Formation and Differential Repair of Covalent DNA Adducts

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Chem. Res. Toxicol. 2009, 22, 81–89

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Formation and Differential Repair of Covalent DNA Adducts Generated by Treatment of Human Cells with (()-anti-Dibenzo[a,l]pyrene-11,12-diol-13,14-epoxide Wendy A. Spencer,† Jaya Singh,† and David K. Orren* Graduate Center for Toxicology, UniVersity of Kentucky College of Medicine, Lexington, Kentucky 40536 ReceiVed May 9, 2008

Dibenzo[a,l]pyrene (DBP) is the most potent tumor initiating polycyclic aromatic hydrocarbon tested to date in rodent tumor models. To investigate how DBP adduct formation and removal might influence carcinogenesis, we have examined the effects of treatment of several nucleotide excision repair (NER)proficient (NER+) and -deficient (NER-) cell lines with the carcinogenic metabolite (()-anti-DBP11,12-diol-13,14-epoxide (DBPDE). The treatment of NER- cells with (()-anti-DBPDE for 0.5, 1, or 2 h yielded similar total adduct levels, indicating that adduct formation was essentially complete during a 2 h treatment period with no additional adducts produced after replacement of media. In all cell lines, treatment with (()-anti-DBPDE generated five major and at least two minor adducts that were chromatographically identical to those formed by direct treatment of 3′-GMP and 3′-AMP with (()anti-DBPDE. When adduct levels were assessed in NER- cells, the number of adducts/109 nucleotides decreased over time, suggesting that DNA replication was ongoing, so we incorporated a normalization strategy based on DNA synthesis. This strategy indicated that DBPDE-DNA adduct levels in NERcells are stable over time. After normalization for DNA synthesis in the NER+ cells, our data indicated that three adducts showed biphasic repair kinetics. A faster rate of removal was observed during the first 6 h following DBPDE removal followed by a slower rate for up to 34 h. Importantly, two of the major guanine adducts were particularly refractory to removal in the NER+ cells. Our results suggest that the extreme carcinogenicity of DBPDE may result from the ability of a substantial percentage of two structurally distinct DBPDE-DNA adducts to escape repair. Introduction 1

Polycyclic aromatic hydrocarbons (PAHs) are environmental carcinogens present in the atmosphere from combustion sources such as cigarette smoke, diesel exhaust, residential heating, and industrial coke production. PAHs can be taken up by cells and then metabolically activated to electrophilic species that react with nucleophilic sites in DNA, primarily forming guanine and adenine adducts (1, 2). If not removed prior to DNA replication, these adducts can result in mutations. If such mutations occur in critical oncogenes or tumor suppressor genes that control cell proliferation, development of cancer may follow. Dibenzo[a,l]pyrene (DBP) is generated by incomplete combustion, albeit at more modest levels as compared to more extensively studied PAHs such as benzo[a]pyrene (BP) (3). Nevertheless, DBP may pose a serious carcinogenic risk to humans as it is the most potent carcinogen of the PAHs tested to date in rodent model systems (4-6). In a study that directly compared the tumor-initiation activities of several PAH compounds applied individually (at 1 nmol) to mouse skin, DBP treatment resulted in 9-fold more tumors per animal than 7,12dimethylbenzanthracene (DMBA), while BP was inactive (5). * To whom correspondence should be addressed. Tel: 859-323-3612. Fax: 859-323-1059. E-mail: [email protected]. † These authors contributed equally to this work. 1 Abbreviations: BP, benzo[a]pyrene; DBP, dibenzo[a,l]pyrene; DBPDE, dibenzo[a,l]pyrene-11,12-diol-13,14-epoxide; DMBA, 7,12-dimethylbenzanthracene; DMSO, dimethylsulfoxide; FBS, fetal bovine serum; NER, nucleotide excision repair; PAH, polycyclic aromatic hydrocarbon; PEI, polyethylene imine; TLC, thin-layer chromatography; XP, xeroderma pigmentosum.

In another study using higher PAH doses, DBP induced significantly more tumors than BP with a shorter latency, while DMBA treatment resulted in more tumors than DBP but a longer latency (4). The lower tumor multiplicity of DBP vs DMBA at the higher doses was attributed to toxicity of DBP at these doses. In addition to being a more potent skin carcinogen than BP and DMBA, DBP was also found to be a stronger mammary carcinogen (4). In support of the link between persistent DNA adduct levels and carcinogenic potential, DBP induced higher (steady-state) DNA adduct levels (up to 9-fold) in rat mammary glands than either BP or DMBA (7). The enzymes primarily responsible for bioactivation of PAHs to DNA-reactive metabolites are the cytochrome P450s, specifically isozymes of the CYP1A gene family (8, 9). Both CYP1A1 (10) and CYP1B1 (11) have been shown to convert several PAHs to DNA reactive metabolites; however, these isozymes differ in their region and stereochemical selectivity of PAH bioactivation (12, 13). The carcinogenic activity of DBP has been primarily attributed to its bioactivation by CYP1A1 or CYP1B1 and epoxide hydrolase to the DNA-reactive fjord region syn- and anti-DBP-11,12-diol-13,14-epoxides (DBPDE) via the intermediate DBP-trans-11,12-dihydrodiol (4, 14). synand anti-DBPDE bind covalently to DNA (15, 16), are extremely potent mutagens in both prokaryotic and eukaryotic test systems (17), and are potent lung, liver, and mammary carcinogens in animal models (18, 19). Thus, the strong carcinogenic potential of DBP would appear to be the result of DBP-DNA adducts generated by these highly reactive DBPDE metabolites.

10.1021/tx8001675 CCC: $40.75  2009 American Chemical Society Published on Web 11/20/2008

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Bulky DNA adducts generated by PAHs are generally removed by nucleotide excision repair (NER) (20, 21). The many protein factors involved in human NER sequentially (i) recognize structurally diverse DNA adducts among a vast excess of undamaged DNA, (ii) catalyze incisions both 5′ and 3′ to the adduct, (iii) excise a ∼30 nt section of the strand containing the adduct, (iv) fill the resulting gap using the undamaged strand as template for repair DNA synthesis, and (5) complete the repair process by ligating the newly synthesized patch to the original DNA. Substantial evidence indicates that the efficiency of NER is determined by the degree of perturbation of the normal helical structure induced by structurally distinct DNA adducts (21-24). Thus, NER may preferentially recognize certain stereochemical conformations assumed by PAH adducts while overlooking others (25). In support of this idea, the efficiency of human NER varied by several fold when examined on different stereochemical configurations of BP-N2-dG adducts (25). NER of PAH adducts is also influenced by local DNA conformation, as exemplified by the moderate excision of BPN6-dA adducts as compared to the lack of excision of benzo[c]phenanthrene N6-dA adducts located within the same sequence context, that is, codon position 61 of the N- or H-ras gene (26). It is notable that, like DBP, benzo[c]phenanthrene contains the fjord type structure, while BP is a bay region PAH. On the basis of the even stronger tumorigenicity of DBP and its metabolites, it can be hypothesized that DBP-DNA adducts may be poorly recognized and repaired by NER. In turn, this would lead to their persistence and thus increase their mutagenic and carcinogenic potential. In this report, we have studied the dynamics of formation and removal of (()-anti-DBPDE-generated DNA adducts in NER-proficient (NER+) and -deficient (NER-) human cell lines using the 32P-postlabeling assay. The extreme sensitivity of 32Ppostlabeling (1 adduct/1010 nucleotides) for the detection of covalent DNA adducts (27) offers opportunities to study the effects of DNA-damaging agents at low, physiologically relevant concentrations. NER-deficient cells were examined to provide a clear representation of the spectrum and levels of damage generated by DBPDE without the confounding factor of removal of adducts during and after the treatment intervals. Our results indicate that treatment of NER+ and NER- human cells with (()-anti-DBPDE resulted in the formation of five major and at least two minor DNA adducts chromatographically comparable to those produced by treatment of the isolated guanine and adenine nucleotides. These major adducts could be easily detected and quantitated at DBPDE doses as low as 1 nM. Using this 1 nM DBPDE treatment on NER+ cells, we monitored changes in the levels of individual adducts over time to determine which adducts are more persistent and thus likely to contribute significantly to the potent carcinogenicity of DBP and its metabolites. Measurement of DNA adducts in three NER+ cell lines indicated differential repair profiles for the individual adducts, with at least two DBP-DNA adducts appearing to be quite poorly repaired. Our results support the concept that inefficient repair of certain DBP-DNA adducts might contribute to the potent carcinogenicity associated with the parent compound.

Experimental Procedures Chemicals and Reagents. (()-anti-DBPDE (>99%) was purchased from the National Cancer Institute Chemical Carcinogen Repository (Kansas City, MO). Chemicals, enzymes, and solvents used for 32P-postlabeling were purchased as described elsewhere (28). Polyethylene imine (PEI) thin-layer chromatography (TLC)

Spencer et al. sheets were generously provided by Dr. Ramesh Gupta (University of Louisville). CyQUANT GR dye for the measurement of DNA synthesis was purchased from Invitrogen Corp. (Carlsbad, CA). Nucleotide 3′-monophosphates and anhydrous dimethylsulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. Louis, MO). Radiolabeled [γ-32P]ATP was from Perkin-Elmer (Waltham, MA). Cell Culture and Treatment with (()-anti-DBPDE. RXRXPA+ cells were NER-deficient human SV40-transformed skin fibroblasts, derived from a xeroderma pigmentosum (XP) patient homozygous for a nonsense mutation in the XPA gene, and were complemented with wild-type XPA cDNA to restore NER capability (29). Although XPA was cloned into the pIND expression vector downstream of the RXR promoter, RXR agonists were not needed to produce levels of XPA protein similar to normal cells (ref 29; our unpublished results). RXR-XPA- cells were the same cell line complemented with empty vector. These isogenic cell lines were kindly provided by Dr. Isabel Mellon (University of Kentucky) and grown in MEM with Earle’s salts supplemented with 10% fetal bovine serum (FBS), 1% nonessential amino acids, 2 mM Lglutamine, and 1% penicillin-streptomycin. 8-D and 1-O cells, generously provided by Dr. Christopher States (University of Louisville), were derived from normal (NER+) and XPA-deficient (NER-) human SV40-transformed fibroblasts, respectively (30). Both cell lines were grown in MEM R-medium supplemented with 10% FBS, 1% HEPES, and 1% penicillin-streptomycin. MCF-7 cells were purchased from American Tissue Culture Collection (Manassas, VA) and were cultivated in DMEM supplemented with 2 mM L-glutamine, 1% penicillin-streptomycin, and 10% FBS. All cell lines were grown at 37 °C in a humidified atmosphere containing 5% CO2. All cell culture media and reagents were purchased from Invitrogen. Cells (4-6 × 106) were plated in 175 cm2 flasks 1 day prior to carcinogen exposure. At the time of treatment, (()-anti-DBPDE (diluted in anhydrous DMSO from stock solutions made in tetrahydrofuran) was added to the medium to a final concentration of 1-20 nM, and incubation was continued for up to 2 h. For repair experiments, cells were washed with PBS after the DBPDE treatment interval and incubated in fresh media for up to 36 h. At the indicated time points, cells were collected by centrifugation and stored at -80 °C prior to DNA isolation. Cell viability and proliferation were measured by trypan blue exclusion and cell counting, respectively. Analysis of DNA Adducts by 32P-Postlabeling. DNA adducts were measured by the nuclease P1 version of the 32P-postlabeling procedure as detailed previously (28). Briefly, DNA was isolated from cells after lysis in Tris-EDTA buffer (pH 8.0) by sequential digestions with RNases A and T1 and then proteinase K. Proteins were removed by organic solvent extractions, and DNA was precipitated from the aqueous fraction with ethanol. DNA (12 µg) was hydrolyzed to deoxynucleotide 3′-monophosphates by incubation with a mixture of micrococcal nuclease and spleen phosphodiesterase (1:5 enzyme to DNA ratio, w/w) for 5 h at 37 °C. Adducts were enriched by selectively hydrolyzing the 3′-monophosphates of nonadducted nucleotides by treatment with nuclease P1 (1:2.5 enzyme to DNA ratio, w/w) for 1 h at 37 °C and 5′-32P-labeled using T4 polynucleotide kinase (New England Biolabs, Beverly, MA) and a molar excess of [γ32P]ATP (6000 Ci/mmol). Labeled adducts were resolved by multidirectional PEI-cellulose TLC as described (28). Solvent systems for TLC are presented in Table 1. Radioactivity associated with individual adducts was visualized and quantified using a Packard Instant Imager. To calculate the relative adduct level, total nucleotides were analyzed in parallel by labeling 2 ng of digested DNA and separating unadducted nucleotides by standard PEI-TLC methods (28). The relative adduct level represents the ratio of (total or individual) adducts to unadducted nucleotides, expressed as adducts per 109 nucleotides. Cellular DNA Synthesis Analysis. To normalize for dilution of DNA adducts due to possible ongoing DNA synthesis, changes in the amount of DNA in cell populations over time were measured using the fluorescence-based CyQUANT GR assay (Invitrogen).

Formation and Repair of DBP-DNA Adducts

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Table 1. Solvent Systems Used for Resolving DBPDE-DNA Adducts by PEI-Cellulose TLCa solvent system I

II

III

composition D1: 1.0 M sodium phosphate, pH 6.0, on 5 cm wickb D3: 4.0 M lithium formate/7.0 M urea, pH 3.5, on 4 cm wickc D4: 0.72 M sodium phosphate/0.45 M Tris-HCl/7.6 M urea, pH 7.3, on 8 cm wickc D5: 1 M sodium phosphate, pH 6.0, on 3 cm wickc D1: 1.0 M sodium phosphate, pH 6.0, on 5 cm wickb D3: 4.0 M lithium formate/7.0 M urea, pH 3.5 D4: isopropanol:4 M NH4OH (0.8:1) D5: 1 M sodium phosphate, pH 6.0, on 3 cm wickc D1: 1.0 M sodium phosphate, pH 6.0, on 5 cm wickb D3: 0.72 M sodium phosphate/0.45 M Tris-HCl/7.6 M urea, pH 7.3 D4: isopropanol:4 M NH4OH (1:1) D5: 1 M sodium phosphate, pH 6.0, on 3 cm wickc

a

All systems were run on D1 opposite D3 template (28). no. 17 wick. c Whatman no. 1 wick.

b

Whatman

Cells (0.64 × 106) were plated in 25 cm2 flasks 1 day prior to carcinogen exposure. Cells were treated with 1 nM anti-DBPDE or vehicle (0.1% DMSO) for 2 h, washed with PBS, and reincubated with fresh media for up to 34 h. At the time of harvest, cells were trypsinized, washed, and collected in 1 mL of PBS and stored at -80 °C. Upon thawing, cell suspensions (10 µL) were placed in a 96 well plates and incubated (1 h at 25 °C) with RNase A (1.35 Kunitz units/mL) in lysis buffer provided with the kit. A CyQUANT GR dye solution was prepared in lysis buffer and added to the wells (100 µL), and fluorescence was measured (ex, 485 nm; em, 530 nM) after 4 min. Changes in the amount of DNA over time were calculated by dividing the fluorescence of DBPDE-treated cells collected at specific times following carcinogen removal by that of cells harvested immediately after the treatment interval. The levels of DNA adducts per nucleotide were then adjusted by these relative values to normalize them for DNA synthesis.

Results 32

P-Postlabeling Analysis of (()-anti-DBPDE-DNA Adducts. To establish the spectrum of covalent DBP-DNA adducts produced by exposure to (()-anti-DBPDE, adduct formation was initially examined in NER- cells that cannot remove bulky adducts typically generated by PAHs. Postlabeling analysis of genomic DNA isolated from NER- (1-O) cells treated for 2 h with (()-anti-DBPDE (up to 20 nM) revealed the formation of multiple DNA adducts. Several different solvent systems for TLC (Table 1) were evaluated to determine the best conditions for separation of these DBP-DNA adducts. These chromatographic conditions were compared with adducts generated from the reaction of purified adenine (dAp) and guanine (dGp) mononucleotide phosphates with (()-anti-DBPDE (Figure 1A-G) as well as genomic DNA. Although some separation between individual adenine and guanine adducts was achieved in all three systems tested, certain adducts did not separate well in the D4 (vertical) direction when using solvent systems II and III. Because of its superior ability in separating DBP-DNA adducts, solvent system I was used in all subsequent studies. Using this TLC solvent system, analysis of genomic DNA from NER- cells treated for 2 h with (()-anti-DBPDE and harvested immediately indicated the formation of five major and two minor structurally distinct DNA adducts (Figure 1H). Direct chromatographic comparison of cellular DNA adducts with adducts obtained from treatment of individual 2′-deoxynucleoside 3′-monophosphates with (()-anti-DBPDE indicated that four of the major adducts (denoted G1-4) were derived from dGp, while another major (denoted A1) and two minor (A2 and

Figure 1. Adduct maps of the reaction of (()-anti-DBPDE (100 µM) with deoxyadenosine-3′-monophosphate (dAp) (A-C) and deoxyguanosine-3′-monophosphate (dGp) (D-F), using 375 µg of dAp or dGp (20 µg/µL) incubated for 2 h at 37 °C in 0.4 mM Tris-HCl buffer (pH 7.4) followed by ethyl acetate extraction and 32P-postlabeling. An aliquot (100 ng) of each nucleotide was used for TLC in three solvent systems detailed in Table 1. (G) Cochromatography of the dAp and dGp adducts using solvent system I. The cellular DNA adduct map (H) is derived from 1-O cells treated with 1 nM (()-anti-DBPDE for 2 h. DNA was isolated, and 12 µg was used for 32P-postlabeling followed by TLC as described in the Experimental Procedures. DNA adducts were visualized using a phosphorimager.

Table 2. Percent Contribution of Individual DNA Adducts to Total Adduct Level Following 1 nM anti-DBPDE Treatment for 2 h adduct (% of total) cell line NER-deficient (NER-)

1-O RXR-XPANER-proficient (NER+) 8-D MCF-7 RXR-XPA+

A1/G2

G3

G4

G1

75 ( 5 11 ( 2 7 ( 2 7 ( 2 72 ( 5 12 ( 1 8 ( 1 8 ( 1 72 ( 7 9 ( 1 6 ( 1 13 ( 1 64 ( 8 12 ( 1 7 ( 1 17 ( 2 73 ( 5 8 ( 1 8 ( 1 11 ( 1

A3) adducts were derived from dAp (Figure 1A,D). These adducts were also chromatographically indistinguishable from those analyzed from purified calf thymus DNA subsequently treated with (()-anti-DBPDE (data not shown) and consistent with those reported by Arif and Gupta (31). DBP-DNA adducts derived from cytidine or thymine nucleotides were not observed to any significant degree following treatment of cells with (()anti-DBPDE (data not shown). Only the major adducts (A1 and G1-4) were quantified for the repair studies below, as the minor adducts could not be reliably measured above the background when using low DBPDE concentrations. Notably, the percentages of each adduct relative to the total number of adducts immediately following DBPDE treatment were very similar between both NER- and NER+ cell lines (Table 2), indicating that formation (and stability) of these individual covalent adducts is relatively consistent. Although adducts A1 and G2 were partially resolved on occasion, they more often completely comigrated, most likely the result of variable PEI-cellulose sheet thickness. By comparing the intensities in the top and bottom halves of the A1/G2 spot in our cochromatogram of purified dAp and dGp nucleotides (Figure 1G) with the individual chromatograms of the dAp and dGp nucleotides (Figure 1A,D, respectively), it can be determined that when these adducts are partially separated, the A1 adducts migrate slightly faster in the vertical direction than G2 adducts. Therefore, we can conclude that for the cellular DNA chromatographed in parallel (Figure 1H), the slower migrating G2 adducts were at least 3-fold more abundant than

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Figure 2. Effect of (()-anti-DBPDE dose (1-20 nM) on total DNA adduct levels in NER+ cells, 8-D (white) and RXR-XPA+ (light gray), and NER- cells, 1-O (dark gray) and RXR-XPA- (black). Each bar is the mean of 3-4 replicates ( SE and represents the total DNA adduct level (G1, A1/G2, G3, and G4 adducts combined).

the A1 adduct just above. Because this analysis was performed in NER- cells immediately after treatment, our results suggest that guanine adducts constitute a significant majority of the DNA adducts generated at the 1 nM (()-anti-DBPDE concentration subsequently used for our repair experiments. Because A1 and G2 adducts could often not be resolved, for our subsequent experiments, they were quantified as a single adduct (A1/G2), and the removal of this combined adduct was assessed. However, it is logical that large decreases in the intensity of combined A1/G2 adduct spot during the repair interval must be primarily due to removal of the predominant G2 adducts. However, we cannot determine the precise repair kinetics of the G2 adducts. Because of its low abundance and small contribution to the A1/G2 spot, removal of the A1 adduct cannot be assessed. Dose Response of anti-DBPDE in NER+ and NER- Cell Lines. To determine a suitable dose for studying adduct repair, two NER- (1-O and RXR-XPA-) and two NER+ (8-D and RXR-XPA+) cell lines were treated with doses ranging from 1 to 20 nM (()-anti-DBPDE for 2 h and then harvested immediately. In both NER+ and NER- cells, total DBP-DNA adduct levels increased in a dose-dependent manner ranging from 386 to 12287 adducts/109 nucleotides at 1 and 20 nM, respectively (Figure 2). Total adduct levels at each individual dose were similar between these cell lines, indicating comparable cellular uptake and detoxification of (()-anti-DBPDE. The observation that NER- and NER+ cells had similar total adduct levels suggested only a minor, at best, contribution of repair during this 2 h treatment interval. The major adduct (A1/G2) constituted 70%, on average, of the total adduct level at each dose. To maintain a measurable level of the major adducts at the lowest possible dose, 1 nM DBPDE was chosen for our repair studies. To test for cytotoxicity of (()-anti-DBPDE at this 1 nM dose, trypan blue exclusion studies were conducted using the NER- cell line 1-O. Although 1-O cells would be expected to be more sensitive to DNA damage as compared to NER+ cells, no effect on cell viability was observed between 2 and 36 h following 1 nM DBPDE treatment for 2 h when compared to vehicle-treated (0.1% DMSO) cells. Thus, the 1 nM DBPDE dose appears to be suitable for both physiological survival and measurement of each major DBP-DNA adduct. Normalization of DNA Adduct Levels for Ongoing DNA Synthesis. When DNA adduct levels were measured in the DBPDE-treated NER- cell lines, RXR-XPA- and 1-O, the number of adducts per unit DNA decreased slowly over time

Figure 3. (A) Effect of (()-anti-DBPDE (1 nM, 2 h of exposure) on DNA synthesis in the NER- cells 1-O (black triangles) and RXRXPA- (gray squares) for up to 34 h following its removal. DNA yield was measured in DBPDE-treated cells using CyQUANT GR intercalating fluorescent dye. Values represent the percent change ((standard error) of DNA yield of treated cells collected at various time points as compared to those collected immediately following DBPDE exposure (0 h). Each result is the average of 6-8 replicates from at least two independent experiments. (B) DBP-DNA total adduct levels without (gray circles) and with (black squares) normalization for DNA synthesis in the NER-deficient cells 1-O and RXR-XPA-. Normalization was achieved using the CyQuant intercalating dye by calculating the ratio of DNA from the DBPDE recovery group (cells treated for 2 h followed by 2-34 h of incubation) to DNA of the control group (cells treated for 2 h without further incubation). DNA adduct levels, 2-34 h following DBPDE removal, were then multiplied by this ratio to normalize for DNA synthesis. Each result is the average of 3-4 replicates from at least two independent experiments, with standard errors for the unnormalized data.

after the treatment interval (Figure 3B, gray lines), apparently as a result of ongoing DNA replication. In agreement with this observation, cell numbers also increased over the 36 h period after DBPDE treatment was initiated; however, because cell division lags behind DNA replication, cell doubling kinetics should not be used to correct for DNA synthesis. To account for ongoing DNA synthesis, we employed a normalization strategy based on quantitation of DNA in cell lysates using a fluorescent dye (CyQUANTGR) that intercalates between the DNA bases. NER- cells were employed to determine if this assay could accurately account for changes in the amount of DNA/cell during the post-treatment (repair) interval and thus be used to normalize our adduct levels in response to ongoing

Formation and Repair of DBP-DNA Adducts

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Table 3. Time-Dependent Decrease of Individual and Total DBP-DNA Adductsa in NER+ (Proficient) Cell Lines G3

G4

G1

total

8-D

cell line

time (h) A1/G2 0 2 6 10 14 22 34

477 ( 44 283 ( 6 277 ( 11 236 ( 19 229 ( 9 140 ( 4 120 ( 8

62 ( 6 39 ( 6 37 ( 2 26 ( 2 29 ( 4 17 ( 1 13 ( 1

42 ( 5 26 ( 3 23 ( 1 16 ( 1 21 ( 1 18 ( 2 15 ( 1

84 ( 9 53 ( 1 62 ( 5 53 ( 2 61 ( 6 43 ( 2 49 ( 2

665 ( 57 401 ( 12 397 ( 15 330 ( 18 339 ( 15 217 ( 4 196 ( 10

RXR-XPA+

0 2 6 10 14 22 34

318 ( 23 291 ( 22 278 228 ( 14 206 ( 23 175 ( 21 126 ( 8

34 ( 1 29 ( 2 28 25 ( 1 26 ( 5 22 ( 3 19 ( 2

38 ( 19 32 ( 3 26 31 ( 1 27 ( 3 23 ( 2 22 ( 2

47 ( 3 40 ( 3 44 50 ( 2 46 ( 6 48 ( 6 48 ( 3

437 ( 28 392 ( 29 376 334 ( 17 305 ( 37 268 ( 32 215 ( 13

MCF-7

0 2 6 10 14 22

595 ( 73 305 ( 8 292 ( 16 255 ( 23 184 ( 9 124 ( 12

112 ( 11 88 ( 5 78 ( 6 66 ( 4 52 ( 5 45 ( 6

62 ( 5 50 ( 1 51 ( 4 53 ( 4 40 ( 5 31 ( 3

158 ( 15 92 ( 5 103 ( 10 116 ( 4 104 ( 12 78 ( 6

926 ( 101 535 ( 13 524 ( 25 490 ( 28 380 ( 28 278 ( 26

a Values expressed are numbers of adducts/109 nucleotides and represent the mean and standard error of 3-4 biological replicates, except for the underlined values (single measurement).

DNA synthesis. In both NER- cell lines, the CyQUANTGR assay showed an increase in total cellular DNA over time reflecting replicative synthesis during the 34 h after removal of DBPDE (Figure 3A). The fluorescent DNA signal measured at individual time points was divided by the fluorescent DNA signal at the end of the treatment period to yield a series of normalization factors for each experiment. At each repair time point, the number of DNA adducts per nucleotide was adjusted using the corresponding normalization factor derived from the measured change in the amount of total DNA in the cell population. After this normalization process, DBP-DNA adduct levels in NER- cells remained relatively stable over time as expected (Figure 3B, black lines), indicating that this method reasonably accounts for the “dilution effect” caused by ongoing DNA synthesis. DNA Adduct Formation and Removal in NER+ Cells. To study the kinetics of NER on DBPDE-DNA adducts, NER+ cells (RXR-XPA+, 8-D, and MCF-7) were treated with 1 nM (()-anti-DBPDE for 2 h and then incubated in fresh media for up to 34 h before harvesting and collection of genomic DNA. 32 P-postlabeling yielded total DNA adduct levels of 437, 665, and 926 adducts/109 nucleotides immediately following the treatment interval (0 h time point) in RXR-XPA+, 8-D, and MCF-7 cells, respectively (Table 3) This moderate variation in the total number of DNA adducts between different cell lines was likely due to differences in carcinogen uptake. However, the proportions of the individual adducts formed were quite comparable between cell lines (Table 2), indicating that at this low dose of DBPDE, the metabolism and adduct formation processes are very similar between different cell types. After correction for ongoing DNA synthesis, the number of total and individual adducts at each time point (Table 3 and Figure 4) is indicative of adduct removal by NER. Kinetics of repair appeared to be similar in 8-D and MCF-7 cells with total adduct levels decreasing by 40-45% by 2 h of carcinogen removal and by 70% after 22 h (Figures 4 and 5). However, in RXRXPA+ cells, repair was much slower (particularly early after carcinogen removal) than in 8-D or MCF-7 cells, with about half of the total adducts remaining after 34 h. This difference in repair kinetics is not entirely unexpected, as these cells were

derived from an NER- cell line complemented with the XPA gene to restore repair capacity. This complementation may not completely return these cells to a normal NER phenotype, and thus, their repair efficiency may be partially compromised. Because the A1/G2 adducts reflect the majority of adducts formed, these overall cellular repair efficiencies are heavily influenced by removal of these specific adducts (see below). It is also extremely important to also examine the repair of individual DBP-DNA adducts, since the efficiency of NER appears to depend on the distinct DNA structures imposed by different adducts. Consistent with this notion, our data demonstrate differential repair kinetics for the individual DBP-DNA adducts (Figures 4 and 6). Levels of the most prominent A1/ G2 adduct mixture declined throughout the time course in all three cell lines tested. By 22 h following DBPDE removal, adduct A1/G2 was removed by >70% in both 8-D and MCF-7 cells. Because the G2 adducts are more than 3-fold more abundant than A1 adducts immediately after DBPDE treatment (Figure 1H), it can be reasoned that the specific removal of G2 adducts contributes significantly to this decrease; unfortunately, we cannot determine whether the less abundant A1 adducts are removed and at what rate. Adduct G3 was repaired with somewhat similar but slightly slower kinetics, being removed by 74 and 54% in 8-D and MCF-7 cells, respectively, at 22 h. RXR-XPA+ cells appeared to have a lower overall repair capacity than either 8-D or MCF-7 cells for the G3 and A1/G2 adducts. In contrast, a large percentage of adduct G1, and possibly G4, appear to be quite refractory to NER in all three cell lines. Only about half of the G1 and G4 adducts were repaired 22 h following carcinogen removal in MCF-7 and 8-D cells. In RXR-XPA+ cells, the G4 adduct was also removed slowly, while the G1 adduct appeared to be almost completely refractory to repair. Thus, individual DBP-DNA adducts are repaired with different kinetics, consistent with the idea that structurally distinct lesions are recognized with varying efficiency by the NER machinery. The efficiency of NER for individual lesions appears to be reasonably consistent between normal cell lines, while the somewhat compromised repair efficiency of the RXR-XPA+ cell line noted above is reflected by consistently lower removal of each individual adduct. It is highly notable that certain DBP-DNA adducts (particularly the G1 adduct) appear to avoid repair and thus persist in DNA. Theoretically, persistence of such adducts may correlate with increased rates of mutation and higher carcinogenic potential of DBP and its metabolites.

Discussion If not removed by cellular repair systems, adducts caused by chemical modification of DNA can cause mutations in critical genes that may eventually result in cellular transformation and, ultimately, cancer (32). PAHs are one of the most studied classes of compounds with respect to metabolic activation (to DNAreactive electrophilic dihydrodiol epoxides), DNA adduct formation, and potential roles as tumor initiators. PAH compounds primarily form adducts with guanine and adenine, and many are strong mutagens as well as potent carcinogens in animal models (33, 34). The intrinsic mutagenicity of PAH adducts is determined by a number of factors, including the NER pathway that recognizes and removes PAH adducts based on how they alter the DNA double helical structure (35). One particular PAH, DBP, has been shown to be an extraordinarily potent carcinogen in several rodent model systems (4-7). A recent report that measured the amounts of certain PAHs in an urban environment detected DBP and suggested that the amounts

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Figure 4. Formation and removal of total individual DNA adducts in 8-D, MCF-7, and RXR-XPA+ cell lines. Cells were treated with (()-antiDBPDE (1 nM, 2 h) and harvested immediately or after the indicated times. For each cell line, the number of each individual DNA adduct was determined at each time point and normalized to account for DNA synthesis. The relative amounts of each adduct (G1, white; A1/G2, dark gray; G3, black; and G4, light gray) as part of the total adduct burden at each time point are presented in the form of a stacked bar graph.

Figure 5. Overall removal of DNA adducts in the NER+ cells 8-D, RXR-XPA+, and MCF-7 for up to 34 h following (()-anti-DBPDE (1 nM, 2 h) exposure. Values (normalized as in Figure 3B) represent the percent of DNA adducts remaining in the recovery group (cells treated with DBPDE for 2 h followed by 2-34 h of recovery time) as compared to the control group (cells treated with DBPDE for 2 h). Each result is the average of 3-4 biological replicates, with the exception of the 6 h time point for RXR-XPA+ cells.

of DBP present posed a higher carcinogenic risk than other detectable PAHs including BP (36). Because the repair efficiency of a DNA adduct impacts it persistence and thus its potential for generating mutations, the potent carcinogenicity of DBP might be due to inefficient removal of one or more covalent adducts generated by DBP metabolites. In this study, we investigated the formation and removal of DNA adducts generated after treatment of several cell lines with the DNA-reactive metabolite (()-anti-DBPDE. To initially examine DBP-DNA adduct formation without potential interference from repair, two NER-deficient (NER-) cell lines, 1-O and RXR-XPA-, were exposed to various doses of (()-anti-DBPDE. It is notable that when the contribution of DNA replication was accounted for, total and individual DNA adduct levels remained relatively constant over time in these NER- cells, attesting to the stability of these covalent DBPDNA adducts. Seven DBP-DNA adducts were identified in both cell lines, with the major adducts being comprised of four dGp and one dAp adduct, while the minor adducts were both dAp derivatives. Our observed collection of DBP-DNA adducts is consistent with others that found covalent linkages of DBPDE intermediates to adenine and guanine bases, presumably at the exocyclic amino groups, as has been demonstrated for other PAH diol epoxides (37-40). Using the methodology currently available to our laboratory, the major dAp adduct (A1) most often comigrated with one of the major dGp adducts (G2),

Figure 6. Removal of individual DNA adducts, G1, A1/G2, G3, and G4, in the NER+ cells 8-D, RXR-XPA+, and MCF-7 for up to 34 h following (()-anti-DBPDE (1 nM, 2 h) exposure. Values (normalized as in Figure 3B) represent the percent of DNA adducts remaining in the recovery group (cells treated with DBPDE for 2 h followed by 2-34 h of recovery time) as compared to the control group (cells treated with DBPDE for 2 h). Each result is the average of 3-4 biological replicates, with the exception of the 6 h time point for RXR-XPA+ cells.

making it difficult to absolutely ascertain the relative contributions of adenine and guanine adducts to the total adduct burden. From instances in which the A1 and G2 adducts were separable, we can deduce that after the initial treatment interval, A1 adducts are much less abundant than G2 adducts (Figure 1H). Thus, it can be concluded that a significant majority of DNA adducts following our (()-anti-DBPDE treatments are formed with guanine. Regarding the prevalence of guanine adducts, our results are consistent with others that have used (()-antiDBPDE for treatment of calf thymus DNA and mouse skin (41). However, reaction of the parent compound, DBP, with DNA in presence of rat liver microsomes was shown to yield A:G ratios of nearly 1:1 (7, 42) while others have reported that (-)anti-DBPDE reacts predominantly with adenine in human epithelial cells and results primarily in mutations at A:T base pairs (43-45). Among these studies, variations in the distributions of adducts are likely due to the different compounds and varying doses used for treatments and possibly influenced by the metabolic capacities of various cell types. Because of the observed predominance of adenine adducts in cells treated with

Formation and Repair of DBP-DNA Adducts

the (-)-anti-DBPDE stereoisomer, we suspect that the (+)-antiDBPDE stereoisomer might be responsible for generating large numbers of guanine adducts at this 1 nM dose. It is also notable that depending upon the treatment interval, measurements of specific adduct levels can be affected by repair in NER+ cells. In contrast to previous studies, our experiments employed very low concentrations of (()-anti-DBPDE and short treatment intervals to directly assess DNA adduct formation and removal under conditions that more closely reflect physiological exposure levels as well as minimize global effects on cells and their metabolic capacities. To our knowledge, this study is also the first to examine intracellular DBP-DNA adduct formation in an NER- background, thus giving a clearer picture of adduct generation without the confounding influence of ongoing repair. Our results indicate that little or no repair occurred during the 2 h treatment interval (Table 2) and thus permitted us a clear picture of the repair efficiencies of total and individual DBP-DNA adducts. When total DBP-DNA adducts are considered, the MCF-7 and 8-D cell lines have markedly similar repair patterns (Figures 4 and 5), suggesting that the general efficiency of DBP-DNA adduct removal is relatively consistent between cell lines with normal NER capacity. Although the RXR-XPA+ cell line also repairs DBP-DNA adducts, adduct removal is significantly slower. Because this cell line was produced by stable transfection of the XPA gene into a previously XPA-deficient and NER-deficient cell line, we attribute the lower efficiency of repair in this cell line to imbalances between ectopic expression of XPA and other protein factors required for NER. When the removal of individual DBP-DNA adducts is compared, marked differences are observed in repair efficiency. Specifically, adducts A1/G2 and G3 were removed somewhat faster than the G4 adduct and much faster than the G1 adduct. Even though the RXR-XPA+ cell line again shows slower repair for each individual adduct than the MCF-7 and 8-D cell lines, the relative repair efficiencies for different adducts are consistent between cell lines. This indicates that the basic mechanism for recognition and removal of different adducts is conserved among these (and probably all) human cell lines. The inefficient removal of the G1 and possibly the G4 adducts may contribute disproportionately to the extreme carcinogenicity of DBP. Because we cannot reliably measure the repair of A1 adducts alone, these adducts may also persist and contribute significantly to mutagenesis and carcinogenesis. Our results with adducts generated by (()-anti-DBPDE are consistent with the concept that the efficiency of NER processes are highly dependent upon structural differences between DNA adducts. In agreement, studies in mouse skin indicate that externally localized DNA adducts of DBPDE seem to persist longer than intercalated adducts (46, 47). It is interesting to note that local DNA sequence context has also been shown to alter PAH DNA adduct conformation, thus potentially altering DNA damage recognition and repair of these adducts (48). Irrespective of whether the levels of DBP-DNA adducts are assessed at the overall or individual adduct level, the kinetics of adduct removal by NER appears to be biphasic, consisting of a relatively fast rate of removal of a significant percentage of adducts over the first 6 h, followed by a very slow rate of removal over the next 16-30 h. Although the reason for this observation is not clear, one possible explanation relates to the established transcription-coupled repair (TCR) and global genome repair (GGR) subpathways of NER. Specifically, the earlier/faster phase could correspond to TCR, while the slower/

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later phase corresponds to GGR. However, because 50% (on average) of the most abundant DBPDE-DNA adduct (A1/G2) is removed in cell lines with normal NER capacity (MCF-7 and 8-D) during this early phase, it would suggest that a disproportionately large amount of these adducts was generated in (the very low percentage of) actively transcribed DNA. Other studies (49-53) support the idea that some bulky DNA adducts are preferentially formed in transcriptionally active and/or linker DNA (with its more open conformation) as compared to less accessible heterochromatic or nucleosomal DNA. Of specific interest here, Quan and States (52) found in NER-deficient cells treated with BP dihydrodiol a markedly higher number of BP adducts in the active p53 gene as compared to the inactive β-globin gene and total genomic DNA, while Obi et al. (53) demonstrated highly preferential formation of BP adducts in active chromatin, particularly when treating with low concentrations of anti-benzo[a]pyrene-7,8-diol-9,10-epoxide. The latter finding may be particularly relevant to our studies that use very low concentrations of DBPDE. However, further studies are needed to examine whether the distribution of DBP-DNA adducts in the genome is heavily biased toward transcriptionally active DNA. It was noteworthy that in our experiments, treatment of the NER- cell lines with low concentrations of (()-anti-DBPDE did not significantly impede DNA synthesis or cell proliferation, at least over the short term. This suggests that these cells were able to replicate and divide in the presence of unrepaired DNA adducts (500-1000/109 nucleotides). At the minimum, this indicates that cells can efficiently circumvent and tolerate persistent bulky adducts. For DBP-DNA adducts, this tolerance may involve bypass of adducts in the template, either by replicative DNA polymerases or by specialized translesion polymerases (54). If such lesion bypass results in nucleotide misincorporation opposite a DBP-DNA adduct, mutations can occur. Thus, the persistence of one or more structurally distinct DBP-DNA adducts (such as G1 adducts) along with an increased frequency of misincorporation opposite these adducts may also contribute to the carcinogenicity of DBP. Acknowledgment. We thank Dr. Ramesh Gupta for his extensive involvement with the establishment of the postlabeling assay in the Orren laboratory, including his contribution of valuable materials and reagents. We thank Drs. Christopher States and Isabel Mellon for providing cell lines for our experiments and the latter also for critical reading of the manuscript. This work was supported by a grant to D.K.O. from the Kentucky Lung Cancer Research Program.

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