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[Cancer Research 64, 7321-7328, October 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

DNA Damage, Repair, and Mutation Induction by (+)-Syn and (–)-Anti-Dibenzo[a,l]Pyrene-11,12-Diol-13,14-Epoxides in Mouse Cells

Jung-Hoon Yoon1, Ahmad Besaratinia1, Zhaohui Feng2, Moon-shong Tang2, Shantu Amin3, Andreas Luch4 and Gerd P. Pfeifer1

1 Beckman Research Institute of the City of Hope, Duarte, California; 2 New York University School of Medicine, Tuxedo, New York; 3 Institute for Cancer Prevention, Valhalla, New York; and 4 Massachusetts Institute of Technology, Cambridge, Massachusetts


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental carcinogens. PAHs are classified into bay and fjord region compounds according to structural differences in the molecule region where enzymatic epoxidation occurs. Dibenzo[a,l]pyrene (DB[a,l]P), one of the fjord region compounds, has been demonstrated to be the most carcinogenic PAH known to date. DB[a,l]P is activated to fjord region (+)-syn and (–)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE) metabolites. In this study, we analyzed mutagenesis induced by (+)-syn- and (–)-anti-DB[a,l]PDE at the cII transgene in Big-Blue mouse cells. The mutant frequency of untreated cells (background level) was 6.53 x 10–5. This level increased 3.7-fold for 20 nmol/L, 5.3-fold for 50 nmol/L, and 7.9-fold for 100 nmol/L (+)-syn-DB[a,l]PDE, respectively. In the case of (–)-anti-DB[a,l]PDE it increased 4.5-fold for 20 nmol/L, 6.7-fold for 50 nmol/L, and 10.6-fold for 100 nmol/L, respectively, indicating that (–)-anti-DB[a,l]PDE is slightly more mutagenic than (+)-syn-DB[a,l]PDE. The mutational spectra of (+)-syn- and (–)-anti-DB[a,l]PDE were quite similar except for several hotspots, specific for either (+)-syn-DB[a,l]PDE or (–)-anti-DB[a,l]PDE. The most frequently induced mutations were A to T transversions, which were 43.9% for (+)-syn- and 38.8% for (–)-anti-DB[a,l]PDE. In addition, G to T transversions were induced significantly, at frequencies of 18.5% by (+)-syn- and 18.1% by (–)-anti-DB[a,l]PDE. Using UvrABC cleavage and ligation-mediated PCR or the terminal transferase-dependent PCR method, we have determined DB[a,l]PDE-DNA adduct formation sites and repair rates in carcinogen-exposed cells. The mutation hotspots coincided with sites of strong adduct formation, but not all of the adduct hotspots were mutational hotspots. Slow adduct removal occurred for both (+)-syn- and (–)-anti-DB[a,l]PDE adducts over a time period of up to 72 hours. The data suggest that, although the (–)-anti-isomer is slightly more mutagenic, DNA adducts of both DB[a,l]PDE stereoisomers may have similar biological properties. We discuss the implications of these findings for human cancer mutagenesis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polycyclic aromatic hydrocarbons (PAHs) are well-characterized environmental pollutants that have been causally linked to the initiation of lung cancer (1 , 2) . Because they are generated via incomplete combustion of organic matter, they are ubiquitously present in the human environment including human foodstuffs (3) . Although gasoline and diesel engines and industrial sources generate a significant amount of PAHs, the background levels in the general environment are rather low (3) . In contrast, carcinogenic PAHs present in tobacco smoke may be inhaled in large amounts depending on individual smoking habits (4, 5, 6) . PAHs are mainly activated by cytochrome P450 enzymes to form vicinal dihydrodiol epoxides ("diol-epoxides") as the ultimate carcinogenic metabolites (7, 8, 9) . Carcinogenic PAHs can be classified into bay and fjord region-containing compounds according to the structural difference in the area of epoxide formation (Fig. 1)Citation . Condensation of an additional benzo ring at position 11–12 in benzo(a)pyrene (B[a]P), the prototypical carcinogenic bay region PAH, produces the fjord region-containing hexacyclic dibenzo[a,l]pyrene (DB[a,l]P; Fig. 1Citation ). The bay region PAH B[a]P is toxicologically well studied, and its derivative, the (+)-anti-B[a]P-7,8-diol-9,10-epoxide (B[a]PDE), has been identified as the most genotoxic B[a]P diol-epoxide isomer in mammalian systems (9 , 10) . B[a]PDE forms covalent DNA adducts primarily at the exocyclic N2 position of 2'-deoxyguanosine (dG). B[a]PDE-N2-dG adducts were found to induce the G to T transversion mutation (11, 12, 13) , which is a molecular signature for cigarette smoke mutagens (2 , 14, 15, 16) .



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  		Fig. 1. Metabolic activation of DB[a,l]P toward stereoisomeric fjord region (+)-syn- and (–)-anti-11,12-diol-13,14-epoxides (DB[a,l]PDE). Metabolic activation is catalyzed by cytochrome P450 enzymes (P450) and microsomal epoxide hydrolase (mEH). The diol-epoxides can react with exocyclic amino groups of 2'-deoxyadenosine (N6-dA) or 2'-deoxyguanosine (N2-dG) in DNA.

 
According to the human p53 tumor mutation database, >30% of the mutations in lung cancer are G to T transversions, and six dominant mutation hotspots have been identified (2) . Five of the six mutational hotspots (codons 157, 158, 245, 248, and 273) are guanines at methylated CpG dinucleotide sites. As demonstrated by mapping of DNA adducts along the human p53 gene, these five hotspots are major binding sites for B[a]PDE and other PAH diol-epoxides (17 , 18) , and B[a]PDE preferentially induces G to T transversions at methylated CpG dinucleotides (11) . The data strongly support the notion that PAHs in cigarette smoke are a major risk factor for lung cancer. The involvement of PAHs in breast cancer etiology has also been suggested (19, 20, 21, 22) , but the available evidence is less clear.

The fjord region PAH DB[a,l]P (Fig. 1)Citation has been characterized as the strongest tumor initiator in mouse skin and rat mammary gland among all of the carcinogenic PAHs tested thus far (23, 24, 25) . DB[a,l]P was found to be several fold more carcinogenic than either 7,12-dimethylbenz[a]anthracene (DMBA) or B[a]P in both tumor models. DB[a,l]P is highly mutagenic in the metabolically competent MCL-5 cell line (26) . In the human mammary carcinoma cell line MCF-7, DB[a,l]P is stereoselectively activated to fjord region (+)-syn- and (–)-anti-DB[a,l]P-11,12-diol-13,14-epoxides (DB[a,l]PDE), which predominantly bind to dA residues in DNA (Fig. 1Citation ; refs. 27 , 28 ). Racemic syn- and anti-DB[a,l]PDE are extraordinary potent mutagens in both prokaryotic and eukaryotic test systems (29) and potent carcinogens in the rat mammary gland (30) . The strong mutagenicity of these compounds may partly result from an increased resistance to enzymatic repair of the lesions induced. As shown by Buterin et al., dA adducts derived from the binding of fjord region diol-epoxides are resistant to repair by nucleotide excision repair enzymes in human cell extracts (31) . In contrast, dG adducts derived from the binding of bay region diol-epoxides such as B[a]PDE are more efficiently removed. The differences in repair are due to structural differences between the two types of adducts (32 , 33) . These findings also may account for the high tumorigenic activities of some fjord region PAHs, because their stable adducts might persist in DNA for a much longer period. In addition, B[a]PDE-N2-dG adducts predominantly induce G to T transversions, whereas fjord region diol-epoxides from benzo[c]phenanthrene (B[c]Ph) or DB[a,l]P mainly induce A to T transversions but also other types of mutations in the supF gene of the pSP189 shuttle vector (34) and in the hprt gene of Chinese hamster V79 cells (35 , 36) . However, the detailed mechanisms of adduct formation, repair, and mutagenesis of DB[a,l]P diol-epoxides in mammalian cells remain to be elucidated. In this study, we have used Big-Blue transgenic mouse cells to analyze the mutation spectra of (+)-syn- and (–)-anti-DB[a,l]PDE stereoisomers in the cII transgene and mapped the DB[a,l]PDE-DNA adduct formation sites using UvrABC cleavage and litigation-mediated PCR. In addition, the repair efficiency of DB[a,l]PDE-DNA adducts was determined by the terminal transferase-dependent PCR method.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Treatment with DB[a,l]PDE.
Embryonic mouse fibroblasts were derived from 13.5-day-old Big Blue mouse embryos. Embryos were minced into small pieces and digested with collagenase overnight. After centrifugation, the pellet (exclusive of cartilage and bones) was resuspended and fibroblasts were cultured in DMEM containing 10% fetal bovine serum. (+)-Syn- and (–)-anti-DB[a,l]PDE were synthesized as described (29 , 37 , 38) .

For the mutagenesis experiments, fibroblasts were grown as monolayers at 20% to 30% confluence. The diol-epoxides were added from a DMSO stock solution to the cells to achieve a final concentration of 20 nmol/L, 50 nmol/L, or 100 nmol/L, and cells were incubated at 37°C for 30 minutes in the dark. At the end of the treatment, the medium was removed, the cells were washed with PBS, and regular DMEM was returned to the cells. They were grown for 6 days to allow mutation fixation. DNA was isolated by standard phenol-chloroform extraction and EtOH precipitation. This DNA was used in the cII mutation assays as described below.

Cytotoxicity Assay.
Cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Approximately 50,000 cells were cultured in a 24-well plate with DMEM containing 10% fetal bovine serum. Before treatment with the diol-epoxides, the culture medium was removed, cells were washed with PBS, and the medium was replaced with 1x Hanks’ balanced salt solution. The compounds were added to the cells to give a final concentration of 0 nmol/L (DMSO only), 10 nmol/L, 20 nmol/L, 40 nmol/L, 0.1 µmol/L, 0.2 µmol/L, 0.4 µmol/L, or 1 µmol/L, respectively. After incubation at 37°C for 30 minutes, the medium was removed, and cells were washed with PBS. Cells were incubated at 37°C for 24 hours in 400 µL of regular DMEM growth medium. After 24-hour incubation, 80 µL of the CellTiter 96 Aqueous One Solution Reagent (Promega, Madison, WI) were directly added into each well of the plate. The plates were incubated at 37°C for 2 hours in a humidified 5% CO2 atmosphere. Then, the absorbance at 490 nm was measured, and the cytotoxicity of the compounds was determined using solvent-treated cells as a control (regarded as 100% viability).

cII Mutation Assay.
The {lambda}-LIZ shuttle vector containing the cII target gene was rescued from total genomic DNA of embryonic mouse fibroblasts by mixing 5 µg DNA aliquots with {lambda} phage packaging extract (Transpack, Stratagene, La Jolla, CA) as described in the Big Blue manual. The cII mutation assay was performed with the G1250 hfl-Escherichia coli host strain (39) . To determine the total titer of packaged phage, 200 µL of the G1250 strain were mixed with 1:100 dilutions of phage, plated on TB1 plates in aliquots, and incubated overnight at 37°C. For mutant selection, 100 µL of the packaged phage were mixed with 200 µL of the G1250 strain, plated on TB1 plates, and incubated at 24°C for 48 hours. At 24°C, {lambda} phages bearing nonmutant cII genes underwent lysogenic growth, but phages with mutant cII genes underwent lytic growth and gave rise to plaques. When incubated at 37°C, non-cII mutants also underwent a lytic cycle due to a temperature-sensitive cI protein. The cII mutant frequency was calculated by dividing the number of mutant plaques by the number of total plaques. Experiments were done in triplicates. It is extremely unlikely that a single mutational event can be scored multiple times in this assay system, because <10% of the total phage genomes obtained after mutagen treatment was packaged into phage. In addition, only a maximum number of four to five population doublings may have occurred within 6 days, and packaging usually captures <0.1% of the {lambda} copies in the DNA (40) .

For sequencing analysis, mutant plaques were selected at random and replated at low density to verify the mutant phenotype and to isolate plaques. Single well-isolated plaques were picked, placed into 25 µL of Tris-EDTA buffer, and boiled for 5 minutes A 433-bp segment containing the cII gene and flanking regions was amplified by PCR with two primers, 5'-CCACACCTATGGTGTATG-3' (positions –68 to –50) and 5'-CCTCTGCCGAAGTTGAGTAT-3' (positions +345 to +365). The PCR products were purified using PCR purification kits (Qiagen, Chatsworth, CA) and were sequenced with a Big Dye terminator cycle sequencing kit (ABI Prism, PE Applied BioSystems, Foster City, CA) on an ABI DNA sequencer. The resulting DNA sequences were analyzed and compared with that of the wild-type cII gene with SeqWeb Version 1.2 software.

Mapping of DB[a,l]PDE-DNA Adducts and Determination of Repair Efficiency In vivo.
DNA adducts in the cII gene were mapped after exposure of embryonic fibroblasts to 0.5, 1, or 2.5 µmol/L of (+)-syn- or (–)-anti-DB[a,l]PDE, respectively, at 37°C for 30 minutes. The genomic DNA was purified and cleaved at DB[a,l]PDE-DNA adducts using the UvrABC nuclease complex of E. coli according to published procedures (11 , 18 , 41 , 42) . Under these conditions, cleavage by UvrABC is quantitative and occurs four nucleotides 3' to a PAH adduct. Sequences of both strands of the cII gene were amplified by litigation-mediated PCR as described previously (11) . For DNA repair assays, the more sensitive terminal transferase-dependent PCR assay was used (43 , 44) . In the repair assays, similar loading of DNA was ensured by exact determinations of DNA amounts in the starting material by agarose gel electrophoresis and by spectrophotometry. Individual terminal transferase-dependent PCR assay reactions were highly reproducible (±10%). The DNA was subjected to repeated primer extension in a mixture of Vent exo- minus DNA polymerase (New England Biolabs, Beverly, MA) and the primer U1 (5'-CCACACCTATGGTGTATG-3'; ref. 45 ). The thermocycler setting was as follows: 2 minutes at 95°C, 2 minutes at 61°C, 3 minutes at 72°C, nine cycles (in which one cycle consisted of 45 seconds at 95°C, 2 minutes at 61°C, and 3 minutes at 72°C), 45 seconds at 95°C, 2 minutes at 61°C, and 10 minutes at 72°C. The extension product was mixed with streptavidin-coupled magnetic beads (Dynal ASA, Oslo, Norway), and binding was achieved by gently rotating the mixture for 45 minutes at room temperature. The streptavidin-bound DNA was denatured by incubation with 0.15 mol/L NaOH at 37°C for 10 minutes. After multiple washes with 1x TE buffer (pH 7.5) in a magnetic particle concentrator (Dynal ASA), the single-stranded DNA was resuspended in 0.1x TE buffer (pH 7.5) and afterward subjected to homopolymeric ribotailing and adapter ligation (46) . The ligation product was washed with TE buffer (pH 8.0) in the magnetic particle concentrator, resuspended in 0.1x TE buffer (pH 8.0), and amplified in a PCR reaction using primer U2 (5'-ATGGTGTATGCATTTATTTGCATA-3') and a custom-made LP25 primer (46) in the presence of Expand Long Polymerase (Roche, Indianapolis, IN). The thermocycler was set for 2 minutes at 95°C, 2 minutes at 62°C, 3 minutes at 72°C, 18 cycles (in which one cycle consisted of 45 seconds at 95°C, 2 minutes at 62°C, and 3 minutes at 72°C), 45 seconds at 95°C, 2 minutes at 62°C, and 10 minutes at 72°C. The PCR products were separated on a 5% polyacrylamide/urea gel by electrophoresis and were electroblotted to a nylon membrane. They were visualized by hybridization with a cII-specific probe. The sites of DNA adduct formation were identified as the locations at which the presence of the lesions stopped the DNA polymerase from progressing. The data were quantitated by phosphorimaging. Repair efficiency was determined from the intensity of adduct-specific bands at various time points relative to the intensity at 0 hour.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cytotoxicity of DB[a,l]PDE in Mouse Embryo Fibroblasts.
To determine the cytotoxicity of the (+)-syn- and (–)-anti-DB[a,l]PDE stereoisomers, mouse embryo fibroblasts derived from BigBlue mice were treated with different concentrations of these compounds (Fig. 2)Citation . Concentrations >200 nmol/L DB[a,l]PDE were severely cytotoxic. The cytotoxicity assay results suggest that (–)-anti-DB[a,l]PDE was slightly more toxic than (+)-syn-DB[a,l]PDE (Fig. 2)Citation , although the difference was not statistically significant (Wilcoxon signed rank test). On the basis of the cytotoxicity results, concentrations of 100 nmol/L or lower were used for the mutagenesis assays.



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  		Fig. 2. Cytotoxicity of (+)-syn- and (–)-anti-DB[a,l]PDE. BigBlue mouse embryo fibroblasts were treated with different concentrations of (+)-syn- and (–)-anti-DB[a,l]PDE for 30 minutes. Viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (see Material and Methods). Experiments were performed in triplicates; bars, ±SD.

 
DB[a,l]PDE-Induced Mutagenesis in the cII Transgene.
Big Blue transgenic mice harbor the {lambda} LIZ vector containing the cII transgene as a mutational reporter. Big Blue mouse embryonic fibroblast cells were treated with 20 nmol/L, 50 nmol/L, and 100 nmol/L of (+)-syn or (–)-anti-DB[a,l]PDE. To allow mutation fixation, the cells were incubated for 6 days after DB[a,l]PDE treatment. Mouse genomic DNA was isolated, packaged into {lambda} phage particles, and then used to determine the mutant frequency and mutational spectra in the cII transgene. The mutant frequency of untreated cells (DMSO only) was 6.53 x 10–5 (Fig. 3)Citation . The mutant frequency of (+)-syn-DB[a,l]PDE-treated cells increased in a dose-dependent manner up to 42.1 x 10–5 (7.9-fold increase) for 100 nmol/L of the diol-epoxide. The mutant frequency of (–)-anti-DB[a,l]PDE-treated cells increased up to 69.2 x 10–5 (10.6-fold increase) for 100 nmol/L of the diol-epoxide (Fig. 3)Citation . Thus, the induced mutant frequency of (–)-anti-DB[a,l]PDE was slightly higher than that of (+)-syn-DB[a,l]PDE (Fig. 3)Citation .



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  		Fig. 3. Mutant frequencies induced by (+)-syn- and (–)-anti-DB[a,l]PDE. BigBlue mouse embryo fibroblasts were treated with different concentrations of (+)-syn- and (–)-anti-DB[a,l]PDE for 30 minutes. After removal of the carcinogen, cells were incubated for 6 days for mutation fixation. Mutations introduced into the cII {lambda} transgene were determined by a plaque formation assay (see Materials and Methods). Experiments were performed in triplicates; bars, ±SD.

 
Fig. 4Citation shows the mutation spectra in the cII transgene obtained with solvent control and with 100 nmol/L of (+)-syn- or (–)-anti-DB[a,l]PDE treatment, respectively. The distribution of mutations was quite similar between (+)-syn- and (–)-anti-DB[a,l]PDE except for several hotspots. Due to the difference in the total number of mutants sequenced, we defined a hotspot as four mutations at an identical site for (–)-anti-DB[a,l]PDE and five mutations at an identical site for (+)-syn-DB[a,l]PDE. Three mutation hotspots, at positions 98, 124, and 193, were in common between both isomers. Positions 1, 26, 41, and 173 were mutation hotspots unique to (+)-syn-DB[a,l]PDE, and positions 25, 53, and 164 were unique to (–)-anti-DB[a,l]PDE. However, it should be noted that the designation of hotspots is somewhat arbitrary, because the difference between having three or four mutation at one site is not statistically significant. Also, a number of mutations at positions 26 and 193 occurred in the control cells and may not be induced by the treatments. In analyzing the types of mutations induced by the compounds, we combined the strand mirror counterparts (i.e., A to T and T to A, G to A and C to T) because the cII transgene is not transcribed, and effects from strand-specific DNA repair cannot be expected. A substantial fraction of the DB[a,l]PDE-induced mutants analyzed harbored A to T transversion mutations, 43.9% in (+)-syn- and 38.8% in (–)-anti-DB[a,l]PDE-treated cells, respectively (P < 0.001 relative to control for both isomers; {chi}2 test; Table 1Citation ). Besides producing an increase in A to T transversions, both stereoisomers specifically induced G to T transversion mutations. This type of mutation was induced at a frequency of 18.5% by (+)-syn- and 18.1% by (–)-anti-DB[a,l]PDE compared with 9.3% in solvent-treated cells (P < 0.025 for both isomers; {chi}2 test).



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  		Fig. 4. Spectrum of spontaneous and DB[a,l]PDE induced mutations in the cII transgene. A, mutations in control- (solvent) treated cells. B, mutations in cells treated with (+)-syn-DB[a,l]PDE. C, mutations in cells treated with (–)-anti-DB[a,l]PDE. Mutations in the cII gene were identified by PCR and DNA sequencing. The number of sequenced mutants was 150 for control treatment, 205 for treatment with (+)-syn-DB[a,l]PDE, and 160 for treatment with (–)-anti-DB[a,l]PDE. Single base deletions are marked by -x-, and single base additions are marked by +N. The nucleotide positions are numbered. A stretch of six guanines (underlined) is characterized by frequent single nucleotide insertions and deletions in control cells. Sites of DB[a,l]PDE DNA adduct formation on the coding strand are indicated by *, and sites of adduct formation on the noncoding strand are indicated by {bullet} (B and C). Data for adduct formation was available for positions 90 to 150 on the coding strand and for positions 60 to 180 on the noncoding strand (see Figs. 5Citation and 6Citation ).

 

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  		Table 1 Types of mutations induced by (+)-syn-DB[a,l]PDE and (–)-anti-DB[a,l]PDE

 
Mapping of DB[a,l]PDE-DNA Adducts by UvrABC Cleavage and Litigation-Mediated PCR.
The occurrence of mutation hotspots might be closely related to selective formation of DB[a,l]PDE adducts. To test this relationship, UvrABC cleavage in combination with the litigation-mediated PCR method (11 , 17 , 18 , 41) was applied for the mapping of DB[a,l]PDE adduct formation sites in the cII transgene. Because of a sensitivity limit with litigation-mediated PCR, a higher dose of DB[a,l]PDE (0.5 µmol/L, 1 µmol/L, and 2.5 µmol/L) was used as compared with the mutagenesis experiments (100 nmol/L). Figs. 5Citation and 6Citation show the mapping of DB[a,l]PDE adducts on the coding strand and noncoding strand of the cII transgene, respectively. The sequence-dependent adduct pattern (i.e., the relative intensity of the bands within one sequencing lane) was not strongly dependent on the concentration of the diol epoxides. Thus, comparisons between adduct and mutation spectra are meaningful even when different concentrations were used, although we cannot entirely exclude the possibility that the sites adducted at the range of concentrations of 500 to 2,500 nmol/L may be different from those adducted at a 100 nmol/L concentration. Almost all of the adducted positions corresponded to either adenines or guanines. An exception was position 97 to 99 (Fig. 6Citation , sequence CAC) where adduct-specific bands were assigned to all three of the bases. This could mean that adducts did form at the cytosines at positions 97 and 99 or, more likely, that the UvrABC nuclease did not make a precise 3' incision at the adenine adduct present at position 98. The patterns of adduct formation by the two isomers were very similar but not completely identical. Several strong adduct formation sites were apparent at different sites for the two isomers, and the strongest sites all corresponded to adenine adducts. Several of the strong adduct formation sites (for example, positions 98, 124, and 173; Figs. 5Citation and 6Citation ) corresponded to sites of frequent mutations. Of particular note, the mutational hotspots at position 173, specific for (+)-syn-DB[a,l]PDE, and at position 164, specific for (–)-anti-DB[a,l]PDE, corresponded to selectively stronger adduct formation sites of the corresponding stereoisomers (Fig. 6Citation , top panel). However, clearly not all sites of adduct formation were mutational hotspots.



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  		Fig. 5. Mapping of DB[a,l]PDE adducts in the cII transgene (coding strand). Big Blue mouse embryonic fibroblasts were treated with (+)-syn- or (–)-anti-DB[a,l]PDE. Genomic DNA was isolated and cleaved at the sites of adducts with UvrABC nuclease. The coding strand sequence was amplified by litigation-mediated PCR. Several strong adduct formation sites (marked with *) were identified. UvrABC cleaves four nucleotides 3' to a PAH adduct. Thus, adduct-specific bands migrate four nucleotides faster than the corresponding band in the Maxam-Gilbert sequencing lanes. Nucleotide positions are indicated by numbers. NO, DNA from solvent-treated cells.

 


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  		Fig. 6. Mapping of DB[a,l]PDE adducts in the cII transgene (noncoding strand). Big Blue mouse embryonic fibroblasts were treated with (+)-syn- or (–)-anti-DB[a,l]PDE. Genomic DNA was isolated and cleaved at the sites of adducts with UvrABC nuclease. Two different portions of the noncoding strand sequence were amplified by litigation-mediated PCR. Several strong adduct formation sites (marked with {bullet}) are identified. UvrABC cleaves four nucleotides 3' to a PAH adduct. Thus, adduct-specific bands migrate four nucleotides faster than the corresponding band in the Maxam-Gilbert sequencing lanes. Nucleotide positions are indicated by the numbers. Bands at positions higher than nucleotide 180 could not be resolved. NO, DNA from solvent-treated cells.

 
Determination of Repair Efficiency of (+)-syn- and (–)-anti-DB[a,l]PDE-DNA Adducts by Terminal Transferase-dependent PCR.
To determine whether DB[a,l]PDE adducts are removed by DNA repair processes, we exposed mouse fibroblasts to a concentration of 200 nmol/L of both (+)-syn and (–)-anti-DB[a,l]PDE and incubated the cells for time periods of up to 72 hours. Because litigation-mediated PCR produces only weak signals at this concentration of the diol-epoxides, and repair assays, in which adduct amounts are progressively reduced with time, would not be reliable, we used terminal transferase-dependent PCR to examine the repair of these adducts (Fig. 7)Citation . This assay is based on the exponential amplification of fragments created by polymerase arrest. Due to the introduction of a ribonucleotide tailing step before PCR amplification, which introduces between two and four additional nucleotides into the PCR products, the amplified products have a corresponding length variability even when only one strong adduct formation site is present. The appearance of faint bands in the untreated samples is probably due to low levels of depurination occurring during DNA isolation (like the presence of adducts, this will lead to appearance of signals at positions of A and G) or is due to polymerase pausing at sites of DNA secondary structure. Pausing within these sequences may be enhanced by the presence of adducts, or the congruence of sites of polymerase pausing and for preferential adduct formation may be inherent to DNA secondary structures that favor both events. Strong sequence-selective polymerase pause sites were seen upon treatment with the diol-epoxides. One intense cluster of bands was seen near position 98, which is a mutational hotspot for both isomers and also corresponds to a strong litigation-mediated PCR band at the same position (Fig. 6)Citation . The disappearance of specific polymerase blocking sites was apparent over the time course (Fig. 7)Citation . At 72 hours, ≥ 90% of the (+)-syn-DB[a,l]PDE-DNA adducts were removed from most sites. Repair occurred with a lower efficiency for the (–)-anti-DB[a,l]PDE-DNA adducts with ~70% to 90% adduct removal after 72 hours. Repair was not much sequence-dependent for (+)-syn-DB[a,l]PDE adducts (<1.5-fold difference at each time point), but some site-to-site variation with greater differences was observed for (–)-anti-DB[a,l]PDE-DNA adducts (Fig. 7)Citation .



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  		Fig. 7. Determination of repair efficiency of (+)-syn- and (–)-anti-DB[a,l]PDE-DNA adducts in mouse embryonic fibroblasts. Repair was analyzed by terminal transferase-dependent PCR after exposure of the cells to 200 nmol/L of the diol-epoxides and incubation for various periods of time. Maxam-Gilbert sequencing lanes are shown as position markers. Note that an adduct-derived band represented by a polymerase stop signal migrates 3 to 4 nucleotides slower than the corresponding Maxam-Gilbert band due to the ribonucleotide-tailing step in terminal transferase-dependent PCR. NO, DNA from solvent-treated cells. Signals derived from four areas of polymerase stop signals (adduct sites) were quantitated using a phosphorimager and plotted as a function of repair time. Experiments were performed in triplicates; bars, ±SD.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We report a detailed comparison of the mutagenicity of the two isomeric fjord region diol-epoxides from DB[a,l]P that are stereoselectively formed in cells in culture and in vivo (Fig. 1)Citation . Although (–)-anti-DB[a,l]PDE was slightly more mutagenic than (+)-syn-DB[a,l]PDE, the mutational spectra produced by these two compounds were very similar. The majority of the induced mutations were transversions, with A to T transversions being the preferred type of mutation for both stereoisomers, and G to T transversions also being significantly induced. The distribution of mutational hotspots differed at several positions along the cII mutational reporter gene. Some of these differences (positions 164 and 173) could be ascribed to different DNA adduct levels forming at specific sites. Data for adduct formation was available for positions 90 to 150 on the coding strand and for positions 60 to 180 on the noncoding strand. litigation-mediated PCR amplification of other parts of the sequence was unsuccessful, or the bands could not be sufficiently resolved (higher than position 180). For the sequences for which both adduct and mutation data were available, the major mutation hotspots (positions 98, 124, 164, and 173) were sites of strong adduct formation, but not all of the strong adduct formation sites were mutational hotspots(Figs. 4Citation to 6Citation ). The reasons for the latter observations can be, for example, that adducts forming at certain sequences, in particular at the third codon position, may not produce a mutation that is selected; there may be correct polymerase bypass of adducts at certain positions leading to no mutation; and other error-free pathways may operate on these lesions. Also, in many cases mutations did occur at the sites of adduct formation but were not mutation hotspots according to the definition applied. Adducts produced by both DB[a,l]PDE isomers were repaired at a rather slow rate, and repair was not much DNA sequence dependent.

It has been proposed that carcinogenic PAHs are involved in breast tumorigenesis (19, 20, 21, 22 , 47) . Many PAHs are known mammary carcinogens in rodents, and DB[a,l]P is the most potent carcinogen ever tested in the rat mammary gland (24 , 25) . DB[a,l]P has been detected in cigarette smoke, although the concentrations are very low and rather uncertain (48, 49, 50) . Cigarette smoking is an established cause of a variety of cancer types, but its role in breast cancer etiology is not clear with a relative risk of <2-fold in most studies (22 , 51) . PAH-like bulky DNA adducts have been consistently found in both normal and breast tumor tissues (47 , 52, 53, 54) . It is likely that specific mammary carcinogens in tobacco smoke can reach breast tissue, although the dose will be much lower than in the lung. Besides cigarette smoke, DB[a,l]P might be present within other PAH mixtures, such as in particulate matter in polluted air, combustion products, and in grilled or barbecued food, but unambiguous determinations are still missing. Selective metabolic activation and/or retention of the carcinogen in breast tissue may make these sources of DB[a,l]P a potentially important exposure factor.

The mutation spectra of the DB[a,l]PDE isomers, as shown here, are similar to those of isomeric 3,4-diol-1,2-epoxides of B[c]Ph (B[c]PhDE), another fjord region compound, which has been studied in the supF gene of the pSP189 shuttle vector (34) and in the hprt gene in Chinese hamster V79 cells (36) . In the supF study, similar frequencies of A to T (35%) and G to T (29%) mutations were observed (34) . In the hprt mutation study, A to T transversions predominated (55% to 60%) in the case of (–)-anti-B[c]PhDE-induced mutations. In contrast, the (+)-anti-enantiomer, which is metabolically and toxicologically without relevance, gave an equal proportion (~35%) of both types of transversions (36) . In addition, there were relatively more (62% versus 55%) A to T transversions at the lower doses of (–)-anti-B[c]PhDE (defined as 10 to 500 nmol/L) as compared with the higher doses (defined as 1 to 3 µmol/L). Because we derived mutation spectra only for the 100 nmol/L DB[a,l]PDE treatments, we do not know if higher doses of these diol-epoxides would increase the relative frequency of G to T transversions over the 18% to 19% level observed at 100 nmol/L. Such studies would be seriously hampered by the increased cytotoxicity of DB[a,l]PDE at the higher concentrations.

The mutagenicity of racemic syn- and anti-DB[a,l]PDE was examined in four his- strains of Salmonella typhimurium and in Chinese hamster V79 cells (29) . In all five of the test systems, these compounds were much more potent than any of the bay or fjord region diol-epoxides tested before (29 , 55) . The specific mutagenicity observed with anti-DB[a,l]PDE in strain TA104 exhibited the highest value ever found with any compound in any his-strains of S. typhimurium. A similar result was obtained with this compound in V79 cells. In both mutagenicity assays, syn-DB[a,l]PDE was moderately less active as compared with its anti-diastereomer (~2-fold). More recently, the mutagenic pattern induced by enantiomerically pure (–)-anti-DB[a,l]PDE at the hprt locus of V79 was investigated (35) . Although the number of sequenced mutants was lower than in this study, increases in A to T and A to G mutations, as well as G to T transversions were observed. Unlike in our study, A to T transversions were not particularly pronounced (~25%). The frequencies of G to T transversions were 9% at 1 nmol/L, 31% at 2 nmol/L, and 44% at 10 nmol/L (–)-anti-DB[a,l]PDE, respectively. Thus, the relative level of G to T transversions was found to be increasing with increasing doses of the diol-epoxide (compare B[c]PhDE above). However, at low doses of this compound, A to T transversions were prevailing (27% A to T versus 9% G to T at 1 nmol/L diol-epoxide; ref. 35 ).

In our previous studies (11 , 56) , we showed that anti-B[a]PDE preferentially formed adducts with dG at methylated CpG dinucleotides, and these adducts induced G to T base substitutions. Most of the anti-B[a]PDE-induced mutation hotspots were at methylated CpG sites, and these mutation hotspots corresponded to the major anti-B[a]PDE-N2-dG adduct formation sites in the cII gene (11) . In contrast, both (+)-syn- and (–)-anti-DB[a,l]PDE predominantly induced A to T transversions and also produced several hotspots of this mutation type at the cll gene in mouse cells. Both isomers also induced a considerable level of G to T transversions, the only other type of mutation that was significantly more frequent relative to the background control (P < 0.025; {chi}2 test). About 37% of these G to T transversions occurred at methylated CpG sites compared with 17% in control-treated cells, although G to T transversions increased also at non-CpG sequences. Thus, fjord region PAHs could theoretically contribute to the G to T mutational signature, which is present in lung cancers from smokers (2) . However, A to T transversions, the main DB[a,l]PDE-induced mutation type, are not particularly common in lung cancers from smokers (only 4% to 6%; ref. 2 ), which would argue against a major specific involvement of DB[a,l]P or structurally related PAHs in tobacco-related lung tumors. A to T transversions are relatively more common in small cell lung cancers (12%) and in cancers of the larynx and esophagus (8% to 9%; ref. 2 ). In breast cancer, A to T transversions are also rare (4%, according to the International Agency for Research on Cancer p53 mutation database, version R8; ref. 57 ). Therefore, the currently available human cancer mutation data, although limited mostly to the p53 gene, do not suggest a major involvement of DB[a,l]P or related fjord region PAHs in human cancer etiology. This might be due to the rather low concentrations of these compounds in cigarette smoke as compared with bay region PAHs such as B[a]P (49) .


   ACKNOWLEDGMENTS
 
We thank Steven Bates for culture of mouse embryo fibroblasts.


   FOOTNOTES
 
Grant support: National Cancer Institute (CA84469; G. P. Pfeifer).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, CA. Phone: 626-301-8853; Fax: 626-358-7703; E-mail: gpfeifer{at}coh.org

Received 3/29/04. Revised 8/ 3/04. Accepted 8/16/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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