclei produce similar overall profiles of stable and depurinating BP-DNA adducts as rat liver microsomes (4, 6, 7) and mouse skin (6, 7), with 70-80% of the adducts as depurinating adducts formed by oneelectron oxidation and the remaining stable adducts arising from both syn- and anti-BPDE, although another unknown stable adduct is present. Thus, one-electron oxidation is the predominant mechanism of formation of BP-DNA adducts in isolated rat liver nuclei. The results presented here also show that when microsomes are used to activate BP, and presumably other PAH, the DNA adducts formed are comparable to those observed in the more physiologically relevant nuclei.
Acknowledgment. This research was supported by U.S. Public Health Service Grants R01 CA25176, R01 CA44686, and P01 CA49210 from the National Cancer Institute and NCI Laboratory Cancer Research Center Support (Core) Grant CA36727.
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