Differential Removal of DNA Adducts Derived from anti-Diol Epoxides

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Chem. Res. Toxicol. 2005, 18, 655-664

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Differential Removal of DNA Adducts Derived from anti-Diol Epoxides of Dibenzo[a,l]pyrene and Benzo[a]pyrene in Human Cells† Kristian Dreij,‡ Albrecht Seidel,§ and Bengt Jernstro¨m*,‡ The Institute of Environmental Medicine, Division of Biochemical Toxicology, Karolinska Institutet, Box 210, S-17177 Stockholm, Sweden, and Biochemical Institute for Environmental Carcinogens, Prof. Dr. Gernot Grimmer Foundation, Lurup 4, D-22927 Grosshansdorf, Germany Received October 21, 2004

The polycyclic aromatic hydrocarbons (PAHs) dibenzo[a,l]pyrene (DBP) and benzo[a]pyrene (BP) are widespread environmental contaminants and potent carcinogens. The fjord-region DBP is considerably more carcinogenic than the bay-region BP. This fact can be ascribed to differences in DNA binding efficiency of their ultimate carcinogenic diol epoxide (DE) intermediates, differences in structural features of the DNA adducts, and differences in DNA adduct recognition and the subsequent lesion removal by nucleotide excision repair (NER). We have compared the formation and removal of adducts as a function of time formed by the carcinogenic metabolites (-)-anti-DBPDE and (+)-anti-BPDE in A549 human epithelial lung carcinoma cells. Cells were exposed to 0.1 or 1.0 µM (-)-anti-DBPDE and (+)-anti-BPDE, respectively. Adducts were measured at various post-treatment times (up to 6 h) by enzymatic DNA hydrolysis and a HPLC procedure that allows monitoring of all cis- and trans-nucleoside adducts of dA and dG. Treatment with 0.1 µM (-)-anti-DBPDE resulted in an initial increase of adducts to a maximal level of 144 pmol adducts/mg of DNA after 1 h of incubation. This was followed by an apparent, although not statistically significant, slow removal of adducts. After 6 h of incubation, at least 80% seems to remain. In cells treated with 1.0 µM (+)-anti-BPDE, the maximal level of 140 pmol adducts/mg of DNA was reached within 20 min of exposure. The formation was followed by an initial rapid decline in the adduct level (1.54 pmol adducts/mg of DNA/min) and a later statistically significant slower rate (0.14 pmol adducts/mg of DNA/ min) of adduct removal. After 1 h of incubation, about 45% of the adducts are removed followed by 75% at 6 h. The biphasic pattern of BPDE removal has been observed previously in mammalian cells and, at least in part, may reflect the action of transcription-coupled repair (TCR) and the subsequent global genomic repair (GGR). Comparing the rate of removal of adducts derived from BPDE with those of DBPDE, the latter are obviously more refractory to the NERcoupled repair than the former. Furthermore, the apparent resistance of adducts from DBPDE to be eliminated may reflect the ability of such adducts to escape recognition and/or the subsequent removal by the NER machinery. Further analysis of DNA adduct distribution as a function of incubation time reveals that the dA/dG adduct ratio for BPDE was independent of time (4% dA, 96% dG), whereas the corresponding ratio for DBPDE was significantly increased from 2.9 (74% dA, 26% dG) at 20 min to 4.0 (80% dA, 20% dG) after 6 h of incubation. The results presented here on DNA adduct removal in mammalian cells are in part consistent with recent results on NER-coupled activity on bay- and fjord-region DE-modified oligonucleotides in vitro and further substantiate the hypothesis that the high carcinogenicity of the nonplanar PAHs arise from the ability of the preferentially formed dA adducts to escape recognition by surveillance systems and the subsequent NER-coupled lesion removal.

Introduction Reactive endogenous and exogenous chemicals and their potential to damage DNA play a major role in causing the mutations that are responsible for the initiation and progression of tumors (1-3). Early critical steps in the protection against mutagenicity and the subsequent initiation of cancer is a rapid DNA adduct/

damage response mediated by various checkpoint protein kinases (e.g., ATM, ATR) that coordinate damage recognition and DNA repair with the action of cell cycle checkpoints (4, 5). To handle the great number of different types of DNA adducts/damage that may arise in a cell, a versatile and sophisticated cellular machinery comprised of a number of different pathways has been developed (6).

† Part of this study was presented at the 19th International Symposium on Polycyclic Aromatic Compounds, Amsterdam, The Netherlands, 21-25 September, 2003 * To whom correspondence should be addressed. Phone: +46-852487576. Fax: +46-8-343849. E-mail: [email protected]. ‡ Karolinska Institutet. § Prof. Dr. Gernot Grimmer Foundation.

Some potentially harmful adducts are the more bulky ones derived from the reaction of DNA with diol epoxide (DE)1 metabolites of polycyclic aromatic hydrocarbons (PAHs) (7, 8). PAHs are widespread environmental pollutants known to induce mutations and tumors in

10.1021/tx0497090 CCC: $30.25 © 2005 American Chemical Society Published on Web 03/24/2005

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Figure 1. Structure and preferred conformation of (+)-antiBPDE (top) and (-)-anti-DBPDE (bottom) used in this study.

experimental animals and, most probably, also in humans (7, 9-11). One critical mutation avoidance system is the DNA mismatch repair that has been shown to be involved in DE-DNA adduct-induced apoptosis (12). The major DNA repair strategy responsible for eliminating bulky DNA adducts is the nucleotide excision repair (NER) pathway (13-16). NER is further divided into two subpathways, termed global genomic repair (GGR) and transcription-coupled repair (TCR). The former deals with adducts throughout the genome, whereas TCR is highly selective for adducts in the transcribed DNA strand in expressed genes (16, 17). This highly specific function of TCR is reflected in a higher rate of adduct removal relative to GGR (18, 19). Most carcinogenic PAH DEs show one structural feature, a bay- and/or a fjord-region. In contrast to the planar and rigid structure of the bay-region DEs, the fjord-region DEs are distorted and more flexible due to steric hindrance (Figure 1) (20-22). Furthermore, fjord-region PAH DEs in general have a significantly higher tumorigenic activity than the bay-region compounds (8, 23, 24). The most carcinogenic PAH identified in the environment (25, 26) so far is dibenzo[a,l]pyrene (DBP) (IUPAC nomenclature; dibenzo[def,p]chrysene or naphtho[1,2,3,4-pqr]tetraphene) which is several orders of magnitude more potent in rodents than the prototype PAH, benzo[a]pyrene (BP) (Figure 1) (27-29). The first studies on differences in DNA adduct formation between bay- and fjord-region DEs showed that fjordregion DEs generally bind more extensively to DNA than the bay-region analogues and predominantly react with adenine residues rather than guanine in DNA (30, 31). Additionally, more recent studies on site-specifically modified oligonucleotides indicate that adducts derived from fjord-region DEs, being more flexible and twisted, distort DNA less than the more rigid bay-region DEs (32-35). These facts may contribute to the great differ1 Abbreviations: DE, diol epoxide; PAHs, polycyclic aromatic hydrocarbons; NER, nucleotide excision repair; GGR, global genomic repair; TCR, transcription-coupled repair; DBP, dibenzo[a,l]pyrene; BP, benzo[a]pyrene; B[g]C, benzo[g]chrysene; (-)-anti-DBPDE, (11R,12S)dihydroxy-(13S,14R)-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene; (+)-anti-BPDE, (7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; ATCC, American type culture collection; CD, circular dichroism; PBS, phosphate-buffered saline.

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ence in carcinogenic potency of fjord- and bay-region DEs, respectively. Differences in efficiency of DNA-adduct recognition and repair have been proposed to be another critical factor in explaining the carcinogenic potency difference (36, 37). In fact, extensive practical and theoretical studies have in many cases formed a solid foundation for relating NER-coupled repair with adduct structural features (35) For instance, in vitro experiments using well-defined DEmodified oligonucleotides and human cell extracts have shown that adenine adducts derived from several fjord PAH DEs, are refractory to NER-mediated repair. In contrast, analogous adducts derived from the bay-region BPDE are more easily recognized and removed by NER (36). Differential repair of fjord- vs bay-region DE-DNA adducts in mammalian systems is less known. However, available information indicates that human fibroblasts in culture remove DE-DNA adducts derived from benzo[g]chrysene (B[g]C), a fjord-region PAH, at lower rates than adducts from BPDE (38). In addition, adducts derived from DBPDE in MCF-7 cells seems to be removed slowly (39). Finally, studies in mouse skin indicate that externally localized DNA adducts of both BPDE and DBPDE are removed more rapidly than adducts with internal localization (40, 41). Preliminary results from our group indicated that DNA adducts derived from both BPDE and DBPDE are removed at substantial rates in cultured mammalian cells (42). The aim of the present study was to further investigate and establish the proposed difference in NER-coupled removal of DNA adducts derived from fjord- or bay-region DEs. We report results showing a marked difference in removal rate of DNA adducts derived from (-)-antiDBPDE compared to (+)-anti-BPDE in A549 human epithelial lung carcinoma cells. In addition, we show that structurally different adducts (i.e., cis vs trans and dG vs dA) (24, 30) in some cases also differ in rate of elimination. The results will be discussed within the context of adduct specificity and conformational heterogeneity.

Materials and Methods Chemicals. Synthesis of the anti-diol epoxides of DBP and BP used in this study was performed according to literature methods (43, 44). Caution: Diol epoxides from polycyclic aromatic hydrocarbons are carcinogens, and thus experimental handling must be carried out under special safety conditions, e.g., those outlined in the NCI guidelines. Oligonucleotides were obtained from Cybergene AB. Chemicals, DNase I, and alkaline phosphatase (type VII-S) were purchased from Sigma-Aldrich Sweden AB, nuclease P1 was from Roche Diagnostics Scandinavia AB, and snake venom phosphodiesterase I was from Amersham Biosciences. Preparation of Standards. Preparation of the nucleoside diol epoxide adducts used as HPLC standards and their structural characterizations were performed essentially as described (42). In brief, enantiomerically pure (+)-anti-BPDE or (-)-anti-DBPDE was incubated with oligonucleotides 5′d(CG)6 or 5′-d(AT)6 for at least 15 h in room temperature or in a refrigerator. To compare the distribution of adducts from cellular DNA, modified DNA standards were also prepared. In brief, 1 mg of calf thymus DNA was incubated with 200 nmol of (+)-anti-BPDE or (-)-anti-DBPDE under similar conditions as the oligonucleotides. The extent of diol epoxide modification of 5′-d(CG)6, 5′-d(AT)6, or calf thymus DNA was estimated spectrophotometrically using 350nm ) 29 000 M-1 cm-1 for BPDE

DNA Adducts of DBPDE and BPDE in Human Cells (45) and 340nm ) 20 000 M-1 cm-1 for DBPDE (43). DE-modified oligonucleotides and DNA were subjected to enzymatic hydrolysis and subsequent HPLC analysis to obtain individual nucleoside adducts (see below). Cell Line. The human A549 lung epithelial carcinoma cells were obtained from American Type Culture Collection (ATCC) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum, Na-pyruvate (1 mM), penicillin (100 U/mL), and streptomycin (100 µg/mL) (all from Gibco, Midlothian, Scotland). The cells were maintained at 37 °C in a humified 5% CO2 atmosphere. Quantification of Cellular DNA. An aliquot (∼2 × 105 cells) of the cell suspension was separately washed with phosphate-buffered saline (PBS), and pelleted cells were frozen until total amount of cellular DNA was estimated. The frozen cell pellet was resuspended in 500 µL TEN buffer (10 mM TrisHCl, 2 M NaCl, 1 mM EDTA, 0.1% Triton x100). Aliquots (125 or 250 µL) were mixed with 5 µL of Hoechst 33258 (100 µg/mL), and TEN buffer was added to a total volume of 1 mL. DNA concentration was determined by measuring fluorescence (λex ) 360 nm, λem ) 460 nm). Samples were compared with calf thymus DNA standard to calculate micrograms of DNA/milliliter (46, 47). Exposure of Cells with DE. Prior to exposure, 1.5 × 106 cells per dish (ø ) 10 cm) were plated and incubated at 37 °C for 72 h as described above but in the presence of 5% fetal calf serum. Subsequently, the cell medium was replaced with 10 mL of exposure medium (1.8 mM KCl, 0.5 mM MgCl2, 0.14 M NaCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, 1 mM CaCl2, 5.5 mM glucose, pH 7.2), and the confluent cells (∼107 cells) were incubated with 0.1 µM (-)-anti-DBPDE or 1.0 µM (+)-anti-BPDE (added in 10 µL DMSO) for 20 min. These concentrations were selected in order to obtain similar levels of DNA adducts, since previous work indicate that DBPDE binds about 10-fold as efficiently as BPDE in cultured cells (48). The exposure medium was then replaced with cell medium, and the cells were incubated for up to 6 h. No effects on cell viability as revealed by trypan blue exclusion and FACS analysis were observed under the experimental conditions used here. In fact, the population in subG0/G1, representing apoptotic and/or necrotic cells, was below 4% for up to 12 h post-treatment.2 At selected time points, incubation was terminated by removal of the cell medium and washing with PBS. Following treatment with trypsin/EDTA, the cells were resuspended and washed twice in PBS, and the cell pellet was frozen (-20 °C) until analysis. All buffers and cell media were kept at 37 °C. Determination of Intracellular Lifetime of BPDE and DBPDE. To determine the intracellular lifetime of BPDE and DBPDE, cells were incubated with 1.0 µM (+)-anti-BPDE or 0.1 µM (-)-anti-DBPDE for up to 6 h and metabolism analyzed essentially as described (49). In brief, trypsinized cells were added to an equal volume of acetone immediately followed by addition of alkaline mercaptoethanol in order to trap unreacted DE. Subsequently, the lyzed cells were centrifuged, and the supernatant was twice extracted with ethyl acetate. The organic phase was evaporated to dryness under N2 and analyzed with HPLC (44). Purification of DNA and Enzymatic Digestion. DNA from ∼2 × 107 cells (corresponding to ∼500 µg) was isolated using a GenomicPrep Cell and Tissue DNA Isolation Kit (Amersham Biosciences, Sweden), according to the manual, and quantitated by UV absorbance at 260 nm ( ) 6600 M-1 cm-1). The recovery of cellular DNA was about 60-70%. Digestion of DBPDE- or BPDE-modified DNA/oligonucleotides was basically performed as previously described with some modifications (42). In brief, purified DNA was incubated for at least 10 h with 50 units of DNase I at 37 °C in 50 mM Na-acetate buffer, 5 mM MgCl2, pH 5.0. Subsequently, the temperature was increased to 50 °C and the incubation continued for 2-3 h in the presence 2 Bajak, E., Dreij, K., Gusnanto, A., Stockling, K., Jernstro ¨ m, B., and Cotgreave, I. Manuscript in preparation.

Chem. Res. Toxicol., Vol. 18, No. 4, 2005 657 of 5 mM ZnCl2, 10 units of nuclease P1, 0.3 units of snake venom phosphodiesterase I, and 5 units of alkaline phosphatase. We noted that larger amounts of snake venom phosphodiesterase I from Amersham Biosciences were required than the corresponding enzyme previously available from Sigma-Aldrich. We further noted that complete hydrolysis of adducted oligonucleotides does not require DNase I or nuclease P1 treatment. Adduct Characterization. Circular dichroism (CD) spectra were recorded on a Jasco J-720 spectropolarimeter. The HPLCpurified nucleoside adducts derived from enantiomeric pure (-)-anti-DBPDE were dissolved in MeOH and measured in a 1 cm cuvette at 20 °C. DNA Adduct Analysis by HPLC. DNA adducts derived from (-)-anti-DBPDE and (+)-anti-BPDE were analyzed by HPLC as previously described with some modifications (42, 50). In brief, the solvent system used was 0.1 M triethylammonium acetate (TEAA), pH 7.0 (solvent A), and acetonitrile (solvent B) delivered at 1.5 mL/min. The DBPDE samples were eluted with a linear gradient (10-26% B for 15 min) followed by isocratic elution (26% B for 30 min). The BPDE samples were eluted with a linear gradient (10-30% B for 11 min) followed by isocratic elution (30% B for 14 min). The effluent was monitored by UV at 260 nm and by fluorescence (DBPDE, λex ) 340 nm, λem ) 410 nm; BPDE, λex ) 344, λem ) 398 nm). The relative fluorescence intensity per picomole (RI/pmol) for dA and dG adducts from DBPDE and BPDE, respectively, were obtained by analyzing known amounts of individual nucleoside adducts by HPLC and fluorescence detection.

Results and Discussion HPLC Analysis of Nucleoside Adducts. Calf thymus DNA or oligonucleotides (5′-d(CG)6 or 5′-d(AT)6) were incubated with enantiomeric pure (-)-anti-DBPDE and (+)-anti-BPDE and enzymatically hydrolyzed to deoxyribonucleosides. The modified enzymatic hydrolysis procedure resulted in a reduction of incubation time from >30 h to about 12 h with sustained efficacy of the hydrolysis. The improved HPLC method (see above) employed for the analysis of nucleoside adducts and their isolation/purification allowed better separation and identification of the different nucleoside adducts. Examples of HPLC elution patterns from (-)-anti-DBPDE and (+)-anti-BPDE are shown in Figures 2 and 3, respectively. Panel A represents the dG-N2 adduct distribution, panel B the distribution of dA-N6 adducts, panel C the distribution of products in modified calf thymus DNA, and panel D the adduct distribution in cellular DNA (see below). On the basis of previous studies, it is safe to assume that (-)-anti-DBPDE and (+)-anti-BPDE, respectively, react predominantly with the exocyclic amino groups of dA or dG and yield both cis- and trans-adducts, thus giving rise to two adducts with each nucleotide (31, 51-54). This is also what we observed with the oligonucleotides 5′-d(CG)6 (peaks I and II in Figures 2A and 3A) and 5′-d(AT)6 (peaks III and IV in Figures 2B and 3B). The elution pattern obtained with DNA (panel C in Figures 2 and 3) is, in principle, a combination of the oligonucleotide distribution patterns and, with the exception of the peak eluting at 35 min in Figure 2C, with no other major peaks. Consistent with the results of others (31, 52, 53, 55), we observed a pronounced preference for adduct formation on dA for DBPDE and dG for BPDE, with dA/dG adduct ratios of 2.9 (74% dA, 26% dG) and 0.04 (4% dA, 96% dG), respectively. No or very little fluorescent material was eluting prior to the first peak, indicating that the enzymatic digestion method used resulted in complete nucleoside adduct hydrolysis.

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Figure 2. Reverse-phase HPLC elution profiles of the enzymatically hydrolyzed products of reaction of (-)-anti-DBPDE with 5′-d(GC)6 (A), 5′-d(AT)6 (B), calf thymus DNA (C), and cellular DNA (D). The adducts are labeled in terms of their elution order: I, (-)-cis-dG; II, (-)-trans-dG; III, (-)-cis-dA; IV, (-)-trans-dA. The effluent was monitored by fluorescence emission at 410 nm (λex ) 340 nm).

Figure 3. Reverse-phase HPLC elution profiles of the enzymatically hydrolyzed products of reaction of (+)-anti-BPDE with 5′-d(GC)6 (A), 5′-d(AT)6 (B), calf thymus DNA (C), and cellular DNA (D). The adducts are labeled in terms of their elution order: I, (+)-cis-dG; II, (+)-trans-dG; III, (+)-trans-dA; IV, (+)-cis-dA. The effluent was monitored by fluorescence emission at 398 nm (λex ) 344 nm).

Adduct Characterization. The individual peaks obtained with (-)-anti-DBPDE-modified and digested 5′-d(CG)6 or 5′-d(AT)6 and HPLC were isolated and subjected to light absorbance spectroscopy and circular dichroism (CD) measurements (results not shown). Com-

parison with our recent CD results on (()-anti-DBPDE modified oligonucleotides (42) and results by Ruan et al. (54) on (()-anti-DBPDE-dA monophosphate adducts allowed us to identify and assign the absolute structure of each product (Table 1). It should be noted that the adduct

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Figure 4. Light absorbance spectrum of dG adducts derived from (-)-anti-DBPDE. Numbers 1 and 2 denote peak maxima presented in Table 2. Table 1. Absolute Structure of Nucleoside Adducts Derived from (-)-anti-DBPDE and (+)-anti-BPDE N2-dG adducts DBPDE BPDE a

N6-dA adducts

Ia

II

III

IV

(-)-cis (+)-cis

(-)-trans (+)-trans

(-)-cis (+)-trans

(-)-trans (+)-cis

The numerals denote HPLC peaks in Figures 2 and 3.

Table 2. Peak Maxima of dA and dG Adducts Derived From (-)-anti-DBPDE

dA (-)-cis (-)-trans dG (-)-cis (-)-trans

peak max 1 (nm)

peak max 2 (nm)

326 323

340 337

326 323

340 337

elution order observed with nucleoside adducts in this study is the same as that observed for nucleotide adducts (31, 54). Regarding the dG adducts of (-)-anti-DBPDE, we based adduct identification and structural assignment on our previous CD results on (()-anti-DBPDE-modified oligonucleotides (42) together with CD results obtained with other fjord-region DEs derived from benzo[c]phenanthrene and benzo[c]chrysene, as well as bay-region DEs (56-58). In the case of (+)-anti-BPDE-modified nucleosides, the structural characterization was based on previously published results (59). The UV absorbance spectra of (-)-anti-DBPDE-dA and -dG adducts yield some interesting information. Both trans-dA and -dG adducts show absorption maxima at 323 and 337 nm (Figure 4 and Table 2, see also ref 54), respectively, whereas the absorption of both cis-adducts are shifted 3 nm to longer wavelengths. As with the dA adducts of DBPDE (54), the shift in the cis-adduct relative to the trans-adduct is most probably due to a closer average distance between the aromatic benzo[e]pyrenyl residue and the guanine base in dG. The implication of these structural differences with regard to DBPDE-dG adducts in DNA is not yet known. Adduct Formation and Removal in A549 Cells. To determine the recovery of DNA from the cultured cells in this study, the DNA content was measured. According to ATCC, A549 is a hypotriploid human cell line and is expected to contain >20 µg DNA/106 cells. This is fully consistent with our result of approximately 25 µg of DNA/ 106 cells. Cells were incubated with 1.0 µM (+)-anti-

Figure 5. Time-dependent removal of DNA adducts from (+)-anti-BPDE (0) and (-)-anti-DBPDE (9) in A549 cells. Each result is the average of at least three separate duplicate experiments, (SD. *p e 0.05 compared to the second time point as determined by the Student t-test.

BPDE or 0.1 µM (-)-anti-DBPDE for up to 6 h. These DE concentrations are expected to yield approximately the same number of DNA adducts (48). The relative short incubation time was chosen to avoid dilution of DEmodified DNA due to cell replication and to avoid unwanted effects on cell viability.1 Following DE exposure, cellular DNA was isolated and enzymatically digested to nucleosides. Figures 2 and 3 (panel D) show the distribution of adducts following incubation with DBPDE and BPDE for 1 h, respectively. Generally, we observed a pronounced preference for adduct formation on dA for DBPDE and dG for BPDE in the cells (compare panels D in Figures 2 and 3) as with calf thymus DNA and oligonucleotides (see above). As evident from Figure 2, the four expected DBPDE adducts (I-IV) were clearly observed with complete resolution. As shown in Figure 3, and in contrast to the adduct distribution with calf thymus DNA, the BPDE-cis-dG adduct (peak I) is barely detectable in the cellular system. Whether this reflects that very small amounts of this adduct are formed in A549 cells or that the adduct is removed very fast and efficiently is not known. In this context it is of interest to note that BPDE-cis-dG adducts are preferred substrates in in vitro NER assays (36). As also shown in Figure 3, the ratio between BPDE-trans- and -cis-dA adducts in cellular DNA relative to calf thymus DNA is reduced (compare peaks III and IV). The effect of incubation time on adduct levels of (+)-anti-BPDE and (-)-anti-DBPDE is shown in Figure 5. In accordance with our expectation using 1.0 µM BPDE and 0.1 µM DBPDE, the maximal adduct levels were about the same, 140 and 144 pmol of adducts/mg of DNA for BPDE and DBPDE, respectively. Exposing the cells to 1.0 µM (+)-anti-BPDE resulted in rapid adduct formation (completed within 20 min of exposure). This is consistent with the rapid cellular uptake of BPDE (t1/2 ) 1.03 min) (48) and its intracellular lifetime (t1/2 ≈ 30 min) obtained in this study (see Materials and Methods). The formation of adducts was followed by an initial rapid decline in the overall adduct level (1.54 pmol/mg of DNA/ min) and a later statistically significant slower rate (0.14 pmol/mg of DNA/min) of adduct removal (p e 0.05)

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(Figure 5). After 1 h of incubation, about 45% of the adducts have been eliminated, and after 6 h of incubation, about 75% have been deleted. In addition to providing insights into the kinetics of BPDE-adduct removal, these results show that A549 cells are capable of adduct recognition and subsequent NER. The apparent biphasic kinetics of BPDE-adduct removal has previously been observed (60-62) and probably reflects to some extent the initial fast and shortterm recruitment and action of TCR and the succeeding steady action of GGR (Figure 5). It is highly unlikely that the entire proportion of adducts removed during the rapid phase is related to TCR, since this requires that 25% of the adducts are localized in approximately 5% of the genome being transcriptionally active. Accordingly, other regions of the genome or particular sequences may carry adducts that are more easily recognized and rapidly removed by NER. In this context, it is interesting to note a previous study from Kaneko et al. (63). These investigators exposed normal human fibroblasts to 1.5 µM (+)-anti-BPDE for 15 min followed by up to 24 h of further incubation. They showed an initial fast reduction of DNA adducts with 56% removed after 8 h followed by a slower rate with 67% removed after 24 h. Interestingly, Kaneko et al. also observed that adduct removal was more efficient in linker DNA than in core DNA, which after 8 h of incubation became refractory to further excision. Studies on the distribution of adducts derived from BPDE in the eukaryotic genome have been performed and demonstrate that the frequency of adducts in linker DNA is 3-4-fold higher than in nucleosomal DNA (64). Furthermore, the adduct frequency in genetically active DNA relative to silent DNA has been estimated to be 5-fold higher (65, 66). In Figure 5 it is evident that, in contrast to BPDE, maximal levels of adducts derived from DBPDE were not observed until after about 1 h of incubation. The difference is most likely due to the higher lipophilicity of DBPDE in conjunction with its lower solvolytic reactivity (cf. Sundberg et al. (48)), leading to a longer intracellular lifetime for DBPDE. The intracellular t1/2 ≈ 180 min for DBPDE relative to ≈30 min for BPDE (see Materials and Methods). The increase in DBPDE-derived adducts was followed by an apparent slow but not statistically significant removal of adducts (on average, 0.11 pmol/mg of DNA/min). After 6 h of incubation, at least 80% of the maximal adduct level remains or a 3-fold higher level relative to BPDE. From Figure 5 it can be concluded that overall NER-coupled removal of adducts derived from the fjord-region DBPDE is significantly less efficient than elimination of adducts derived from the bay-region BPDE. The results presented here are consistent with others on NER-coupled repair of fjord-region DEs. Lloyd et al. (38) demonstrated in cultured human fibroblasts that DNA adducts derived from benzo[g]chrysene DEs (B[g]CDE) were removed at a lower rate than adducts from BPDE. However, these investigators proposed that adduct persistence possibly was due to the high intracellular lifetime of B[g]CDE and, thus, a constant ongoing formation of adducts. As we determined the lifetime of the DEs used here, we can rule out such an explanation in this study. Results from a similar study in human MCF-7 cells (39) demonstrated low rates of formation as well as removal of adducts derived from (-)-anti-DBPDE. Previous results from Wei et al. (67-69) show dosedependent differences in mutational profiles in mam-

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malian cells exposed to bay- or fjord-region diol epoxides. Interestingly, the ratio between mutations at dA and dG increases at lower levels of diol epoxide exposure. It is likely that these results reflect differences in adduct recognition and repair. Distribution of DNA Adducts in A549 Cells as a Function of Time. (+)-anti-BPDE. Figure 6 show the results on the time-dependent relative distribution of DNA adducts derived from (+)-anti-BPDE (panel A) and (-)-anti-DBPDE (panel B) in A549 cells. As evident from the results in panel A, the distribution of the different BPDE adducts is, with the possible exception of cis-dA adducts (insert panel A), constant during the experiment. These results demonstrate that, regardless of known structural differences, adducts of (+)-anti-BPDE are removed at similar rates. Buterin et al. (36) observed recently in in vitro NER assays a marked difference in removal efficiency of different BPDE-derived adducts. In addition, experiments using well-defined DE-modified oligonucleotides have clearly demonstrated a correlation between certain adduct structural properties and NER efficiency (70-72). Our data in Figures 5 and 6 imply that the features of the BPDE adducts when localized in chromosomal DNA, despite large structural differences and expected distortional effects on DNA helix conformation, are of less importance for efficient recognition and NER-coupled repair in A549 cells. This is very surprising, since extensive studies on DE-modified oligonucleotides in conjunction with molecular dynamics simulations have provided detailed information on adduct structure and, in many cases, marked effects on helix conformation (35). Geacintov et al. have classified PAH DE adducts into five different categories, depending on the target for adduct formation (dA or dG) and if the reaction involves cis- or trans-addition (34). In the present study, (+)-antiBPDE yields two dG adducts, S-trans-BPDE-N2-dG and R-cis-BPDE-N2-dG (minor product in the cells) and two dA adducts, S-trans-BPDE-N6-dA and R-cis-BPDEN6-dA. The trans-dG adduct belongs to the category minor groove conformation, and the pyrenyl chromophore is located in the minor groove and directed toward the 5′-end of the helix. Although the adduct causes helix bending, no effect on hydrogen-bonding is evident. The cis-dG adduct belongs to the category base-displaced intercalation, and in this case the aromatic and rigid chromophore is intercalated and the modified guanine displaced into the minor groove. This type of adduct is, of course, associated with loss of proper base-pairing and the helix is severely distorted. The trans-dA adduct can be placed in the category distorting intercalation from the major groove, and available information indicates that the chromophore is intercalated on the 3′-side of the modified dA. However, the pyrenyl residue of BPDE is not parallel to the adjacent base pairs and therefore distorts the helix conformation. The R-cis-BPDE-N6-dA adduct is also categorized as distorting intercalation from the major groove with the BP ring system intercalated toward the 5′-side of the lesion site without disrupting the flanking Watson-Crick base pairs (73). The results in Figure 6 on the relative distribution of this adduct indicate that this adduct is eliminated at a higher rate than the other adducts (adduct level at 6 h compared to 20 min, p ) 0.054). At present, we cannot provide an explanation why the structurally different BPDE adducts formed in our cell system are not reflected in more pronounced effects on recognition/excision. Accordingly,

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Figure 6. Time-dependent relative distribution of DNA adducts from cells exposed to (+)-anti-BPDE (A) and (-)-anti-DBPDE (B). Each result is the average of at least three separate duplicate experiments, (SD. *p e 0.05, **p e 0.01, and ***p e 0.005 determined by Student’s t-test.

information on how BPDE adducts (and similar adducts from the rigid bay-region DEs) influence the structure and conformation of the chromatin and how these changes are related to DNA damage sensing (i.e. ATM mediated phosphorylation of histone H2AX) (74) and further downstream signaling is urgently required. (-)-anti-DBPDE. Turning the attention to panel B (Figure 6), the time-dependent relative distribution of the R-cis-DBPDE-N2-dG and S-trans-DBPDE-N6-dA adducts is constant. However, the S-trans-DBPDE-N2-dG and R-cis-DPDE-N6-dA demonstrate significant changes in distribution. This observation demonstrates that adduct removal is taking place, although at a low rate. The S-trans-DBPDE-N2-dG adduct seems to be more efficiently removed than R-cis-DPDE-N6-dA. The structural features of the (-)-anti-DBPDE adducts have not been studied in detail, but information obtained by molecular dynamics calculations and by high-resolution NMR on analogous fjord-region DEs allows categorization. Given that the information obtained with less complex fjord-region DEs can be extended to the larger DBPDE, the S-trans-DBPDE-N2-dG may be placed in the category intercalation from the minor groove without base displacement and the adduct formation is not associated with impaired Watson-Crick base pairing.

The insertion of the bulky benzo[e]pyrenyl residue between adjacent base pairs, however, is expected to cause partial helix unwinding. This distortion seems to be recognized and the adduct removed by NER. This is consistent with the multipartite model of GGR-NER recently proposed by Geacintov et al. (34). Structural information on the R-cis-DBPDE-N2-dG adduct is not yet available and urgently needed. The S-trans-DBPDEN6-dA adduct can be categorized as intercalation from the major groove and is likely to be intercalated on the 3′-side of the modified adenine. Due to the bulkiness of the adduct, stretching and unwinding of the helix is expected. As evident from Figure 6B, the relative contribution of the R-cis-DBPDE-N6-dA adduct increases significantly as a function of time. This adduct relative to the corresponding trans-adduct is characterized by a closer average distance between the aromatic benzo[e]pyrenyl residue and the adenine base in dA (54). It is possible that this structural feature renders the adduct less recognizable and more persistent. Detailed structural information on the R-cis-DBPDE-N6-dA adduct in DNA is lacking. The structural differences among the DBPDE adducts seems to be, in contrast to adducts derived from BPDE, reflected in differential repair, since the ratio between

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Table 3. Effect of Incubation Time on Ratio between dA/dG Adducts Derived from (-)-anti-DBPDE and (+)-anti-BPDE in A549 Cells DBPDE BPDE

20 min

1h

3h

6h

2.9 ( 0.6 0.04 ( 0.03

3.4 ( 0.5 0.05 ( 0.03

3.5 ( 0.5 0.04 ( 0.02

4.0 ( 0.6* 0.03 ( 0.01

*p e 0.05 using the Student t-test, n g 3, (SD.

dA/dG adducts is significantly dependent on incubation time, going from 2.9 (74% dA, 26% dG) at 20 min up to 4.0 (80% dA, 20% dG) after 6 h of incubation (Table 3). This is an interesting observation, since the exceptional high carcinogenicity of fjord PAHs is believed to arise from these compounds’ preference to form dA adducts (30, 31). Regarding (+)-anti-BPDE, the ratio of adducts appear to be independent of time (4% dA, 96% dG). Some of the results presented here are consistent with in vitro findings (35, 36). However, considering the exceedingly complex molecular machinery mobilized in cells in response to DNA adduct/damage, a full explanation for the marked difference in overall rate of removal of adducts of DBPDE and BPDE cannot be provided. However, the structural features of the adducts are expected to play a pivotal role in adduct recognition and the subsequent mobilization of the repair machinery. Compatible with the structural differences of BPDE and DBPDE adducts in mammalian cells are the numerous previous results on the impact of cell cycle regulatory proteins (38, 39, 75-79) and more recent results on the response of Mdm2. The adduct derived from BPDE causes phosphorylation of Mdm2 and p53 stabilization at concentrations severalfold lower than DBPDE (80). Dipple et al. (75) coined the expression “stealth carcinogens” for adducts escaping the cellular recognition/repair machinery. DBPDE is hitherto the most powerful member of this category with the consequence that its DNA adducts are not recognized by different sensors and therefore undergoing replication along with formation of mutations and eventually tumors.

Conclusion The time-dependent formation and removal of adducts derived from the ultimate carcinogens (-)-anti-DBPDE and (+)-anti-BPDE in A549 human epithelial lung carcinoma cells has been studied. Treatment with (-)-antiDBPDE resulted in an initial increase of adducts to a maximal level after 1 h of incubation followed by a slow and, apparently, steady removal of adducts. After 6 h of incubation, at least 80% of the adducts remain. In cells treated with (+)-anti-BPDE, the maximal level of adducts was reached within 20 min of exposure. The formation was followed by an initial rapid decline in the adduct level (55% remain after 1 h of incubation) and a later slower removal rate (25% remain after 6 h incubation). The overall rate of removal of adducts derived from (-)-anti-DBPDE relative to those of (+)-anti-BPDE clearly demonstrates that the former are more refractory to NER-coupled repair than the latter. Further analysis of DNA adduct distribution as a function of incubation time reveals that the dA/dG adduct ratio for (+)-anti-BPDE was independent of time, whereas the corresponding ratio for (-)-anti-DBPDE was significantly increased (from 2.9 at 20 min to 4.0 after 6 h of incubation). Our results on DNA-adduct removal in mammalian cells further substantiate the hypothesis that the high carcinogenicity of

the nonplanar fjord-region PAHs relative to the more rigid bay-region ones arise from the former’s ability to preferentially form dA adducts. Such adducts are more prone to escape recognition by the cellular surveillance systems and the subsequent NER-coupled lesion removal.

Acknowledgment. The authors want to thank Prof. Nicholas Geacintov for most valuable comments on this paper and the following funding bodies for support: The Swedish Research Council for Environment, Agricultural Sciences and Special Planning (FORMAS), the Swedish Animal Welfare Agency, and AstraZeneca.

References (1) Harris, C. C. (1991) Chemical and physical carcinogenesis: Advances and perspectives for the 1990s. Cancer Res. 51, 50235044. (2) Vogelstein, B., and Kinzler, K. W. (1992) Carcinogens leave fingerprints. Nature 355, 209-210. (3) Friedberg, E. C., McDaniel, L. D., and Schultz, R. A. (2004) The role of endogenous and exogenous DNA damage and mutagenesis. Curr. Opin. Genet. Dev. 14, 5-10. (4) Rouse, J., and Jackson, S. P. (2002) Interfaces between the detection, signaling, and repair of DNA damage. Science 297, 547-551. (5) Zhou, B. B., and Elledge, S. J. (2000) The DNA damage response: Putting checkpoints in perspective. Nature 408, 433439. (6) Lindahl, T., and Wood, R. D. (1999) Quality control by DNA repair. Science 286, 1897-1905. (7) Sims, P., and Grover, P. L. (1974) Epoxides in polycyclic aromatic hydrocarbon metabolism and carcinogenesis. Adv. Cancer Res. 20, 165-274. (8) Szeliga, J., and Dipple, A. (1998) DNA adduct formation by polycyclic aromatic hydrocarbon dihydrodiol epoxides. Chem. Res. Toxicol. 11, 1-11. (9) Dipple, A. (1985) Polycyclic aromatic hydrocarbons: An introduction. In Polycyclic Hydrocarbons and Carcinogenesis (Harvey, R. G., Ed.) pp 1-17, American Chemical Society, Washington, DC. (10) Thakker, D. R., Yagi, H., Levin, W., Wood, A. W., Conney, A. H., and Jerina, D. M. (1985) Polycyclic aromatic hydrocarbons: Metabolic activation to ultimate carcinogens. In Bioactivation of Foreign Compounds (Anders, M. W., Ed.) pp 177-242, Academic Press, London. (11) IARC. (1986) Tobacco Smoking. In Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, IARC Publishers, Lyon, France. (12) Wu, J., Zhu, B. B., Yu, J., Zhu, H., Qiu, L., Kindy, M. S., Gu, L., Seidel, A., and Li, G. M. (2003) In vitro and in vivo modulations of benzo[c]phenanthrene-DNA adducts by DNA mismatch repair system. Nucleic Acids Res. 31, 6428-6434. (13) Wood, R. D. (1999) DNA damage recognition during nucleotide excision repair in mammalian cells. Biochimie 81, 39-44. (14) Sancar, A. (1996) DNA excision repair. Annu. Rev. Biochem. 65, 43-81. (15) Braithwaite, E., Wu, X., and Wang, Z. (1999) Repair of DNA lesions: Mechanisms and relative repair efficiencies. Mutat. Res. 424, 207-219. (16) Mitchell, J. R., Hoeijmakers, J. H., and Niedernhofer, L. J. (2003) Divide and conquer: Nucleotide excision repair battles cancer and aging. Curr. Opin. Cell. Biol. 15, 232-240. (17) Hanawalt, P. C., Donahue, B. A., and Sweder, K. S. (1994) Repair and transcription. Collision or collusion? Curr. Biol. 4, 518-521. (18) Bohr, V. A., Smith, C. A., Okumoto, D. S., and Hanawalt, P. C. (1985) DNA repair in an active gene: Removal of pyrimidine dimers from the DHFR gene of CHO cells is much more efficient than in the genome overall. Cell 40, 359-369. (19) Mellon, I., Spivak, G., and Hanawalt, P. C. (1987) Selective removal of transcription-blocking DNA damage from the transcribed strand of the mammalian DHFR gene. Cell 51, 241-249. (20) Carrell, C. J., Carrell, T. G., Carrell, H. L., Prout, K., and Glusker, J. P. (1997) Benzo[a]pyrene and its analogues: Structural studies of molecular strain. Carcinogenesis 18, 415-422. (21) Katz, A. K., Carrell, H. L., and Glusker, J. P. (1998) Dibenzo[a,l]pyrene (dibenzo[def,p]chrysene): Fjord-region distortions. Carcinogenesis 19, 1641-1648. (22) Cho, K.-B., Dreij, K., Jernstro¨m, B., and Gra¨slund, A. (2003) Conformations of benzene- and dibenzo[a,l]pyrene diol epoxides

DNA Adducts of DBPDE and BPDE in Human Cells

(23)

(24)

(25)

(26)

(27)

(28)

(29)

(30)

(31)

(32)

(33)

(34)

(35)

(36)

(37)

(38)

studied by density functional theory: Ground states, transition states, dynamics, and solvent effects. Chem. Res. Toxicol. 16, 590597. Jerina, D. M., Sayer, J. M., Agarwal, S. K., Yagi, H., Levin, W., Wood, A. W., Conney, A. H., Pruess-Schwartz, D., Baird, W. M., Pigott, M. A., and Dipple, A. (1986) Reactivity and tumorigenicity of bay-region diol epoxides derived from polycyclic aromatic hydrocarbons. Adv. Exp. Med. Biol. 197, 11-30. Jerina, D. M., Chadha, A., Cheh, A. M., Schurdak, M. E., Wood, A. W., and Sayer, J. M. (1991) Covalent bonding of bay-region diol epoxides to nucleic acids. In Biological Reactive Intermediates IV. Molecular and cellular effects and their impact on human health (Witmer, C. M., Snyder, R., Jollow, D. J., Kalf, G. S., Kocsis, J. J., and Sipes, I. G., Eds.) pp 533-553, Plenum Press. Sauvain, J. J., Vu Duc, T., and Huynh, C. K. (2001) Development of an analytical method for the simultaneous determination of 15 carcinogenic polycyclic aromatic hydrocarbons and polycyclic aromatic nitrogen heterocyclic compounds. application to diesel particulates. Fresenius’ J. Anal. Chem. 371, 966-974. Seidel, A., Frank, H., Behnke, A., Schneider, D., and Jacob, J. (2004) Determination of dibenzo[a,l]pyrene and other fjord-region PAH isomers with MW 302 in environmental samples. Polycycl. Arom. Compd. 24, 759-771. LaVoie, E. J., He, Z. M., Meegalla, R. L., and Weyand, E. H. (1993) Exceptional tumor-initiating activity of 4-fluorobenzo[j]fluoranthene on mouse skin: Comparison with benzo[j]fluoranthene, 10fluorobenzo[j]fluoranthene, benzo[a]pyrene, dibenzo[a,l]pyrene and 7,12-dimethylbenz[a]anthracene. Cancer Lett. 70, 7-14. Cavalieri, E. L., Higginbotham, S., RamaKrishna, N. V., Devanesan, P. D., Todorovic, R., Rogan, E. G., and Salmasi, S. (1991) Comparative dose-response tumorigenicity studies of dibenzo[a,l]pyrene versus 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene and two dibenzo[a,l]pyrene dihydrodiols in mouse skin and rat mammary gland. Carcinogenesis 12, 1939-1944. Higginbotham, S., RamaKrishna, N. V., Johansson, S. L., Rogan, E. G., and Cavalieri, E. L. (1993) Tumor-initiating activity and carcinogenicity of dibenzo[a,l]pyrene versus 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene at low doses in mouse skin. Carcinogenesis 14, 875-878. Dipple, A., Pigott, M. A., Agarwal, S. K., Yagi, H., Sayer, J. M., and Jerina, D. M. (1987) Optically active benzo[c]phenanthrene diol epoxides bind extensively to adenine in DNA. Nature 327, 535-536. 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. Cosman, M., Laryea, A., Fiala, R., Hingerty, B. E., Amin, S., Geacintov, N. E., Broyde, S., and Patel, D. J. (1995) Solution conformation of the (-)-trans-anti-benzo[c]phenanthrene-dA ([BPh]dA) adduct opposite dT in a DNA duplex: Intercalation of the covalently attached benzo[c]phenanthrenyl ring to the 3′-side of the adduct site and comparison with the (+)-trans-anti-[BPh]dA opposite dT stereoisomer. Biochemistry 34, 1295-1307. Suri, A. K., Mao, B., Amin, S., Geacintov, N. E., and Patel, D. J. (1999) Solution conformation of the (+)-trans-anti-benzo[g]chrysene-dA adduct opposite dT in a DNA duplex. J. Mol. Biol. 292, 289-307. Geacintov, N. E., Cosman, M., Hingerty, B. E., Amin, S., Broyde, S., and Patel, D. J. (1997) NMR solution structures of stereoisometric covalent polycyclic aromatic carcinogen-DNA adduct: Principles, patterns, and diversity. Chem. Res. Toxicol. 10, 111146. Geacintov, N. E., Naegeli, H., Dinshaw, J. P., and Broyde, S. (2005) Structural aspects of polycyclic aromatic carcinogendamaged DNA and its recognition by NER proteins. In DNA Damage Recognition (Siede, W., Kow, Y. W., and Doetsch, P. W., Eds.) Marcel Dekker Inc., New York. Buterin, T., Hess, M. T., Luneva, N., Geacintov, N. E., Amin, S., Kroth, H., Seidel, A., and Naegeli, H. (2000) Unrepaired fjord region polycyclic aromatic hydrocarbon-DNA adducts in ras codon 61 mutational hot spots. Cancer Res. 60, 1849-1856. Huang, X., Kolbanovskiy, A., Wu, X., Zhang, Y., Wang, Z., Zhuang, P., Amin, S., and Geacintov, N. E. (2003) Effects of base sequence context on translesion synthesis past a bulky (+)-trans-antiB[a]P-N2-dG lesion catalyzed by the Y-family polymerase pol kappa. Biochemistry 42, 2456-2466. Lloyd, D. R., and Hanawalt, P. C. (2002) p53 controls global nucleotide excision repair of low levels of structurally diverse benzo[g]chrysene-DNA adducts in human fibroblasts. Cancer Res. 62, 5288-5294.

Chem. Res. Toxicol., Vol. 18, No. 4, 2005 663 (39) Luch, A., Kudla, K., Seidel, A., Doehmer, J., Greim, H., and Baird, W. M. (1999) The level of DNA modification by (+)-syn-(11S,12R,13S,14R)- and (-)-anti-(11R,12S,13S,14R)-dihydrodiol epoxides of dibenzo[a,l]pyrene determined the effect on the proteins p53 and p21WAF1 in the human mammary carcinoma cell line MCF7. Carcinogenesis 20, 859-865. (40) Suh, M., Ariese, F., Small, G. J., Jankowiak, R., Hewer, A., and Phillips, D. H. (1995) Formation and persistence of benzo[a]pyrene-DNA adducts in mouse epidermis in vivo: Importance of adduct conformation. Carcinogenesis 16, 2561-2569. (41) Jankowiak, R., Ariese, F., Hewer, A., Luch, A., Zamzow, D., Hughes, N. C., Phillips, 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. (42) Dreij, K., Bajak, E., Sundberg, K., Seidel, A., Gusnanto, A., Cotgreave, I., and Jernstro¨m, B. (2004) DNA adducts of benzo[a]pyrene- and dibenzo[a,l]pyrene-diol epoxides in human lung epithelial cells: Kinetics of adduct-removal, effects on cell cycle check points and gene expression. Polycycl. Arom. Compd. 24, 549-566. (43) Luch, A., Glatt, H., Platt, K. L., Oesch, F., and Seidel, A. (1994) Synthesis and mutagenicity of the diastereomeric fjord-region 11,12-dihydrodiol 13,14-epoxides of dibenzo[a,l]pyrene. Carcinogenesis 15, 2507-2516. (44) Jernstro¨m, B., Funk, M., Frank, H., Mannervik, B., and Seidel, A. (1996) Glutathione S-transferase A1-1-catalysed conjugation of bay and fjord region diol epoxides or polycyclic aromatic hydrocarbons with glutathione. Carcinogenesis 17, 1491-1498. (45) Weinstein, I. B., Jeffrey, A. M., Jennette, K. W., Blobstein, S. H., Harvey, R. G., Harris, C., Autrup, H., Kasai, H., and Nakanishi, K. (1976) Benzo[a]pyrene diol epoxides as intermediates in nucleic acid binding in vitro and in vivo. Science 193, 592-595. (46) Labarca, C., and Paigen, K. (1980) A simple, rapid, and sensitive DNA assay procedure. Anal. Biochem. 102, 344-352. (47) Downs, T. R., and Wilfinger, W. W. (1983) Fluorometric quantification of DNA in cells and tissue. Anal. Biochem. 131, 538547. (48) Sundberg, K., Dreij, K., Seidel, A., and Jernstro¨m, B. (2002) Glutathione conjugation and DNA adduct formation of dibenzo[a,l]pyrene and benzo[a]pyrene diol epoxides in V79 cells stably expressing different human glutathione transferases. Chem. Res. Toxicol. 15, 170-179. (49) Burke, M. D., Vadi, H., Jernstro¨m, B., and Orrenius, S. (1977) Metabolism of benzo[a]pyrene with isolated hepatocytes and the formation and degradation of DNA-binding derivatives. J. Biol. Chem. 252, 6424-6431. (50) Ponte´n, I., Kim, S. K., Gra¨slund, A., Norde´n, B., and Jernstro¨m, B. (1994) Spectroscopic studies of the trans adducts derived from (+)- and (-)-anti-benzo[a]pyrene-7,8-dihydrodiol-9, 10-epoxide and the oligonucleotide 5′-d(CCTATAGATATCC). Carcinogenesis 15, 2207-2213. (51) Li, K. M., George, M., Gross, M. L., Seidel, A., Luch, A., Rogan, E. G., and Cavalieri, E. L. (1999) Structure elucidation of the adducts formed by fjord-region dibenzo[a,l]pyrene 11,12-dihydrodiol 13,14-epoxides and deoxyadenosine. Chem. Res. Toxicol. 12, 758-767. (52) Koreeda, M., Moore, P. D., Wislocki, P. G., Levin, W., Yagi, H., and Jerina, D. M. (1978) Binding of benzo[a]pyrene 7,8-diol-9,10-epoxides to DNA, RNA, and protein of mouse skin occurs with high stereoselectivity. Science 199, 778-781. (53) Meehan, T., and Straub, K. (1979) Double-stranded DNA steroselectively binds benzo[a]pyrene diol epoxides. Nature 277, 410-412. (54) Ruan, Q., Kolbanovskiy, A., Zhuang, P., Chen, J., Krzeminski, J., Amin, S., and Geacintov, N. E. (2002) Synthesis and characterization of site-specific and stereoisomeric fjord dibenzo[a,l]pyrene diol epoxide-N(6)-adenine adducts: Unusual thermal stabilization of modified DNA duplexes. Chem. Res. Toxicol. 15, 249-261. (55) Arif, J. M., and Gupta, R. C. (1997) Microsome-mediated bioactivation of dibenzo[a,l]pyrene and identification of DNA adducts by 32P-postlabeling. Carcinogenesis 18, 1999-2007. (56) Agarwal, S. K., Sayer, J. M., Yeh, H. J. C., Pannell, L. K., Hilton, B. D., Pigott, M. A., Dipple, A., Yagi, H., and Jerina, D. M. (1987) Chemical characterization of DNA adducts derived from the configurationally isomeric benzo[c]phenanthrene-3,4-diol 1,2epoxides. J. Am. Chem. Soc. 109, 2497-2504. (57) Szeliga, J., Page, J. E., Hilton, B. D., Kiselyov, A. S., Harvey, R. G., Dunayevskiy, Y. M., Vouros, P., and Dipple, A. (1995) Characterization of DNA adducts formed by anti-benzo[g]chrysene 11, 12-dihydrodiol 13, 14-epoxide. Chem. Res. Toxicol. 8, 10141019.

664

Chem. Res. Toxicol., Vol. 18, No. 4, 2005

(58) Pradhan, P., Jernstro¨m, B., Seidel, A., Norde´n, B., and Gra¨slund, A. (1999) Studies on the adduct heterogeneity of benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide stereoisomers covalently bound to deoxyribooligonucleotides by induced circular dichroism and light absorption spectroscopy. Chem. Res. Toxicol. 12, 403-411. (59) Cheng, S. C., Hilton, B. D., Roman, J. M., and Dipple, A. (1989) DNA adducts from carcinogenic and noncarcinogenic enantiomers of benzo[a]pyrene dihydrodiol epoxide. Chem. Res. Toxicol. 2, 334-340. (60) Shinohara, K., and Cerutti, P. A. (1977) Excision repair of benzo[a]pyrene-deoxyguanosine adducts in baby hamster kidney 21/ C13 cells and in secondary mouse embryo fibroblasts C57BL/6J. Proc. Natl. Acad. Sci. U.S.A. 74, 979-983. (61) Feldman, G., Remsen, J., Shinohara, K., and Cerutti, P. (1978) Excisability and persistence of benzo[a]pyrene DNA adducts in epithelioid human lung cells. Nature 274, 796-798. (62) Yang, L. L., Maher, V. M., and McCormick, J. J. (1980) Errorfree excision of the cytotoxic, mutagenic N2-deoxyguanosine DNA adduct formed in human fibroblasts by (()-7β,8R-dihydroxy9R,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. Proc. Natl. Acad. Sci. U.S.A. 77, 5933-5937. (63) Kaneko, M., and Cerutti, P. A. (1982) Excision of benzo[a]pyrene diol epoxide I adducts from nucleosomal DNA of confluent normal human fibroblasts. Chem. Biol. Interact. 38, 261-274. (64) Kootstra, A., Slaga, T. J., and Olins, D. E. (1979) Interaction of benzo[a]pyrene diol-epoxide with nuclei and isolated chromatin. Chem. Biol. Interact. 28, 225-236. (65) Obi, F. O., Ryan, A. J., and Billett, M. A. (1986) Preferential binding of the carcinogen benzo[a]pyrene to DNA in active chromatin and the nuclear matrix. Carcinogenesis 7, 907-913. (66) Mironov, N. M., Grover, P. L., and Sims, P. (1983) Preferential binding of polycyclic hydrocarbons to matrix-bound DNA in ratliver nuclei. Carcinogenesis 4, 189-193. (67) Wei, S. J., Chang, R. L., Wong, C. Q., Bhachech, N., Cui, X. X., Hennig, E., Yagi, H., Sayer, J. M., Jerina, D. M., and Preston, B. D. (1991) Dose-dependent differences in the profile of mutations induced by an ultimate carcinogen from benzo[a]pyrene. Proc. Natl. Acad. Sci. U.S.A. 88, 11227-11230. (68) Wei, S. J., Chang, R. L., Bhachech, N., Cui, X. X., Merkler, K. A., Wong, C. Q., Hennig, E., Yagi, H., Jerina, D. M., and Conney, A. H. (1993) Dose-dependent differences in the profile of mutations induced by (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase gene in Chinese hamster V-79 cells. Cancer Res. 53, 3294-3301. (69) Wei, S. J., Chang, R. L., Cui, X. X., Merkler, K. A., Wong, C. Q., Yagi, H., Jerina, D. M., and Conney, A. H. (1996) Dose-dependent differences in the mutational profiles of (-)-(1R,2S,3S,4R)-3,4-

Dreij et al.

(70)

(71)

(72) (73)

(74) (75)

(76)

(77)

(78)

(79)

(80)

dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene and its less carcinogenic enantiomer. Cancer Res. 56, 3695-3703. Zou, Y., Liu, T. M., Geacintov, N. E., and Van Houten, B. (1995) Interaction of the UvrABC nuclease system with a DNA duplex containing a single stereoisomer of dG-(+)- or dG-(-)-anti-BPDE. Biochemistry 34, 13582-13593. Hess, M. T., Gunz, D., Luneva, N., Geacintov, N. E., and Naegeli, H. (1997) Base pair conformation-dependent excision of benzo[a]pyrene diol epoxide-guanine adducts by human nucleotide excision repair enzymes. Mol. Cell. Biol. 17, 7069-7076. Zou, Y., Luo, C., and Geacintov, N. E. (2001) Hierarchy of DNA damage recognition in Escherichia coli nucleotide excision repair. Biochemistry 40, 2923-2931. Mao, B., Gu, Z., Gorin, A., Chen, J., Hingerty, B. E., Amin, S., Broyde, S., Geacintov, N. E., and Patel, D. J. (1999) Solution structure of the (+)-cis-anti-benzo[a]pyrene-dA ([BP]dA) adduct opposite dT in a DNA duplex. Biochemistry 38, 10831-10842. Fernandez-Capetillo, O., Lee, A., Nussenzweig, M., and Nussenzweig, A. (2004) H2AX: The histone guardian of the genome. DNA Repair 3, 959-967. Khan, Q. A., Vousden, K. H., and Dipple, A. (1997) Cellular response to DNA damage from a potent carcinogen involves stabilization of p53 without induction of p21(waf1/cip1). Carcinogenesis 18, 2313-2318. Binkova, B., Giguere, Y., Rossner, P., Jr., Dostal, M., and Sram, R. J. (2000) The effect of dibenzo[a,l]pyrene and benzo[a]pyrene on human diploid lung fibroblasts: The induction of DNA adducts, expression of p53 and p21(WAF1) proteins and cell cycle distribution. Mutat. Res. 471, 57-70. Mahadevan, B., Luch, A., Seidel, A., Pelling, J. C., and Baird, W. M. (2001) Effects of (-)-anti-11R,12S-dihydrodiol 13S,14R-epoxide of dibenzo[a,l]pyrene on DNA adduct formation and cell cycle arrest in human diploid fibroblasts. Carcinogenesis 22, 161-169. Khan, Q. A., and Anderson, L. M. (2001) Hydrocarbon carcinogens evade cellular defense mechanism of G1 arrest in nontransformed and malignant lung cell lines. Toxicol. Appl. Pharmacol. 173, 105-113. Wang, A., Gu, J., Judson-Kremer, K., Powell, K. L., Mistry, H., Simhambhatla, P., Aldaz, C. M., Gaddis, S., and MacLeod, M. C. (2003) Response of human mammary epithelial cells to DNA damage induced by BPDE: Involvement of novel regulatory pathways. Carcinogenesis 24, 225-234. Pa¨a¨ja¨rvi, G., Jernstro¨m, B., Seidel, A., and Stenius, U. (2004) Exposure of mammalian cells to diol epoxides from benzo[a]pyrene and dibenzo[a,l]pyrene and effects on Mdm2 and p53. Polycycl. Arom. Compd. 24, 537-548.

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